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• Research highlights
  - Nat Struct Mol Biol 18(7):747 (2011)
  Article preview View full access options Nature Structural & Molecular Biology | Research Highlights Research highlights Journal name:Nature Structural & Molecular BiologyVolume: 18,Page:747Year published:(2011)DOI:doi:10.1038/nsmb0711-747Published online06 July 2011 Inhibiting amyloid formation Amyloid fibril deposits are associated with a number of neurodegenerative conditions. The relationship between the formation of such fibrils and the etiology of the diseases is not totally clear, but there has been considerable progress in understanding amyloid structures. This information comes mainly from X-ray crystallography of fibrils formed by short segments from the full-length proteins. Such fibrils have properties similar to those from amyloids formed with the full-length polypeptides. A common structural feature observed is the so-called steric zipper, in which β-sheets are held together by their interdigitated side chains; in the full-length amyloids, these short segments would stack to form the cross-β spine of the growing filament. Now Eisenberg and colleagues have used this detailed structural knowledge to design peptides that bind to the end of the fibrils and block them from further growth. The authors chose as targets the fibrils formed by peptides from ta! u protein and from prostatic acid phosphatase (PAP). Fibrils derived from these polypeptides have been respectively implicated in Alzheimer's disease and shown to increase infection by HIV-1. The inhibitory peptides were computationally designed to bind tightly to the ends of the steric zippers, with the interfaces maximized for hydrogen bonding and hydrophobic interactions. To achieve this, the authors used both D-amino acids and non-natural L-amino acids. They then found that the inhibitory peptides can efficiently block fibril formation by the full-length polypeptides in vitro. In addition, the inhibitor against PAP fibrils could also suppress the increase in HIV infectivity caused by the fibrils in human cells in culture. The predicted mode of interaction of the peptides with the fibrils was supported by electron microscopy imaging and by NMR spectroscopy. In addition to opening a new avenue for potential therapeutic intervention, the work supports the assertion that st! eric zippers are the structural spine of native amyloid fibril! s. (Nature doi:10.1038/nature10154, published online 15 June 2011) IC Nuclear membrane protein import Nuclear pore complexes (NPCs) have a cylindrical central channel, in which nucleoporins (Nups) with disordered phenylalanine-glycine (FG)-rich regions provide the selectivity barrier. Some nuclear membrane proteins reach the inner nuclear membrane (INM) by diffusion through the pore membrane and adjacent lateral channels, and accumulate by binding nuclear structures. Other nuclear membrane proteins have a nuclear localization signal (NLS) and bind to transport factors karyopherin-α and karyopherin-β1 to pass the NPC and reach the INM. Veenhoff and colleagues now reveal new insights into the transport pathway of these NLS-containing nuclear membrane proteins. By using green fluorescent protein reporters of an integral INM protein from budding yeast in confocal fluorescence microscopy and immunoelectron microscopy analyses, the authors showed that the NLS-containing domain is sufficient for accumulation at the INM. The long linker region between the NLS and transmembrane dom! ain is unstructured, as demonstrated by NMR analysis, and reporter proteins containing synthetic unfolded linker regions of sufficient length were efficiently transported to the INM. Remarkably, reporters containing a synthetic linker, an NLS and either one or all ten transmembrane segments of the endoplasmic reticulum protein Sec61 were efficiently imported to the INM. Transport of the reporters across the NPC was dependent on FG-rich regions of nucleoporins of the central channel, suggesting that nuclear membrane proteins pass through the central channel of the NPC. Trapping of reporter proteins at the Nup Nsp1–containing central channel of the NPC specifically blocked transport of membrane proteins but not soluble proteins. Together, these findings suggest a transport mechanism that is likely to exist in parallel with a previously proposed route based on diffusion and nuclear retention. (Science doi:10.1126/science.1205741, published online 9 June 2011) AH Article preview Read the full article * Instant access to this article: US$32 Buy now * Subscribe to Nature Structural & Molecular Biology for full access: Subscribe * Personal subscribers: Log in Additional access options: * Login via Athens * Login via your Institution * Purchase a site license * Use a document delivery service * British Library Document Supply Centre * Infotrieve * Thompson ISI Document Delivery * You can also request this document from your local library through inter-library loan services. Additional data
• Unraveling the mechanism of BRCA2 in homologous recombination
  - Nat Struct Mol Biol 18(7):748-754 (2011)
  Nature Structural & Molecular Biology | Review Unraveling the mechanism of BRCA2 in homologous recombination * William K Holloman1Journal name:Nature Structural & Molecular BiologyVolume: 18,Pages:748–754Year published:(2011)DOI:doi:10.1038/nsmb.2096Published online06 July 2011 Abstract * Abstract * Author information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg BRCA2 is the product of a breast cancer susceptibility gene in humans and the founding member of an emerging family of proteins present throughout the eukaryotic domain that serve in homologous recombination. The function of BRCA2 in recombination is to control RAD51, a protein that catalyzes homologous pairing and DNA strand exchange. By physically interacting with both RAD51 and single-stranded DNA, BRCA2 mediates delivery of RAD51 preferentially to sites of single-stranded DNA (ssDNA) exposed as a result of DNA damage or replication problems. Through its action, BRCA2 helps restore and maintain integrity of the genome. This review highlights recent studies on BRCA2 and its orthologs that have begun to illuminate the molecular mechanisms by which these proteins control homologous recombination. View full text Figures at a glance * Figure 1: DNA repair by homologous recombination. Schematics illustrate how the repair of DNA with a DSB or ssDNA gap can be initiated by homologous recombination. Both pathways begin with invasion of a ssDNA tract (1a or 1b) into a homologous DNA sequence. In DSB repair, the D-loop formed after invasion of the ssDNA tail (2a) is extended by DNA synthesis (3a) until it can pair with, or capture, the second end through an exposed complementary sequence. The 3′ strand of the second end serves as a primer for fill-in synthesis (4a). Additional processing of the intermediates and several subsequent steps result in a repaired chromosome. In the other pathway, termed post-replication repair, an ssDNA gap generated during DNA replication of a damaged template (1b) invades the undamaged homologous sequence present in the sister chromatid (2b). By branch migration through a short tract, the 3′ end of the broken strand switches templates (3b) and then serves as a primer for fill-in synthesis (4b). After additional steps, the inte! rmediate is resolved and repair is completed. * Figure 2: BRCA2 organization and DBD domain structure. () BRCA2 is shown schematically with protein interaction motifs and domains identified on top. The acronyms are as described in the text. CTRM stands for 'C-terminal RAD51-binding motif'. The elements are illustrated as gray and black boxes. The DBD is broken down into the helix-rich domain (hatches) and the three oligonucleotide-binding folds (ovals). The approximate regions of interaction with the various proteins and DNA are shown underneath. () The 800-residue DBD is shown schematically with the helix-rich domain (HD) followed by the oligonucleotide-binding folds. OB2 and OB3 are packed in tandem whereas OB1 is packed with OB2 in the opposite orientation. The tower domain emerges from OB2 and has a three-helix bundle (3HB) on top. ssDNA (black squiggle) interacts with OB2 and OB3. DSS1 (gray squiggle) interacts with the helix-rich domain and OB1 on the opposite face of the domain. Author information * Abstract * Author information Affiliations * Department of Microbiology and Immunology, Weill Cornell Cancer Center, Weill Cornell Medical College, New York, New York, USA. * William K Holloman Competing financial interests The author declares no competing financial interests. Corresponding author Correspondence to: * William K Holloman Author Details * William K Holloman Contact William K Holloman Search for this author in: * NPG journals * PubMed * Google Scholar Additional data
• The structural basis of modularity in ECF-type ABC transporters
  - Nat Struct Mol Biol 18(7):755-760 (2011)
  Nature Structural & Molecular Biology | Article The structural basis of modularity in ECF-type ABC transporters * Guus B Erkens1, 2 * Ronnie P-A Berntsson1, 2 * Faizah Fulyani1, 2 * Maria Majsnerowska1, 2 * Andreja Vujičić-Žagar1, 2 * Josy ter Beek1, 2 * Bert Poolman1, 2 * Dirk Jan Slotboom1, 2 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:755–760Year published:(2011)DOI:doi:10.1038/nsmb.2073Received12 January 2011Accepted21 April 2011Published online26 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Energy coupling factor (ECF) transporters are used for the uptake of vitamins in Prokarya. They consist of an integral membrane protein that confers substrate specificity (the S-component) and an energizing module that is related to ATP-binding cassette (ABC) transporters. S-components for different substrates often do not share detectable sequence similarity but interact with the same energizing module. Here we present the crystal structure of the thiamine-specific S-component ThiT from Lactococcus lactis at 2.0 Å. Extensive protein-substrate interactions explain its high binding affinity for thiamine (Kd ~10−10 M). ThiT has a fold similar to that of the riboflavin-specific S-component RibU, with which it shares only 14% sequence identity. Two alanines in a conserved motif (AxxxA) located on the membrane-embedded surface of the S-components mediate the interaction with the energizing module. Based on these findings, we propose a general transport mechanism for ECF transp! orters. View full text Figures at a glance * Figure 1: The structure of ThiT. () Architecture of ABC and ECF transporters. The three types of ABC transporters are SBD-dependent ABC-type importers (left), ABC exporters (center) and the recently discovered ECF transporters (right). The NBDs (blue) are conserved among all ABC transporters; the TMDs (various colors) are different. Substrates are indicated as black dots. () Surface (left) and secondary structure ribbon representation (right) of ThiT. In the surface model, hydrophobic residues are gray and hydrophilic residues are green; positively charged residues are blue and negatively charged residues are red (the latter are not visible in this orientation). The ribbon is colored from N-terminal blue to C-terminal red. The bar indicates the approximate position of the lipid membrane (35 Å). The membrane topology for ThiT is depicted below and colored as in the ribbon model. H1–H6, helices 1–6; L1–L5, loops 1–5. () The dimer of ThiT in the asymmetric unit. One monomer is colored purple, the seco! nd gray and bound thiamine in black. The dashed lines indicate the position of the membrane for both monomers. () The unusual structure of helix 4, colored green; the rest of ThiT is depicted in gray. The lines indicate the vertical position of the secondary structure elements. * Figure 2: The high-affinity thiamine-binding site. () Electron density for thiamine shown in gray mesh (2Fo – Fc map contoured at 1.5σ), with the modeled thiamine molecule. () Residues forming hydrogen bonds and aromatic interactions with thiamine. Carbon atoms of the thiamine molecule and side chains of the binding residues are shown in green and blue, respectively. Hydrogen bonds are indicated by the red dashes. Tyr146 interacts with thiamine through a water molecule (black asterisk). () Ribbon and sliced-surface models of the ThiT structure with the conserved amino acids in ThiT homologs colored according to their conservation score. The arrow indicates the access to the cavity that can accommodate phosphate moieties of TMP and TPP. * Figure 3: Transport of [3H]thiamine in E. coli and L. lactis cells. () Thiamine uptake by recombinant E. coli cells. E. coli cells expressing ThiT and EcfAA′T from L. lactis (•) or ThiT alone (○), and control cells containing an empty expression plasmid (▪), were assayed for thiamine uptake. All cells were energized with glucose. () Thiamine uptake by recombinant L. lactis cells. De-energized control cells (harboring an empty plasmid but containing chromosomal copies of the genes thiT (also known as llmg_0334) and ecfAA′T (cbiO, cbiO, cbiQ2) (○), de-energized cells expressing ThiT from a plasmid (∇), energized control cells (•) and energized cells expressing ThiT (▴) were assayed for thiamine uptake. Thiamine binding, rather than transport, was observed in the de-energized cells. The levels of binding depended on the expression levels of ThiT. In the energized cells harboring the empty plasmid, rapid thiamine uptake was observed. In energized cells overexpressing ThiT, the offset on the y axis—indicative of binding—incr! eased, rather than the uptake rate. All experiments were conducted at least in duplicate. The error bars indicate the range () or s.d. (). * Figure 4: Interaction of ThiT with the energizing module. () Superposition of the RibU structure in gray (PDB 3P5N)5 on the ThiT structure (colored as in Fig. 1b). The dashed line indicates the proposed interface with the energizing module. The helices are numbered from N terminus to C terminus as in Figure 1b. () The ThiT structure as seen from the interface with the energizing module. The surface of the L1 loop region is highlighted in blue and indicated by the dashed circle. Rearrangement of the L1 loop would expose the bound thiamine (black sticks) to the lateral EcfT interface. The alanine motif in helix 1 that is shared by all S-components in L. lactis is colored red. () Thiamine uptake by recombinant E. coli cells coexpressing EcfAA′T with wild-type ThiT (○), A15W ThiT (n) or A19W ThiT (▾). Thiamine uptake by a control strain harboring an empty plasmid is indicated by the black circles (•).The error bars indicate the upper and lower measured values. The inset shows western blot analyses of the expression levels of Hi! s-tagged EcfT (using antibodies directed against the His tag) and Strep-tagged ThiT (using antibodies against the Strep tag). () Pull-out experiment. EcfAA′T with a His tag on EcfT was coexpressed with Strep-tagged wild-type ThiT, or variants A15W or A19W in E. coli as in . Membranes were solubilized and the complexes were purified using nickel-affinity chromatography. The elution fractions were analyzed by SDS-PAGE followed by western-blot analysis using antibodies against ThiT-strep. * Figure 5: Working model for substrate translocation and interaction with EcfA. () Model for coupling helix interaction in ECF transporters. The S-component is colored red, EcfT is orange and the EcfA subunits are blue. () Specific interactions between the L1 loop and helices 5 and 6. Glu38 in L1 forms a salt bridge with Lys121 in helix 5 and a hydrogen bond with Tyr122; the side chains of Ser154 and Thr158 in helix 6 form hydrogen bonds with backbone NH groups in loop L1. In addition, Trp34 in L1 makes an aromatic interaction with Tyr74 in L3 (not shown). Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3RLB * 3RLB Referenced accessions Protein Data Bank * 3P5N * 3P5N Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * University of Groningen, Groningen Biomolecular Science and Biotechnology Institute, Groningen, The Netherlands. * Guus B Erkens, * Ronnie P-A Berntsson, * Faizah Fulyani, * Maria Majsnerowska, * Andreja Vujičić-Žagar, * Josy ter Beek, * Bert Poolman & * Dirk Jan Slotboom * University of Groningen, Zernike Institute for Advanced Materials, Groningen, The Netherlands. * Guus B Erkens, * Ronnie P-A Berntsson, * Faizah Fulyani, * Maria Majsnerowska, * Andreja Vujičić-Žagar, * Josy ter Beek, * Bert Poolman & * Dirk Jan Slotboom Contributions G.B.E., R.P.-A.B. and D.J.S. designed the experiments. G.B.E., R.P.-A.B., F.F., M.M., A.V.-Z. and J.t.B. conducted the experiments. G.B.E., R.P.-A.B., B.P. and D.J.S. analyzed the data. G.B.E., B.P. and D.J.S. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Dirk Jan Slotboom Author Details * Guus B Erkens Search for this author in: * NPG journals * PubMed * Google Scholar * Ronnie P-A Berntsson Search for this author in: * NPG journals * PubMed * Google Scholar * Faizah Fulyani Search for this author in: * NPG journals * PubMed * Google Scholar * Maria Majsnerowska Search for this author in: * NPG journals * PubMed * Google Scholar * Andreja Vujičić-Žagar Search for this author in: * NPG journals * PubMed * Google Scholar * Josy ter Beek Search for this author in: * NPG journals * PubMed * Google Scholar * Bert Poolman Search for this author in: * NPG journals * PubMed * Google Scholar * Dirk Jan Slotboom Contact Dirk Jan Slotboom Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–8 and Supplementary Table 1 Additional data Entities in this article * Thiamine transporter ThiT llmg_0334 Lactococcus lactis subsp. cremoris (strain MG1363) * View in UniProt * View in Entrez Gene * Lmo1429 protein lmo1429 Listeria monocytogenes * View in UniProt * View in Entrez Gene * Biotin transporter BioY RCAP_rcc03249 Rhodobacter capsulatus (strain ATCC BAA-309 / NBRC 16581 / SB1003) * View in UniProt * View in Entrez Gene * Energy-coupling factor transporter transmembrane protein EcfT cbiQ2 Lactococcus lactis subsp. cremoris (strain MG1363) * View in UniProt * View in Entrez Gene * Riboflavin transporter RibU Staphylococcus aureus (strain TCH60) * View in UniProt * Energy-coupling factor transporter ATP-binding protein EcfA2 cbiO Lactococcus lactis subsp. cremoris (strain MG1363) * View in UniProt * View in Entrez Gene * Energy-coupling factor transporter ATP-binding protein EcfA1 cbiO Lactococcus lactis subsp. cremoris (strain MG1363) * View in UniProt * View in Entrez Gene
• Chfr and RNF8 synergistically regulate ATM activation
  - Nat Struct Mol Biol 18(7):761-768 (2011)
  Nature Structural & Molecular Biology | Article Chfr and RNF8 synergistically regulate ATM activation * Jiaxue Wu1 * Yibin Chen1 * Lin-Yu Lu1 * Yipin Wu2 * Michelle T Paulsen3 * Mats Ljungman3 * David O Ferguson2 * Xiaochun Yu1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:761–768Year published:(2011)DOI:doi:10.1038/nsmb.2078Received05 December 2010Accepted20 April 2011Published online26 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Protein ubiquitination is a crucial component of the DNA damage response. To study the mechanism of the DNA damage–induced ubiquitination pathway, we analyzed the impact of the loss of two E3 ubiquitin ligases, RNF8 and Chfr. Notably, DNA damage–induced activation of ATM kinase is suppressed in cells deficient in both RNF8 and Chfr (double-knockout, or DKO), and DKO mice develop thymic lymphomas that are nearly diploid but harbor clonal chromosome translocations. Moreover, DKO mice and cells are hypersensitive to ionizing radiation. We present evidence that RNF8 and Chfr synergistically regulate histone ubiquitination to control histone H4 Lys16 acetylation through MRG15-dependent acetyltransferase complexes. Through these complexes, RNF8 and Chfr affect chromatin relaxation and modulate ATM activation and DNA damage response pathways. Collectively, our findings demonstrate that two chromatin-remodeling factors, RNF8 and Chfr, function together to activate ATM and mainta! in genomic stability in vivo. View full text Figures at a glance * Figure 1: The DNA damage–induced ATM signaling pathway is impaired in DKO cells. () Domain architecture of mouse RNF8 and Chfr. (,) RNF8 and Chfr synergistically regulate ATM autophosphorylation and the ATM-dependent signaling pathway after DNA damage. MEFs () and thymocytes () were treated with 10 Gy ionizing radiation (IR) or without radiation, lysed and subjected to western blotting using antibodies to the indicated proteins. 'p' prefix denotes phosphorylation; pS1987, pS15 and pS345 are phosphorylated serines. () Chfr and RNF8 synergistically regulate ATM kinase activity. In vitro kinase assay mixture was separated by SDS-PAGE and subjected to western blotting using antibodies to the indicated proteins. Coomassie brilliant blue staining of GST-p53 is shown below as a loading control. (,) RNF8 and Chfr DKO abrogates G1/S () and G2/M () checkpoint activation. BrdU assay (for G1/S checkpoint) and pH3 assay (for G2/M checkpoint) were performed and mean values were calculated from three independent experiments. Error bars represent s.e.m. * Figure 2: RNF8 and Chfr double-deficient mice develop T-cell lymphoma. () Lymphoma-free survival rate of mice over 6 months. () Massive thymic lymphomas harvested from DKO mice. () Thymocytes from wild-type mice and tumors analyzed by flow cytometry. Upper right quadrant represents CD4+ CD8+ cells. () Hematoxylin-and-eosin–stained sections from DKO thymic lymphomas and wild-type thymus. Arrows indicate mitotic figures. * Figure 3: Thymic lymphomas from DKO mice harbor clonal translocations. () A representative metaphase spread of lymphoma 1, with DAPI staining. () Spectral karyotype (SKY) image of the metaphase spread, with translocations indicated. () Representative karyotype from lymphoma 1. () Summary of chromosome translocations in DKO lymphomas. * Figure 4: DKO mice and MEFs are hypersensitive to ionizing radiation. () Survival of mice treated with 8 Gy ionizing radiation at 8 weeks. () MEFs were treated with ionizing radiation at the indicated doses and counted after 10 d. The ratio of ionizing radiation–treated over mock-irradiated cells of the same genotype is plotted. Results represent the mean values from three independent experiments. Error bars represent s.e.m. () Chromosome breaks in mitotic DKO MEFs were more numerous after ionizing-radiation treatment. MEFs were treated with 1 Gy of ionizing radiation. Chromosome breaks were counted in each metaphase spread (100 per genotype). * Figure 5: RNF8 and Chfr synergistically regulate histone ubiquitination and acetylation. (,) H2A and H2B ubiquitination (Ub) and H4K16 acetylation (Ac) are lower in DKO MEFs and in thymocytes from DKO mice. Soluble and chromatin fractions were extracted from MEFs () or thymocytes () and analyzed by western blotting using antibodies to the indicated proteins. () H4K16 acetylation is downregulated in DKO lymphomas. Chromatin fractions from three different DKO lymphomas and wild-type thymus were analyzed by western blotting using antibodies to the indicated proteins. (,) Chromatin relaxation is impaired in DKO cells. MNase sensitivity assay was performed (), and average length of oligonucleosome was calculated from three independent experiments (). Error bars represent s.e.m. () Chromatin-associated MOF and Tip60 are less abundant in DKO MEFs. The soluble and chromatin fractions were prepared from MEFs and analyzed by western blotting using antibodies to the indicated proteins. * Figure 6: MRG15 links histone H2B ubiquitination and H4K16 acetylation. () MRG15 associates with Ub-H2B. Proteins pulled down by GST-tagged recombinant proteins of MOF or Tip60 complexes were analyzed by immunoblotting with antibody to Ub-H2B. Coomassie brilliant blue (CBB) stain of recombinant proteins (bands marked by asterisks) is shown below a loading control. () A region within residues 82–262 of MRG15 recognizes Ub-H2B. Full-length MRG15 (FL) and deletion mutants D1–D6 (schematic, top) were isolated by GST pull-down and analyzed by immunoblotting with antibody to Ub-H2B. CBB staining of all the GST-MRG15 variants is shown below as a loading control. () Chromatin-associated MRG15 is markedly reduced in DKO MEFs. The soluble and chromatin fractions were prepared from MEFs and subjected to western blotting with antibodies to indicated proteins. () Depletion of MRG15 affects H4K16 acetylation (Ac). Chromatin-bound proteins from control siRNA or MRG15 siRNA-treated U2OS cells were analyzed by western blotting. () Chromatin-associated MOF an! d Tip60 were decreased in MRG15-depleted cells. Soluble and chromatin fractions were extracted from U2OS cells treated with control siRNA or MRG15 siRNA and analyzed by western blotting. () The ATM-dependent DNA damage response pathway is impaired in MRG15-depleted cells. Control siRNA– or MRG15 siRNA–treated U2OS cells were treated with 10 Gy ionizing radiation or without radiation. One hour after radiation treatment, cell lysates were analyzed by western blotting. pS1981, pS345 and pT68 denote phosphorylated serines and threonine. * Figure 7: Suppression of histone acetylation rescues ATM-dependent DNA damage response in DKO MEFs. () TSA treatment restores the ATM-dependent signaling pathway in DKO cells. Cells were treated with TSA and ionizing radiation (IR) as described in Online Methods. Cell lysates were subjected to western blotting with antibodies to the indicated proteins. Ac, acetylation; pS1987 denotes a phosphorylated serine. () TSA treatment reduces ionizing radiation–induced chromosome breaks in DKO cells. Cells were treated with TSA and ionizing radiation as described in Online Methods. Chromosome breaks were counted in metaphase spreads, with 100 metaphases evaluated for each genotype. () TSA treatment increases DKO cell survival after DNA damage. Cells were treated with TSA and ionizing radiation followed by colony-formation assay. Results were calculated from three independent experiments. Error bars represent s.e.m. () TSA treatment partially restores ionizing radiation–induced 53BP1 foci formation in RNF8 KO and DKO cells. Cells were treated with TSA and ionizing radiation as de! scribed in Online Methods. Cells were fixed and immunostained with antibodies to indicated proteins (top; γH2AX, phosphorylated H2AX), and cells containing 53BP1 foci were counted (bottom). Error bars represent s.e.m. from three independent experiments. * Figure 8: A model of histone modifications, chromatin relaxation and ATM activation after DNA damage. RNF8 and Chfr are important for histone ubiquitination and acetylation that in turn induce chromatin relaxation, which facilitates ATM activation and ATM-dependent DNA damage response after DNA damage. Author information * Abstract * Author information * Supplementary information Affiliations * Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan, USA. * Jiaxue Wu, * Yibin Chen, * Lin-Yu Lu & * Xiaochun Yu * Department of Pathology, University of Michigan Medical School, Ann Arbor, Michigan, USA. * Yipin Wu & * David O Ferguson * Department of Radiation Oncology, University of Michigan Medical School, Ann Arbor, Michigan, USA. * Michelle T Paulsen & * Mats Ljungman Contributions J.W. performed most experiments. Y.C. analyzed the MRG15-related protein-protein interactions. L.-Y.L., M.T.P., M.L., Y.W. and D.O.F. provided technical support for various assays. X.Y. designed the experiments. X.Y. and J.W. wrote the manuscript. All the authors read and approved the final manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Xiaochun Yu Author Details * Jiaxue Wu Search for this author in: * NPG journals * PubMed * Google Scholar * Yibin Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Lin-Yu Lu Search for this author in: * NPG journals * PubMed * Google Scholar * Yipin Wu Search for this author in: * NPG journals * PubMed * Google Scholar * Michelle T Paulsen Search for this author in: * NPG journals * PubMed * Google Scholar * Mats Ljungman Search for this author in: * NPG journals * PubMed * Google Scholar * David O Ferguson Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaochun Yu Contact Xiaochun Yu Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figs. 1-7, Supplementary Table 1 and Supplementary Methods Additional data Entities in this article * Mediator of DNA damage checkpoint protein 1 Mdc1 Mus musculus * View in UniProt * View in Entrez Gene * Nibrin Nbn Mus musculus * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase Chk2 Chek2 Mus musculus * View in UniProt * View in Entrez Gene * T-cell surface glycoprotein CD4 Cd4 Mus musculus * View in UniProt * View in Entrez Gene * Breast cancer type 1 susceptibility protein homolog Brca1 Mus musculus * View in UniProt * View in Entrez Gene * Probable histone acetyltransferase MYST1 Myst1 Mus musculus * View in UniProt * View in Entrez Gene * Ubiquitin-conjugating enzyme E2 N Ube2n Mus musculus * View in UniProt * View in Entrez Gene * Tumor suppressor p53-binding protein 1 Trp53bp1 Mus musculus * View in UniProt * View in Entrez Gene * Histone acetyltransferase KAT5 Kat5 Mus musculus * View in UniProt * View in Entrez Gene * V(D)J recombination-activating protein 2 Rag2 Mus musculus * View in UniProt * View in Entrez Gene * V(D)J recombination-activating protein 1 Rag1 Mus musculus * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase RNF8 RNF8 Homo sapiens * View in UniProt * View in Entrez Gene * Serine-protein kinase ATM ATM Homo sapiens * View in UniProt * View in Entrez Gene * Histone H2A.x H2AFX Homo sapiens * View in UniProt * View in Entrez Gene * Mediator of DNA damage checkpoint protein 1 MDC1 Homo sapiens * View in UniProt * View in Entrez Gene * Serine-protein kinase ATM Atm Mus musculus * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase RNF8 Rnf8 Mus musculus * View in UniProt * View in Entrez Gene * Mortality factor 4-like protein 1 Morf4l1 Mus musculus * View in UniProt * View in Entrez Gene * Histone H4 Mus musculus * View in UniProt * E3 ubiquitin-protein ligase HERC2 HERC2 Homo sapiens * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase RNF168 RNF168 Homo sapiens * View in UniProt * View in Entrez Gene * Ubiquitin-conjugating enzyme E2 N UBE2N Homo sapiens * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase CHFR Chfr Mus musculus * View in UniProt * View in Entrez Gene * Tumor suppressor p53-binding protein 1 TP53BP1 Homo sapiens * View in UniProt * View in Entrez Gene * Breast cancer type 1 susceptibility protein BRCA1 Homo sapiens * View in UniProt * View in Entrez Gene * BRCA1-A complex subunit RAP80 UIMC1 Homo sapiens * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase RAD18 RAD18 Homo sapiens * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase Chk1 Chek1 Mus musculus * View in UniProt * View in Entrez Gene * Cellular tumor antigen p53 Trp53 Mus musculus * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase CHFR CHFR Homo sapiens * View in UniProt * View in Entrez Gene * Histone H2A.x H2afx Mus musculus * View in UniProt * View in Entrez Gene
• ATRX ADD domain links an atypical histone methylation recognition mechanism to human mental-retardation syndrome
  - Nat Struct Mol Biol 18(7):769-776 (2011)
  Nature Structural & Molecular Biology | Article ATRX ADD domain links an atypical histone methylation recognition mechanism to human mental-retardation syndrome * Shigeki Iwase1, 2 * Bin Xiang3, 4 * Sharmistha Ghosh5 * Ting Ren1, 2 * Peter W Lewis6 * Jesse C Cochrane7 * C David Allis6 * David J Picketts8 * Dinshaw J Patel9 * Haitao Li3, 4 * Yang Shi1, 2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:769–776Year published:(2011)DOI:doi:10.1038/nsmb.2062Received20 December 2010Accepted18 March 2011Published online12 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg ATR-X (alpha-thalassemia/mental retardation, X-linked) syndrome is a human congenital disorder that causes severe intellectual disabilities. Mutations in the ATRX gene, which encodes an ATP-dependent chromatin-remodeler, are responsible for the syndrome. Approximately 50% of the missense mutations in affected persons are clustered in a cysteine-rich domain termed ADD (ATRX-DNMT3-DNMT3L, ADDATRX), whose function has remained elusive. Here we identify ADDATRX as a previously unknown histone H3–binding module, whose binding is promoted by lysine 9 trimethylation (H3K9me3) but inhibited by lysine 4 trimethylation (H3K4me3). The cocrystal structure of ADDATRX bound to H31–15K9me3 peptide reveals an atypical composite H3K9me3-binding pocket, which is distinct from the conventional trimethyllysine-binding aromatic cage. Notably, H3K9me3-pocket mutants and ATR-X syndrome mutants are defective in both H3K9me3 binding and localization at pericentromeric heterochromatin; thus, we h! ave discovered a unique histone-recognition mechanism underlying the ATR-X etiology. View full text Figures at a glance * Figure 1: Methylation at K4 and K9 inversely regulates interaction between ADDATRX and histone H3. () ATR-X mutations (circles) are predominantly found in either the ADD or the sucrose nonfermenting (SNF2)-type ATP-dependent chromatin-remodeling domain. aa, amino acid. () ADDATRX interacts with the unmodified H3 peptide (residues 1–21, lane 3), and the interaction is enhanced by K9 methylation (lanes 4–6). (–) Experimental ITC titration curves for unmodified H3 are shown () (top, peptide to buffer control) and for H3K9me3 peptide (). The fitting curves and the calculated binding affinities are listed in and . Methylation at K4 () and K9 () had entirely different effects on the binding affinity. Detailed peptide sequences and complete ITC fitting parameters are summarized in Supplementary Table 1. * Figure 2: Atrx is recruited to PCH by Suv39h1- and Suv39h2-mediated H3K9 trimethylation. () Atrx fails to locate on PCH in Suv39h1 and Suv39h2 double knockout (DKO) cells. (,) Suv39h1-WT but not a catalytically inactive mutant Suv39h1-H324L restores the PCH localization of Atrx in DKO cells. Suv39h1-WT (images a–d) and Suv39h1-H324L (images e–h) were expressed in DKO cells. Quantification of presence or absence of H3K9me3 signal in the Suv39h1-positive cells (, left) and punctate PCH localization or diffused distribution of Atrx (, right). The ratio of interphase cells showing designated staining pattern is represented as percentage of total interphase cells. () Mecp2 is localized at PCH in both WT- (images a–c) and DKO- (images d–f) MEF cells. () Schematic diagram of ATRX proteins used for testing their localization. () Deletion of PxVxL motif resulted in partial loss of PCH localization. Subnuclear distribution of ATRX proteins represented in (Fig. 5f) was quantified. () Targeting of Atrx (images b and f) to Daxx-positive PML bodies (images a and e) is! not affected in Suv39h DKO cells (images e to h). * Figure 3: Molecular basis for H3K9me3 recognition by ADDATRX. () Ribbon representation of ADDATRX domain in complex with H31–15K9me3 peptide. ADDATRX domain is a hybrid of a GATA-like zinc finger (cyan) and a PHD finger (magenta). Three zinc ions are depicted as spheres, and the H3 peptide is colored yellow, with residues Lys4 and K9me3 shown in stick representation. () Surface electrostatic view of ADDATRX domain in complex with H31–15K9me3 peptide, showing negatively charged (red) and positively charged (blue) potentials. The Fo – Fc omit map was contoured at the 3.5-σ level for the bound H3 peptide. () Stereo view of the K9me3-binding pocket. The GATA-like segment Tyr203–Ser210 and the PHD finger segment Gln219–Glu225 are color-coded in cyan and magenta, respectively. Blue dotted lines refer to nonconventional C-H:O hydrogen bonds. () A snug fit of the bulky trimethyllysine group inserted into the reader pocket of ADDATRX. * Figure 4: Effect of H3 sequence context on ADDATRX binding. () Details of H3-ADDATRX interaction. Histone H3 Ala1–Ser10 containing the K9me3 modification is depicted as yellow sticks; ADDATRX as light blue ribbons; residues that interact with H3 peptide as pink sticks; hydrogen bonds as dotted red lines; and three zinc ions and water molecules as large blue and small red spheres, respectively. () ITC titration fitting curves and the binding constants of synthetic H3 peptide variants to WT-ADDATRX. AAH3, H3 N-terminal double alanine extension; R2me2s, Arg2 symmetrical dimethylation; R2me2a, Arg2 asymmetrical dimethylation; K9me3, Lys9 trimethylation; K9ac, Lys9 acetylation; n.d., not determined. Detailed peptide sequences and complete ITC fitting parameters are summarized in Supplementary Table 1. * Figure 5: Mutations in ADDATRX compromise histone binding and targeting of ATRX onto PCH. () Alignment of the ADD domains. Hs, human; Dr, Danio rerio (zebrafish); Xt, Xenopus tropicalis (frog). Conserved cysteines are shown as red letters. () Mutations in the K9me3 pocket limit the binding of ADDATRX to H3 peptides methylated at Lys9. Mutant proteins were subjected to peptide-binding assays and detected by coomassie staining. () Mutations in the K9me3 pocket disrupt binding to nucleosomes. GST alone (lane 1) or GST-ADDATRX WT (lane 2) as well as mutants (lanes 3–6) were incubated with nucleosomes. (,) Four mutations resulted in defective H3 peptide binding () and nucleosome binding (). () Representative images of subnuclear distribution of recombinant ATRX proteins in NIH3T3 cells. (,) Quantification of the distribution patterns is shown for the four K9me3-pocket mutants () and additional four syndrome-associated mutants (). * Figure 6: Model of the mechanism underlying ATR-X syndrome that is caused by the mutations in ADDATRX. Atrx is recruited on PCH by means of an interaction between ADD and H3K9me3 that is generated by Suv39h HMTs. Physical associations of Hp1-Atrx and Mecp2-Atrx support the PCH anchoring of Atrx. Mutations in ADD disrupt H3K9me3 binding, then Atrx falls off from PCH. Loss of Atrx from PCH may lead to compromised higher-order structure of PCH, followed by chromosome missegregation and apoptosis in neuroprogenitor cells. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3QLN * 3QLC * 3QL9 * 3QLA * 3QLN * 3QLC * 3QL9 * 3QLA Referenced accessions Entrez Nucleotide * NM_000489.3 Protein Data Bank * 1KNE * 3G7L * 3B95 * 1KNE * 3G7L * 3B95 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Division of Newborn Medicine, Department of Medicine, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA. * Shigeki Iwase, * Ting Ren & * Yang Shi * Epigenetics Program, Department of Medicine, Children's Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA. * Shigeki Iwase, * Ting Ren & * Yang Shi * Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China. * Bin Xiang & * Haitao Li * School of Medicine, Tsinghua University, Beijing, China. * Bin Xiang & * Haitao Li * Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA. * Sharmistha Ghosh * Laboratory of Chromatin Biology and Epigenetics, Rockefeller University, New York, New York, USA. * Peter W Lewis & * C David Allis * Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA. * Jesse C Cochrane * Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. * David J Picketts * Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. * Dinshaw J Patel Contributions S.I., H.L., D.J. Patel and Y.S. participated in the experimental design, contributed to the concept and wrote the paper. S.I. did pulldown assays, SPR assays and localization analysis. B.X. crystallized the ADDATRX–H3K9me3 complex and participated in calorimetric assays. H.L. carried out crystallographic and calorimetric studies. P.W.L., T.R. and J.C.C. carried out or helped with pulldown assays. S.G. helped with measuring SPR. C.D.A. supervised pulldown assays. D.J. Picketts provided important reagents and participated in manuscript preparation. Competing financial interests Y.S. is a cofounder of Constellation Pharmaceuticals. D.J. Patel is on the Epigenetics Advisory Board of Epinova-Glaxo. The remaining authors declare no competing financial interests. Corresponding authors Correspondence to: * Haitao Li or * Dinshaw J Patel or * Yang Shi Author Details * Shigeki Iwase Search for this author in: * NPG journals * PubMed * Google Scholar * Bin Xiang Search for this author in: * NPG journals * PubMed * Google Scholar * Sharmistha Ghosh Search for this author in: * NPG journals * PubMed * Google Scholar * Ting Ren Search for this author in: * NPG journals * PubMed * Google Scholar * Peter W Lewis Search for this author in: * NPG journals * PubMed * Google Scholar * Jesse C Cochrane Search for this author in: * NPG journals * PubMed * Google Scholar * C David Allis Search for this author in: * NPG journals * PubMed * Google Scholar * David J Picketts Search for this author in: * NPG journals * PubMed * Google Scholar * Dinshaw J Patel Contact Dinshaw J Patel Search for this author in: * NPG journals * PubMed * Google Scholar * Haitao Li Contact Haitao Li Search for this author in: * NPG journals * PubMed * Google Scholar * Yang Shi Contact Yang Shi Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (14M) Supplementary Figures 1–9, Supplementary Table 1 and Supplementary Methods Additional data Entities in this article * Chromobox protein homolog 5 Cbx5 Mus musculus * View in UniProt * View in Entrez Gene * Histone-lysine N-methyltransferase SUV39H2 Suv39h2 Mus musculus * View in UniProt * View in Entrez Gene * Death domain-associated protein 6 Daxx Mus musculus * View in UniProt * View in Entrez Gene * Methyl-CpG-binding protein 2 Mecp2 Mus musculus * View in UniProt * View in Entrez Gene * DNA (cytosine-5)-methyltransferase 3A DNMT3A Homo sapiens * View in UniProt * View in Entrez Gene * DNA (cytosine-5)-methyltransferase 3-like DNMT3L Homo sapiens * View in UniProt * View in Entrez Gene * Histone-lysine N-methyltransferase SUV39H1 Suv39h1 Mus musculus * View in UniProt * View in Entrez Gene * Transcriptional regulator ATRX Atrx Mus musculus * View in UniProt * View in Entrez Gene * Histone-lysine N-methyltransferase EHMT1 EHMT1 Homo sapiens * View in UniProt * View in Entrez Gene * Chromo domain-containing protein 1 chp1 Schizosaccharomyces pombe (strain ATCC 38366 / 972) * View in UniProt * View in Entrez Gene * Heterochromatin protein 1 Su(var)205 Drosophila melanogaster * View in UniProt * View in Entrez Gene * Transcriptional regulator ATRX ATRX Homo sapiens * View in UniProt * View in Entrez Gene * Transcription intermediary factor 1-alpha TRIM24 Homo sapiens * View in UniProt * View in Entrez Gene * Zinc finger protein DPF3 DPF3 Homo sapiens * View in UniProt * View in Entrez Gene * Histone H3.3 Mus musculus * View in UniProt * DNA (cytosine-5)-methyltransferase 3B DNMT3B Homo sapiens * View in UniProt * View in Entrez Gene * Chromobox protein homolog 5 CBX5 Homo sapiens * View in UniProt * View in Entrez Gene * Protein PML PML Homo sapiens * View in UniProt * View in Entrez Gene
• Combinatorial readout of histone H3 modifications specifies localization of ATRX to heterochromatin
  - Nat Struct Mol Biol 18(7):777-782 (2011)
  Nature Structural & Molecular Biology | Article Combinatorial readout of histone H3 modifications specifies localization of ATRX to heterochromatin * Sebastian Eustermann1, 3 * Ji-Chun Yang1, 3 * Martin J Law2 * Rachel Amos2 * Lynda M Chapman1 * Clare Jelinska2 * David Garrick2 * David Clynes2 * Richard J Gibbons2 * Daniela Rhodes1 * Douglas R Higgs2 * David Neuhaus1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:777–782Year published:(2011)DOI:doi:10.1038/nsmb.2070Received01 December 2010Accepted15 April 2011Published online12 June 2011Corrected online19 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Change history * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Accurate read-out of chromatin modifications is essential for eukaryotic life. Mutations in the gene encoding X-linked ATRX protein cause a mental-retardation syndrome, whereas wild-type ATRX protein targets pericentric and telomeric heterochromatin for deposition of the histone variant H3.3 by means of a largely unknown mechanism. Here we show that the ADD domain of ATRX, in which most syndrome-causing mutations occur, engages the N-terminal tail of histone H3 through two rigidly oriented binding pockets, one for unmodified Lys4 and the other for di- or trimethylated Lys9. In vivo experiments show this combinatorial readout is required for ATRX localization, with recruitment enhanced by a third interaction through heterochromatin protein-1 (HP1) that also recognizes trimethylated Lys9. The cooperation of ATRX ADD domain and HP1 in chromatin recruitment results in a tripartite interaction that may span neighboring nucleosomes and illustrates how the 'histone-code' is interpr! eted by a combination of multivalent effector-chromatin interactions. View full text Figures at a glance * Figure 1: The ADD domain of ATRX is required for localization in vivo. () Missense mutations (small circles) associated with ATRX syndrome cluster in the highly conserved ADD and SWI-SNF ATPase domains of ATRX protein. Residue numbering is shown for human (h) and mouse ATRX (m); mutants are named using mouse numbering. () Localization of fusion protein pATRX-GFP1 (here called wild type (WT); see Supplementary Methods) to pericentric heterochromatin foci as marked by the DNA stain ToPro3. Panels show representative examples of cells transfected with the indicated mutant proteins. () Quantification of ATRX localization. Each bar represents the ratio of fluorescence intensity at enriched foci relative to background (averaged across 60 cells per construct in three independent transfections, error bars are 95% confidence limits; see Supplementary Methods). Absence of localization results in a homogenous distribution within the nucleus, corresponding to a ratio of 1 (dashed gray line). Wild-type protein is highly enriched at foci (ratio 9.6 ± 2.0), ! whereas mutations in the ADD domain or HP1 interaction motif show reduced localization (2.3 ± 0.2 for C240G and 5.1 ± 0.9 for V580E, respectively). Double mutation delocalizes ATRX to background levels, as for the EGFP control. * Figure 2: Recognition of an N-terminal H3 tail by the ADD domain of ATRX. () Two views of human ATRX ADD domain (surface representation) in complex with K9me3 H3 peptide (orange sticks). The ADD domain holds the GATA-like and PHD zinc fingers in a rigid mutual orientation, facilitating combinatorial readout of the H3 modifications; the PHD zinc finger recognizes H3K4me0, whereas K9me3 is inserted in a hydrophobic pocket formed by parts of the GATA-like zinc finger and the interfinger linker. The interface is formed by H3 residues 1–9; peptide residues 11–15 (not shown) are disordered. () Cartoon representation of the same complex. Residues 3–5 of the H3 peptide extend the β-sheet of the PHD zinc finger. Q219P is a syndrome-associated mutation located between the Lys4 and K9me3 pockets. (,) Detailed views of the H3K4 and H3K9me3 recognition pockets. () Sequence of the N-terminal tail of histone H3, showing posttranslational modifications assessed in this study. () ITC data of the indicated H3 peptides (residues 1–15) added to the wild-type! or Q219P mutant ADD domain (100 μM for the upper two curves, 50 μM for the lower two). Left, raw data; right, fitted curves (see also Table 1). () Superimposed 15N-1H HSQC spectra of free ATRX ADD, ATRX ADD mixed 1:1 with unmodified H31–15, and ATRX ADD mixed 1:1 with H31–15K9me3. Some perturbations are unique to H31–15K9me3, showing that this modification is specifically recognized; insets show three particularly clear cases. Amide group chemical shift differences for the ADD domain between these two complexes are plotted below as a function of sequence and mapped to the surface of the structure; this shows the site of K9me3 recognition. * Figure 3: Structure-guided mutations reveal tripartite chromatin interaction of ATRX in vivo. () Localization of pATRX-GFP1 fusion protein, comparing wild-type with ADD mutants that selectively target the Lys4 (E218A) and K9me3 (Y203K) interactions, or both (Q219P). () As for , but in combination with the V580E mutation. () Quantification of ATRX localization averaged over 60 cells as described for Figure 1; error bars are 95% confidence limits. Each bar represents the ratio of intensity at enriched foci relative to background, and absence of localization corresponds to a ratio of 1 (dashed gray line). The impact of these mutations on pericentric enrichment is summarized by the crosses below the histogram, showing which protein interactions are affected. () Schematic summarizing interactions of ATRX that have been identified and tested in vitro and in vivo: the ADD domain reads both H3K4me0 and H3K9me3, and in addition, an HP1 dimer recognizes H3K9me3. Pericentric heterochromatin provides an array of H3 tails highly enriched in K9me3 as well as HP1, permitting an ADD! domain and HP1 to bind adjacent tails and hence cooperate in ATRX recruitment. HP1 is drawn bridging nucleosomes, as described in a recent report28, although different arrangements of the tails may actually occur. ATRX has recently been shown to be required for localization of Daxx–H3.3 at heterochromatin and to catalyze incorporation of H3.3 (refs. 10,12). * Figure 4: ADD K9me3 recognition provides mechanistic evidence for HP1 independent recruitment of ATRX in mitosis. () Three superimposed 15N-1H HSQC spectra. Gray, free ATRX ADD; red, ATRX ADD bound to H31–15 peptide with K9me3; blue, ATRX ADD bound to H3 peptide with K9me3, S10P and K14Ac. ATRX ADD binds both peptides with similar affinities (see Table 1) and in the same conformation, as shown by the closely corresponding peak positions in the two bound-state 15N-1H HSQC spectra. In combination, these data suggest strongly that ATRX ADD binding is independent of the Ser10 and Lys14 modification status of H3 histone tails. () Competition experiments with HP1β. Unlabeled HP1β was titrated to the peptide–protein complexes described in . The panel shows a series of 15N-1H HSQC spectra recorded at the given ADD:peptide:HP1β stoichiometries. The region selected (amide resonance of Gly277) is highlighted in panel by a black box and reports the peptide-binding status of ADD ATRX; the color code is as for . The H3K9me3 peptide was competed off its binding site in ATRX ADD upon addition of! excess HP1. Phosphorylation of Ser10 and acetylation of Lys14 interfere with HP1 binding as reported previously23. Consequently, ATRX ADD remains bound to the triply modified H3 peptide as excess HP1 is added. Accession codes * Abstract * Accession codes * Change history * Author information * Supplementary information Primary accessions Protein Data Bank * 2LBM * 2LBM Change history * Abstract * Accession codes * Change history * Author information * Supplementary informationCorrigendum 19 June 2011In the version of this article initially published online, the Kd for the binding of the triply modified H3 peptide to the ATRX ADD domain was reported incorrectly in the text. The correct value is 0.59 µM. Additionally, the residues in insets A and B of Figure 2g were incorrectly labeled. The correct residue labels are Ser206 in inset A and Asp207 in inset B. These errors have been corrected for the print, PDF and HTML versions of this article. Author information * Abstract * Accession codes * Change history * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Sebastian Eustermann & * Ji-Chun Yang Affiliations * Medical Research Council Laboratory of Molecular Biology, Cambridge, UK. * Sebastian Eustermann, * Ji-Chun Yang, * Lynda M Chapman, * Daniela Rhodes & * David Neuhaus * Medical Research Council Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Headington, Oxford, UK. * Martin J Law, * Rachel Amos, * Clare Jelinska, * David Garrick, * David Clynes, * Richard J Gibbons & * Douglas R Higgs Contributions S.E. and J.-C.Y. carried out NMR experiments and structure determination. S.E., J.-C.Y. and D.N. analyzed the structures. S.E. and L.M.C. prepared protein and complex samples. S.E. carried out in vitro binding experiments. M.J.L., R.A., D.C. and D.G. carried out in vivo imaging experiments. S.E. and C.J. carried out in vitro experiments with mutants. S.E., D.R., D.N., R.J.G. and D.R.H. designed the experiments and wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Douglas R Higgs or * David Neuhaus Author Details * Sebastian Eustermann Search for this author in: * NPG journals * PubMed * Google Scholar * Ji-Chun Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Martin J Law Search for this author in: * NPG journals * PubMed * Google Scholar * Rachel Amos Search for this author in: * NPG journals * PubMed * Google Scholar * Lynda M Chapman Search for this author in: * NPG journals * PubMed * Google Scholar * Clare Jelinska Search for this author in: * NPG journals * PubMed * Google Scholar * David Garrick Search for this author in: * NPG journals * PubMed * Google Scholar * David Clynes Search for this author in: * NPG journals * PubMed * Google Scholar * Richard J Gibbons Search for this author in: * NPG journals * PubMed * Google Scholar * Daniela Rhodes Search for this author in: * NPG journals * PubMed * Google Scholar * Douglas R Higgs Contact Douglas R Higgs Search for this author in: * NPG journals * PubMed * Google Scholar * David Neuhaus Contact David Neuhaus Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Change history * Author information * Supplementary information PDF files * Supplementary Text and Figures (5M) Supplementary Figures 1–8, Supplementary Table 1 and Supplementary Methods Additional data Entities in this article * Death domain-associated protein 6 DAXX Homo sapiens * View in UniProt * View in Entrez Gene * Serine/threonine-protein kinase 12 AURKB Homo sapiens * View in UniProt * View in Entrez Gene * Transcriptional regulator ATRX ATRX Homo sapiens * View in UniProt * View in Entrez Gene * DNA (cytosine-5)-methyltransferase 3-like DNMT3L Homo sapiens * View in UniProt * View in Entrez Gene * Chromobox protein homolog 1 CBX1 Homo sapiens * View in UniProt * View in Entrez Gene * DNA (cytosine-5)-methyltransferase 3A DNMT3A Homo sapiens * View in UniProt * View in Entrez Gene * Transcriptional regulator ATRX Atrx Mus musculus * View in UniProt * View in Entrez Gene * Histone-lysine N-methyltransferase SUV39H1 Suv39h1 Mus musculus * View in UniProt * View in Entrez Gene * Histone H3.3 Homo sapiens * View in UniProt * Chromobox protein homolog 5 CBX5 Homo sapiens * View in UniProt * View in Entrez Gene * Histone-lysine N-methyltransferase SUV39H2 Suv39h2 Mus musculus * View in UniProt * View in Entrez Gene
• Phospholipid-dependent regulation of the motor activity of myosin X
  - Nat Struct Mol Biol 18(7):783-788 (2011)
  Nature Structural & Molecular Biology | Article Phospholipid-dependent regulation of the motor activity of myosin X * Nobuhisa Umeki1, 3 * Hyun Suk Jung2, 3 * Tsuyoshi Sakai1, 3 * Osamu Sato1 * Reiko Ikebe1 * Mitsuo Ikebe1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:783–788Year published:(2011)DOI:doi:10.1038/nsmb.2065Received08 August 2010Accepted01 April 2011Published online12 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Myosin X is involved in the reorganization of the actin cytoskeleton and protrusion of filopodia. Here we studied the molecular mechanism by which bovine myosin X is regulated. The globular tail domain inhibited the motor activity of myosin X in a Ca2+-independent manner. Structural analysis revealed that myosin X is monomeric and that the band 4.1-ezrin-radixin-moesin (FERM) and pleckstrin homology (PH) domains bind to the head intramolecularly, forming an inhibited conformation. Binding of phosphatidylinositol-3,4,5-triphosphate (PtdIns(3,4,5)P3) to the PH domain reversed the tail-induced inhibition and induced the formation of myosin X dimers. Consistently, disruption of the binding of PtdIns(3,4,5)P3 attenuated the translocation of myosin X to filopodial tips in cells. We propose the following mechanism: first, the tail inhibits the motor activity of myosin X by intramolecular head-tail interactions to form the folded conformation; second, phospholipid binding reverses t! he inhibition and disrupts the folded conformation, which induces dimer formation, thereby activating the mechanical and cargo transporter activity of myosin X. View full text Figures at a glance * Figure 1: Schematic diagram of myosin X constructs used in this study. Amino acid numbers of the constructs are indicated. * Figure 2: The actin-activated ATPase activity of M10full and M10ΔGTD. () Actin-activated ATPase activity of M10full is much lower than of M10ΔGTD. The ATPase activity was measured as described in the Online Methods. Open circles, M10ΔGTD in pCa4; closed circles, M10ΔGTD in EGTA; open squares, M10full in pCa4; closed squares, M10full in EGTA. () Inhibition of the actin-activated ATPase activity of M10ΔGTD by exogenous tail domain. Assay conditions are as described in Online Methods. Values are mean ± s.e.m. from three independent experiments. Closed circles, EGTA; open circles, pCa4. One hundred percent activities in EGTA and pCa4 conditions were 5.83 ± 0.24 s−1 and 4.42 ± 0.36 s−1, respectively. () Determination of the regions in the tail domain that are crucial for inhibition of the actin-activated ATPase activity of M10ΔGTD under EGTA conditions. Open circles, M10PH-FERM; closed circles, M10PEST-MyTH4; open triangles, M10MyTH4-FERM; closed triangles, M10FERM. One hundred percent represents 5.83 ± 0.24 s−1. () Effect of M10tail! on the actin-activated ATPase activity of M10IQ0 under EGTA conditions. One hundred percent represents 6.59 ± 0.32 s−1. Inset shows the pulldown assay (see Supplementary Methods). Values are mean ± s.e.m. from three independent experiments. * Figure 3: Electron microscopy analysis of myosin X constructs. (,) Negatively stained fields of M10IQ0 and M10ΔGTD. Black and white arrowheads point to individual molecules in each field, appearing as single-headed monomers. (,) Fields of M10ΔGTD with the tail domain and M10full, respectively. White and black arrows indicate appearances of wider molecules observed in fields. Note that ~50% of molecules appeared as closely packed wider molecules as a result of intermolecular interactions between the head and the tail domain, consistent with the results shown in Figure 2. Inset images in –, galleries of raw images of individual molecules taken from each construct. () Selected averages of myosin X constructs chosen on the basis of similar oriented views in each construct; M10IQ0 (upper left), M10ΔGTD (upper right), M10ΔGTD + M10tail construct (lower left) and M10full (lower right). Each average consists of 20–50 images. Adjacent schematic drawings show possible interpretations of densities of each construct identified from the aver! ages. () Enlarged drawing of M10full showing main domains of closely packed M10full. Electron microscope specimen preparations of all samples (–) were diluted with 50 mM sodium acetate in the presence of ATP. Scale bars, 50 nm (–) and 20 nm (). * Figure 4: Binding of PtdIns(3,4,5)P3 to myosin X abolishes the tail-induced inhibition of the actin-activated ATPase activity of myosin X. () Effect of phospholipid-containing vesicles on the actin-activated ATPase activity of M10full. Open triangles, PtdIns(4,5)P2; open circles, PtdIns(3,4,5)P3; closed triangles, PtdIns(3,4)P2; closed circles, phosphatidylinositol; open squares, control vesicles. Molar ratio composition of each vesicle is 2:6:2 for PtdIns:phosphatidylcholines:phosphatidylserine. Molar ratio of the control vesicle is 8:2 for phosphatidylcholines:phosphatidylserine. () Reversal of tail-induced inhibition of the actin-activated ATPase activity of M10ΔGTD (0.05 μM) by vesicles containing PtdIns(3,4,5)P3. We used 0.5 μM M10tail. () Effect of Ca2+ on the PtdIns(3,4,5)P3-dependent activation of the actin-activated ATPase activity of full-length myosin X (M10full). () Mutation of PH2 domain in myosin X attenuated the PtdIns(3,4,5)P3-induced activation of actin-activated ATPase activity of M10ΔGTD in the presence of M10tail. We examined the effect of R1231C, R1231C/K1215A or R1231C/F1233A mutations! on the PtdIns(3,4,5)P3-induced activation of actin-activated ATPase activity of M10ΔGTD (0.05 μM) in the presence of M10tail (0.5 μM). The final concentration of PtdIns(3,4,5)P3 in vesicles (molar ratio of PtdIns(3,4,5)P3:PC:PS was 2:6:2) was 40 μM. We used 20 μM actin. Values are mean ± s.e.m. from three independent experiments. * Figure 5: Activation of the actin-gliding velocity of M10full by PtdIns(3,4,5)P3. (,) We determined the actin-gliding activity of M10full in the presence of control vesicle (molar ratio of 8:2 for diC8-phosphatidylcholine:diC8-phosphatidylserine; ) or diC8- PtdIns(3,4,5)P3 (Echelon; ) (see Online Methods). The velocity every 20 s () or 10 s () was measured with randomly selected moving actin filaments. The mean ± s.d. values were 0.11 ± 0.09 μm s−1 (n = 293) for and 0.29 ± 0.09 μm s−1 (n = 286) for . * Figure 6: Induction of myosin X dimer formation by PtdIns(3,4,5)P3 measured by cross-linking. Myc-M10tail/CC or M10tail (10 nM) was subjected to cross-linking with 10 mM EDC for 10 min in the presence of PtdIns(3,4,5)P3-containing vesicles (molar ratio of PtdIns(3,4,5)P3:phosphatidylcholine:phosphatidylserine was 2:6:2) or control vesicles (molar ratio of phosphatidylcholine:phosphatidylserine, 8:2). We used M10tail/CC/LZ as a positive control. We analyzed dimer formation by western blot using anti-Myc antibodies. Right, molecular masses. () Effect of PtdIns(3,4,5)P3 on dimer formation of M10tail/CC. () Role of predicted coiled-coil domain in PtdIns(3,4,5)P3-induced dimer formation. * Figure 7: Phospholipid binding is crucial for the filopodial tip localization of myosin X. COS 7 cells were transfected with GFP-M10full, GFP-M10full (R1231C), GFP-M10fullΔPH/MyTH4/FERM, GFP-M10fullΔMyTH4/FERM or M10fullΔcoil. () Representative images. Scale bars, 10 μm. () Statistical analysis of the number of filopodia per cell with the tip GFP signal. Values are mean ± s.e.m. (GFP-M10full, n = 65; GFP-M10full (R1231C), n = 30; GFP-M10fullΔPH/MyTH4/FERM, n = 23; GFP-M10fullΔMyTH4/FERM, n = 24; M10fullΔcoil, n = 43). Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Entrez Nucleotide * NM_174394 * NM_010864 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Nobuhisa Umeki, * Hyun Suk Jung & * Tsuyoshi Sakai Affiliations * Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts, USA. * Nobuhisa Umeki, * Tsuyoshi Sakai, * Osamu Sato, * Reiko Ikebe & * Mitsuo Ikebe * Division of Electron Microscopic Research, Korea Basic Science Institute, Daejeon, Korea. * Hyun Suk Jung Contributions N.U. performed the ATPase assays of myosin X constructs, cross-linking assay and prepared myosin X proteins for EM experiments. H.S.J. performed EM image analysis and wrote the EM part of the paper. T.S. performed lipid binding assay, cross-linking, cell imaging, measurement of the activity of PH domain mutants and production of some myosin X constructs. O.S. performed the actin gliding assay of myosin X. R.I. prepared myosin X expression constructs. M.I. supervised the whole project and wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Mitsuo Ikebe Author Details * Nobuhisa Umeki Search for this author in: * NPG journals * PubMed * Google Scholar * Hyun Suk Jung Search for this author in: * NPG journals * PubMed * Google Scholar * Tsuyoshi Sakai Search for this author in: * NPG journals * PubMed * Google Scholar * Osamu Sato Search for this author in: * NPG journals * PubMed * Google Scholar * Reiko Ikebe Search for this author in: * NPG journals * PubMed * Google Scholar * Mitsuo Ikebe Contact Mitsuo Ikebe Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–8 and Supplementary Methods Additional data Entities in this article * Myosin-X MYO10 Bos taurus * View in UniProt * View in Entrez Gene * Myosin-Va Myo5a Mus musculus * View in UniProt * View in Entrez Gene * Calmodulin calm2 Xenopus laevis * View in UniProt * View in Entrez Gene * General control protein GCN4 GCN4 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Myosin-VIIa ck Drosophila melanogaster * View in UniProt * View in Entrez Gene
• Recognition of the F&H motif by the Lowe syndrome protein OCRL
  - Nat Struct Mol Biol 18(7):789-795 (2011)
  Nature Structural & Molecular Biology | Article Recognition of the F&H motif by the Lowe syndrome protein OCRL * Michelle Pirruccello1, 2, 3, 5 * Laura E Swan1, 2, 3, 5 * Ewa Folta-Stogniew4 * Pietro De Camilli1, 2, 3 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:789–795Year published:(2011)DOI:doi:10.1038/nsmb.2071Received10 December 2010Accepted15 April 2011Published online12 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Lowe syndrome and type 2 Dent disease are caused by defects in the inositol 5-phosphatase OCRL. Most missense mutations in the OCRL ASH-RhoGAP domain that are found in affected individuals abolish interactions with the endocytic adaptors APPL1 and Ses (both Ses1 and Ses2), which bind OCRL through a short phenylalanine and histidine (F&H) motif. Using X-ray crystallography, we have identified the F&H motif binding site on the RhoGAP domain of OCRL. Missense mutations associated with disease affected F&H binding indirectly by destabilizing the RhoGAP fold. By contrast, a disease-associated mutation that does not perturb F&H binding and ASH-RhoGAP stability disrupted the interaction of OCRL with Rab5. The F&H binding site of OCRL is conserved even in species that do not have an identified homolog for APPL or Ses. Our study predicts the existence of other OCRL binding partners and shows that the perturbation of OCRL interactions has a crucial role in disease. View full text Figures at a glance * Figure 1: Domain structures of INPP5B and OCRL and their F&H motif–containing interactors, and the crystal structure of the human ASH-RhoGAP domain in complex with the F&H peptide from human Ses1. () The ASH domain is red, the RhoGAP domain blue and the Ses1 F&H peptide yellow. Molecular graphics for this and all subsequent figures were generated with PyMOL (Schrödinger). () Close-up view of the interface between the F&H peptide (yellow) and the RhoGAP domain (blue). () F&H peptide sequences used in this study. Phosphoserine residues are circled in green. () GST-ASH-RhoGAP constructs of OCRL were assayed for binding to APPL1 from rat brain homogenate. The GST-negative control is marked as a dash, and the OCRL constructs are either wild-type (WT), or mutated in the F&H binding site (W739A and D743R). As predicted, the mutants disrupt binding to APPL1, without affecting binding to clathrin heavy chain (CHC). * Figure 2: Mutation of the F&H binding site in OCRL disrupts its colocalization with proteins that contain F&H motifs. Colocalization of exogenously expressed OCRL and F&H motif–containing proteins was assessed in human fibroblasts derived from an individual with Lowe syndrome37. Quantification is shown below the images. () Mutation of the F&H interface (W739A) disrupts colocalization of RFP-APPL1 and GFP-OCRL on individual endosomes, although the overall localization of the two proteins is not compromised. Quantification of the colocalization of APPL1 with OCRLWT and OCRLW739A is expressed as the percentage of APPL1 that colocalizes with OCRL. () mTagRFP-T-Ses2 expressed in the absence of OCRL is primarily cytosolic. When co-transfected with GFP-OCRLWT, mTagRFP-T-Ses2 colocalizes with this protein on endosomes and in the Golgi complex area, but this colocalization is disrupted by the W739A mutation in OCRL. The quantification of colocalization is expressed as a function of the colocalization in well-defined puncta, as the main result of the mutation is a diffuse signal of the Ses2 protein! . Error bars, s.e.m. * Figure 3: Conserved surfaces of the ASH-RhoGAP domain map to the F&H-interaction and potential Rab-interaction surfaces. () Surface representation is colored by degree of conservation; darker colors indicate the most conservation. Images were generated using PROTSKIN42. The predicted Rab interaction surface on the ASH domain is indicated by a dashed box.() Sequence alignment highlighting the conservation of the major residues responsible for recognition of the F&H motif. Accession codes for sequences used in the alignment are listed in Supplementary Methods. * Figure 4: Disease-causing mutations that affect F&H binding are global folding mutations. Disease-causing mutations are represented in all structures as spheres. () The mapping of the disease-causing missense mutations onto the ASH-RhoGAP structure, with two mutation networks expanded, one in the ASH domain and the other in the RhoGAP domain. () F&H binding is disrupted by destabilization of OCRL. Inspection of a Coomassie blue–stained gel shows significant degradation as well as the co-purification of a DnaK chaperone for mutants that show a lack of F&H binding. This destabilization is not seen in our designed F&H-binding mutation (W739A), a Rab5-defective mutant (F668V) or a splice-site mutation that results in a lack of expressed protein (A861T). Asterisks, mutations that cause Dent 2 disease. Molecular weights of markers are indicated in kDa. () The GST-ASH-RhoGAP OCRL constructs in were assessed for APPL1 binding using a GST pulldown assay from rat brain followed by western blot for APPL1. * Figure 5: Characterization of a mutation that impairs binding of Rab5. () Residues shown to be important for Rab binding both in previous studies19 and here (Phe668), are represented by gray spheres. () Pulldowns using nucleotide-loaded Rab5 as bait for overexpressed OCRL constructs show that the GTPγS-dependent interaction between Rab5 and OCRL is perturbed by the F668V mutation, but not affected by mutation of the F&H recognition site. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3QIS * 3QIS Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Michelle Pirruccello & * Laura E Swan Affiliations * Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut, USA. * Michelle Pirruccello, * Laura E Swan & * Pietro De Camilli * Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut, USA. * Michelle Pirruccello, * Laura E Swan & * Pietro De Camilli * Program in Cellular Neuroscience Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut, USA. * Michelle Pirruccello, * Laura E Swan & * Pietro De Camilli * W.M. Keck Biotechnology Resource Laboratory, Yale University School of Medicine, New Haven, Connecticut, USA. * Ewa Folta-Stogniew Contributions M.P., L.E.S. and P.D.C. designed research; M.P., L.E.S. and E.F.-S. conducted experiments and contributed new reagents or analytical tools; M.P., L.E.S. and P.D.C. analyzed data; M.P. and P.D.C. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Pietro De Camilli Author Details * Michelle Pirruccello Search for this author in: * NPG journals * PubMed * Google Scholar * Laura E Swan Search for this author in: * NPG journals * PubMed * Google Scholar * Ewa Folta-Stogniew Search for this author in: * NPG journals * PubMed * Google Scholar * Pietro De Camilli Contact Pietro De Camilli Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (6M) Supplementary Figures 1–7 and Supplementary Methods Additional data Entities in this article * Ras-related protein Rab-8A RAB8A Homo sapiens * View in UniProt * View in Entrez Gene * Inositol polyphosphate 5-phosphatase OCRL-1 OCRL Homo sapiens * View in UniProt * View in Entrez Gene * Vesicle-associated membrane protein-associated protein A VAPA Homo sapiens * View in UniProt * View in Entrez Gene * Type II inositol-1,4,5-trisphosphate 5-phosphatase INPP5B Homo sapiens * View in UniProt * View in Entrez Gene * Ras-related C3 botulinum toxin substrate 1 RAC1 Homo sapiens * View in UniProt * View in Entrez Gene * Adaptor protein, phosphotyrosine interaction, PH domain and leucine zipper containing 1 Appl1 Rattus norvegicus * View in UniProt * View in Entrez Gene * Sesquipedalian-1 FAM109A Homo sapiens * View in UniProt * View in Entrez Gene * DCC-interacting protein 13-alpha APPL1 Homo sapiens * View in UniProt * View in Entrez Gene * Ras-related protein Rab-5A RAB5A Homo sapiens * View in UniProt * View in Entrez Gene * Sesquipedalian-2 FAM109B Homo sapiens * View in UniProt * View in Entrez Gene
• Structure-function studies of FMRP RGG peptide recognition of an RNA duplex-quadruplex junction
  - Nat Struct Mol Biol 18(7):796-804 (2011)
  Nature Structural & Molecular Biology | Article Structure-function studies of FMRP RGG peptide recognition of an RNA duplex-quadruplex junction * Anh Tuân Phan1, 2, 6 * Vitaly Kuryavyi1, 6 * Jennifer C Darnell3, 6 * Alexander Serganov1 * Ananya Majumdar5 * Serge Ilin1 * Tanya Raslin1 * Anna Polonskaia1 * Cynthia Chen3 * David Clain3 * Robert B Darnell3, 4 * Dinshaw J Patel1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:796–804Year published:(2011)DOI:doi:10.1038/nsmb.2064Received12 August 2010Accepted30 March 2011Published online05 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg We have determined the solution structure of the complex between an arginine-glycine-rich RGG peptide from the human fragile X mental retardation protein (FMRP) and an in vitro–selected guanine-rich (G-rich) sc1 RNA. The bound RNA forms a newly discovered G-quadruplex separated from the flanking duplex stem by a mixed junctional tetrad. The RGG peptide is positioned along the major groove of the RNA duplex, with the G-quadruplex forcing a sharp turn of R10GGGGR15 at the duplex-quadruplex junction. Arg10 and Arg15 form cross-strand specificity–determining intermolecular hydrogen bonds with the major-groove edges of guanines of adjacent Watson-Crick G•C pairs. Filter-binding assays on RNA and peptide mutations identify and validate contributions of peptide-RNA intermolecular contacts and shape complementarity to molecular recognition. These findings on FMRP RGG domain recognition by a combination of G-quadruplex and surrounding RNA sequences have implications for the rec! ognition of other genomic G-rich RNAs. View full text Figures at a glance * Figure 1: Sequence and NMR spectra of sc1 RNA and RGG peptide. () Sequence of 36-mer sc1 stem-loop RNA and 28-mer FMRP RGG peptide. (,) 1H,15N HSQC spectra of RGG peptide in the free () and sc1 RNA-bound () states. Backbone amide resonance assignments are included in panel . (–) Imino proton spectra (10.0–14.0 p.p.m.) of free sc1 RNA (), sc1 RNA bound to the RGG peptide (), and RGG peptide–sc1 RNA complex () after 16 h in D2O. Spectra were recorded in 50 mM potassium acetate, pH 6.8, at 25 °C. * Figure 2: RNA resonance assignments in the RGG peptide–sc1 RNA complex. () Example of imino proton assignment. Top, reference imino proton NMR spectrum of the complex. Bottom, corresponding filtered spectrum of the complex following site-specific incorporation of 2% 15N-labeled deoxyguanine at the G31 position in the RNA. () Guanine H8 protons were assigned by through-bond connectivities to the guanine imino protons via 13C5 through HMBC experiments. * Figure 3: Identification of G-tetrad alignments and assignment of through-bond correlations in the RGG peptide–sc1 RNA complex. () HNN-COSY contour plot showing through-bond connectivities between amino nitrogens and H8 protons around the G-tetrad. The labeling represents through-bond amino-H8 correlations between adjacent guanines within a G-tetrad plane. () HNN-COSY contour plot showing through-bond connectivities between amino protons and N7 nitrogens around the G-tetrad. The labeling represents through-bond amino-N7 correlations between adjacent guanines within a G-tetrad plane. () Hydrogen-bonding alignment around the G-tetrad and data analysis supporting formation of three G-tetrads. () NOE connectivities between guanine imino and H8 protons around the G-tetrad. The labeling represents through-space imino-H8 correlations between adjacent guanines within a G-tetrad plane. () Identification of intermolecular 1H-15N through-bond correlations in HNN-COSY spectra between guanine H8 protons of RNA and arginine-guanidinium group nitrogens in the complex. Other correlations present in the spectra are i! ntramolecular through-bond connectivities. Peak labeled 'a' is between the H8 proton of G31 and Nɛ nitrogen of Arg10, and peak labeled 'b' is between the H8 proton of G7 and the Nη nitrogen of Arg15. () Identification of intermolecular 1H-1H through-bond correlations in HNN-COSY spectra between guanine H8 protons of RNA and arginine-guanidinium group protons in the complex. Peak labeled 'a' is between the H8 proton of G31 and NɛH proton of Arg10, and peaks labeled 'b' are between the H8 proton of G7 and the NηH amino protons of Arg15. Other correlations present in the spectra are intramolecular through-bond connectivities. * Figure 4: Solution structure of the RGG peptide–sc1 RNA complex and the architecture of the G-quadruplex and duplex-quadruplex junction. () Stereo views of ten superposed refined structures of the RGG peptide–sc1 RNA complex. The bound peptide is colored in red, and the duplex, junction and quadruplex segments are colored in magenta, orange and cyan, respectively. The sugar-phosphate backbone of the RNA is in silver, with phosphorus atoms in yellow. Important arginine (Arg10 and Arg15) side chains are colored in green. () A representative refined structure of the RGG peptide–sc1 RNA complex with the same color coding as in panel . () Location of a potential K+ binding site in the vicinity of the duplex-quadruplex junction following dynamics computations in a water bath containing K+ cations. Note that this K+ cation is anchored through interaction with oxygens from four phosphate groups. * Figure 5: Architecture of the G-quadruplex and duplex-quadruplex junction. () Schematic of the pairing alignments and strand connectivities at the duplex (magenta)–quadruplex (cyan) junction mediated by a mixed tetrad (orange). () Ribbon representation of schematic in panel . (,). Base alignments around the junctional U8•A17•U28•G29 tetrad in the solution structure of the complex. Four refined structures showed the pairing alignment in panel , and six others showing the pairing alignment in panel . () Schematic of pairing alignments and strand connectivities within the G-quadruplex. () Ribbon representation of schematic in panel . * Figure 6: Details of intermolecular peptide-RNA interactions in the solution structure of the RGG peptide–sc1 RNA complex. () Positioning of the Arg10–Arg15 segment of the RGG peptide (stick representation) within the major groove of the duplex segment of the sc1 RNA in the complex (surface representation). () An enlargement of the intermolecular interaction shown in . The peptide residues are labeled from Arg10 to Arg15. (,) Two views highlighting the intermolecular hydrogen bonding interactions between the guanidinium groups of Arg10 and Arg15 with the major-groove edges of G31 and G7, respectively. * Figure 7: Assessment of the molecular determinants of the peptide and of the RNA for the FMRP RGG peptide–sc1 RNA interaction by filter-binding assay. The affinity of interaction of the FMRP RGG box with 35-mer sc1 RNA (red) and mutations therein was determined by filter-binding assay using the indicated concentrations of purified GST-tagged RGG box and 32P end-labeled commercially synthesized RNA. () Mutation of Arg15 to alanine, lysine or leucine abolished sc1 RNA binding by the peptide. () Mutation of the glycines surrounding these crucial arginines. WT RGG: red circles, Kd = 3.8 nM; G12A: open squares, Kd = not determined (ND); G13A: diamonds, Kd = ND; G14A: half-closed squares, Kd = 49.5 nM; G16A: crossed squares, Kd = ND. () Mutation of nucleotides involved in the functional tetrads including U8C and A17U single mutations and U27A U28A double mutation resulted in affinities too low to be measured. () The affinity of the RGG box for synthetic RNAs incorporating deazaguanine—either singly or in combination—in positions G7 and G31 in sc1 RNA was measured to assess the importance of the N7 nitrogen of these guanines.! Mutation of either N7 (G7, open black squares; G31, diamonds) decreased binding by more than two orders of magnitude relative to the WT RNA (red circles), and the double mutation (half-closed black squares) decreased binding to an undetectable level. Accession codes * Abstract * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 2LA5 * 2LA5 Author information * Abstract * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Anh Tuân Phan, * Vitaly Kuryavyi & * Jennifer C Darnell Affiliations * Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York, USA. * Anh Tuân Phan, * Vitaly Kuryavyi, * Alexander Serganov, * Serge Ilin, * Tanya Raslin, * Anna Polonskaia & * Dinshaw J Patel * School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore. * Anh Tuân Phan * Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York, USA. * Jennifer C Darnell, * Cynthia Chen, * David Clain & * Robert B Darnell * Howard Hughes Medical Institute, The Rockefeller University, New York, New York, USA. * Robert B Darnell * Biomolecular NMR Center, Johns Hopkins University, Baltimore, Maryland, USA. * Ananya Majumdar Contributions A.T.P., A.M. and S.I. were responsible for NMR studies, V.K. undertook the computations, and A.S., T.R. and A.P. prepared labeled NMR samples, all under the supervision of D.J.P. C.C., D.C. and J.C.D. did the filter-binding assays under the joint supervision of J.C.D. and R.B.D. The paper was written jointly by D.J.P., A.T.P., V.K., J.C.D. and R.B.D. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Dinshaw J Patel or * Jennifer C Darnell or * Anh Tuân Phan Author Details * Anh Tuân Phan Contact Anh Tuân Phan Search for this author in: * NPG journals * PubMed * Google Scholar * Vitaly Kuryavyi Search for this author in: * NPG journals * PubMed * Google Scholar * Jennifer C Darnell Contact Jennifer C Darnell Search for this author in: * NPG journals * PubMed * Google Scholar * Alexander Serganov Search for this author in: * NPG journals * PubMed * Google Scholar * Ananya Majumdar Search for this author in: * NPG journals * PubMed * Google Scholar * Serge Ilin Search for this author in: * NPG journals * PubMed * Google Scholar * Tanya Raslin Search for this author in: * NPG journals * PubMed * Google Scholar * Anna Polonskaia Search for this author in: * NPG journals * PubMed * Google Scholar * Cynthia Chen Search for this author in: * NPG journals * PubMed * Google Scholar * David Clain Search for this author in: * NPG journals * PubMed * Google Scholar * Robert B Darnell Search for this author in: * NPG journals * PubMed * Google Scholar * Dinshaw J Patel Contact Dinshaw J Patel Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (10M) Supplementary Figures 1–9, Supplementary Tables 1–3, Supplementary Discussion and Supplementary Methods Additional data Entities in this article * Fragile X mental retardation 1 protein FMR1 Homo sapiens * View in UniProt * View in Entrez Gene * Myelin expression factor 2 Myef2 Mus musculus * View in UniProt * View in Entrez Gene * Protein arginine N-methyltransferase 1 PRMT1 Homo sapiens * View in UniProt * View in Entrez Gene * Fragile X mental retardation syndrome-related protein 1 FXR1 Homo sapiens * View in UniProt * View in Entrez Gene * Fragile X mental retardation protein 1 homolog Fmr1 Mus musculus * View in UniProt * View in Entrez Gene * Nucleolin NCL Homo sapiens * View in UniProt * View in Entrez Gene * Fragile X mental retardation syndrome-related protein 2 FXR2 Homo sapiens * View in UniProt * View in Entrez Gene
• Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation
  - Nat Struct Mol Biol 18(7):805-812 (2011)
  Nature Structural & Molecular Biology | Article Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation * Geert van den Bogaart1 * Shashi Thutupalli2 * Jelger H Risselada3 * Karsten Meyenberg4 * Matthew Holt1 * Dietmar Riedel5 * Ulf Diederichsen4 * Stephan Herminghaus2 * Helmut Grubmüller3 * Reinhard Jahn1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:805–812Year published:(2011)DOI:doi:10.1038/nsmb.2061Received23 November 2010Accepted17 March 2011Published online05 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Synaptotagmin-1 triggers Ca2+-sensitive, rapid neurotransmitter release by promoting interactions between SNARE proteins on synaptic vesicles and the plasma membrane. How synaptotagmin-1 promotes this interaction is unclear, and the massive increase in membrane fusion efficiency of Ca2+-bound synaptotagmin-1 has not been reproduced in vitro. However, previous experiments have been performed at relatively high salt concentrations, screening potentially important electrostatic interactions. Using functional reconstitution in liposomes, we show here that at low ionic strength SNARE-mediated membrane fusion becomes strictly dependent on both Ca2+ and synaptotagmin-1. Under these conditions, synaptotagmin-1 functions as a distance regulator that tethers the liposomes too far from the plasma membrane for SNARE nucleation in the absence of Ca2+, but while bringing the liposomes close enough for membrane fusion in the presence of Ca2+. These results may explain how the relatively we! ak electrostatic interactions between synaptotagmin-1 and membranes substantially accelerate fusion. View full text Figures at a glance * Figure 1: Electrostatic repulsion blocks SNARE-mediated membrane fusion at low ionic strength. () Lipid mixing of DiD-labeled liposomes containing syntaxin-1A–SNAP-25 (Sx1ASn25) with OG-PE–labeled liposomes containing synaptobrevin-2 (Sb2). Membrane fusion results in donor quenching. Fusion was measured at the KCl concentrations indicated. () As in , but now for liposomes containing the synaptobrevin-249–96–stabilized acceptor complex34. The osmolarity was preserved with sucrose. Lipid mixing was still blocked without sucrose (dashed green curve), but the liposomes were deformed by osmotic stress (inset, negative-staining EM; scale bar, 100 nm). Synaptobrevin-21–96 inhibited fusion (Rsol; for 150 mM KCl). () As in , but now with pure phosphatidylcholine (PC) liposomes where lipid mixing occurred at both low (green) and normal (black) ionic strength. () Ten percent anionic phosphoserine (PS) blocked fusion at low, but not normal, ionic strength. () SNARE complex formation by FRET35. The soluble SNARE domain of synaptobrevin-2 (100 nM; Rsol; solid curves) or s! yntaxin-1A with SNAP-25 (Qsol; dashed curves) resulted in complex formation regardless of ionic strength. () By contrast, mixing liposomes containing membrane-anchored synaptobrevin-2 and syntaxin-1A resulted in complex formation only at normal but not at low ionic strength regardless of 1 mM Ca2+ (pink). The structure58 shows the dye positions. The FRET efficiencies are underestimated because the ~20% cross-talk is not accounted for. Typical curves of several repeats are shown. Experiments were performed with 4–8 nM liposomes at 20 °C. * Figure 2: At low ionic strength, Ca2+–synaptotagmin-1 triggers lipid mixing. (,) Lipid mixing of synaptobrevin-2–synaptotagmin-1 liposomes with those containing syntaxin-1A–SNAP-25 () and the synaptobrevin-249–96–stabilized acceptor complex34 (). At low ionic strength, 1 mM Ca2+ substantially increased lipid mixing. There was much less fusion with 1 mM Mg2+. No lipid mixing was observed without synaptotagmin-1 (No Syt1) or synaptobrevin-2 (No Syb2). Synaptobrevin-21–96 (Rsol; for 1 mM Ca2+) or SNAP-25 and syntaxin-1A183–262 (Qsol) inhibited fusion. (–) Ca2+-triggered lipid mixing measured by flow cytometry. () Donor and acceptor liposomes (as in ) were mixed in the 23 cm × 100 μm × 80 μm (LWH) flow chamber. The flow speed was kept constant. At 25% of the channel length, Ca2+ was introduced by fast focused mixing. (,) Acceptor fluorescence along the channel with fluorescence microscopy () and binning of the fluorescence (), showing Ca2+-specific lipid mixing. () Lipid mixing at low ionic strength (as in ) at the Ca2+ concentrations i! ndicated, with (solid) or without (dashed) 0.5 mM Mg2+. () As in , but now with the synaptobrevin-249–96–stabilized acceptor complex. () Fluorescence dequenching experiment showing Ca2+-dependent lipid mixing of synaptobrevin-249–96–stabilized acceptor complex liposomes with purified rat synaptic vesicles37. Experiments were performed with 4 nM liposomes at 20 °C. Typical experiments from two to four independent repeats are shown. * Figure 3: Ca2+–synaptotagmin-1 triggers full membrane fusion at low ionic strength. () Content mixing24 with a self-quenching concentration of calcein encapsulated in liposomes with the stabilized Q-SNARE complex. Fusion with empty R-SNARE liposomes resulted in specific fluorescence dequenching. Synaptobrevin-21–96 (Rsol) or the absence of Ca2+ or synaptotagmin-1 (No Syt1) abolished fusion. () Ca2+-dependent SNARE complex formation by FRET as in Figure 1f with membrane-anchored synaptobrevin-2–synaptotagmin-1. (–) SNARE complex formation with FRET. Liposomes containing the stabilized Q-SNARE complex were incubated with synaptobrevin-21–116 liposomes without Ca2+. The synaptobrevin-2 liposomes contained no synaptotagmin-1 (), wild-type (WT) synaptotagmin-1 () or the KAKA mutant (). At the indicated times, synaptobrevin-21–96 was added, either unlabeled (red curve; in case of Texas red–labeled synaptobrevin-21–116) or Texas red labeled (blue; in case of unlabeled synaptobrevin-21–116). Black curve, control without synaptobrevin-21–96. Membra! ne fusion was triggered with Ca2+. Synaptobrevin-21–96 bound to the Q-SNAREs regardless of the presence of synaptobrevin-21–116–synaptotagmin-1 liposomes. () Lipid mixing by wild-type Ca2+–synaptotagmin-1 (AB) and Ca2+-binding disruption mutants to C2B (Ab*), C2A (a*B) or both (a*b*). () SNARE complex formation by FRET for the synaptotagmin-1 mutants paralleled lipid mixing. () Lipid mixing of synaptobrevin-2–synaptotagmin-1 liposomes with 10–20-μm GUVs containing the Q-SNAREs (inset, fluorescence microscopy; scale bar, 50 μm). Disrupting binding of Ca2+ to the C2B domain abolished fusion with GUVs. We used ~4 nM liposomes at 20 °C. Typical curves from two or three independent repeats are shown. * Figure 4: Liposome clustering by synaptotagmin-1. () DLS at low ionic strength. Liposomes with 1:4,000 synaptotagmin-1 mutants were mixed 1:1 with PtdIns(4,5)P2 liposomes (no SNAREs; bar graphs and polydispersities, Supplementary Fig. 4c). The grayscale indicates the liposome cluster size. () Microscale capillary thermophoresis44, 45 tethering experiment. OG-PE–labeled liposomes move toward the heated spot in a capillary (negative thermophoresis). By contrast, DiD-labeled liposomes containing synaptotagmin-1 move away from the heated spot (positive thermophoresis). Tethered liposomes show intermediate thermophoresis. () The change in OG-PE fluorescence after 30 s heating with the focused IR laser (F2 − F1; n = 3, ± s.d.). Tethering was reduced without PtdIns(4,5)P2 or with the KAKA mutant. (,) Docking arrangements with liposomes tethered close () or further away (). () FRET docking46 immediately after mixing DiI-liposomes containing 1:4,000 synaptotagmin-1 with DiD-liposomes. No FRET was observed without Ca2+, and Ca2+! increased FRET. We used 1 mM EDTA to show that this increased FRET was due to close proximity of the membranes. FRET was substantially reduced without PtdIns(4,5)P2. () The acceptor over donor fluorescence from (± s.d., n = 3). () DLS as in , but with 10 nM of the C2AB fragment (see also Supplementary Fig. 4f). Total liposome concentrations were 2–4 nM. Typical data from two or three independent repeats are shown. * Figure 5: Preclustering of liposomes accelerates lipid mixing. () We incubated 150 pM or 2 nM of R-SNARE liposomes for 20 min with the same amounts of Q-SNARE liposomes. Subsequently, lipid mixing was triggered by addition of Ca2+. For the high (4 nM total) liposome concentrations, the lipid mixing efficiency of wild-type synaptotagmin-1 was comparable to that of the KAKA mutant. By contrast, for the low liposome concentrations (300 pM), the fusion efficiency was substantially lower for the KAKA mutant compared to the wild type. () Lipid mixing of full-length synaptotagmin-1–synaptobrevin-2 liposomes with liposomes containing the Q-SNAREs of constitutive exocytosis (syntaxin-4 and SNAP-23). We saw no fusion in the absence of Ca2+ (No Ca2+), and fusion could be blocked with synaptobrevin-21–96 (Rsol). All fusion experiments were performed at 20 °C. Typical curves from two independent repeats are shown. * Figure 6: Models of distance regulation by synaptotagmin-1. () Molecular dynamic simulations to estimate the maximal distances between the various domains. In the simulations, we pulled pairwise on various membrane-binding sites of the C2AB domain: the N terminus, the polybasic lysine patch (KKKK) and the Ca2+-binding sites of the C2A and C2B domains. The two conserved arginines (R398 and R388) are shown in brown; the linker is in black. The N terminus is connected to the transmembrane helix with a 61-residue linker that can extent to ~23 nm. Maximal distances are indicated; these are approximate for the tethering distances of the membranes but do not take into account additional interactions such as membrane insertion and bending. () Model of synaptotagmin-1–mediated lipid mixing. In the absence of Ca2+, synaptotagmin-1 (gray) tethers to anionic membranes, and particularly to PtdIns(4,5)P2 (orange) through its polybasic lysine patch. The distance is too far for SNAREs to form complexes (synaptobrevin-2: blue; syntaxin-1A: red; SNA! P-25: green). Step A: in the presence of Ca2+ (purple), a transitional conformation change occurs and synaptotagmin-1 binds the membrane by its calcium binding pockets and basic residues on the C2AB domain9. This drives the membranes together and SNARE complex formation can occur. Step B: the formation of SNARE complexes drives membrane fusion. Author information * Abstract * Author information * Supplementary information Affiliations * Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. * Geert van den Bogaart, * Matthew Holt & * Reinhard Jahn * Department of Dynamics of Complex Fluids, Max Planck Institute for Dynamics and Self-Organization, Göttingen, Germany. * Shashi Thutupalli & * Stephan Herminghaus * Department of Theoretical and Computational Biophysics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. * Jelger H Risselada & * Helmut Grubmüller * Institute for Organic and Biomolecular Chemistry, Georg-August University, Göttingen, Germany. * Karsten Meyenberg & * Ulf Diederichsen * Facility for Electron Microscopy, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. * Dietmar Riedel Contributions S.T. and S.H. performed the flow cytometry experiments. J.H.R. and H.G. performed the MD simulations. M.H. purified the synaptic vesicles. Thermophoresis data were from K.M. and U.D. D.R. performed the EM. G.v.d.B. performed all other experiments. G.v.d.B. and R.J. designed the study and wrote the paper. All authors discussed the results and commented on the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Reinhard Jahn Author Details * Geert van den Bogaart Search for this author in: * NPG journals * PubMed * Google Scholar * Shashi Thutupalli Search for this author in: * NPG journals * PubMed * Google Scholar * Jelger H Risselada Search for this author in: * NPG journals * PubMed * Google Scholar * Karsten Meyenberg Search for this author in: * NPG journals * PubMed * Google Scholar * Matthew Holt Search for this author in: * NPG journals * PubMed * Google Scholar * Dietmar Riedel Search for this author in: * NPG journals * PubMed * Google Scholar * Ulf Diederichsen Search for this author in: * NPG journals * PubMed * Google Scholar * Stephan Herminghaus Search for this author in: * NPG journals * PubMed * Google Scholar * Helmut Grubmüller Search for this author in: * NPG journals * PubMed * Google Scholar * Reinhard Jahn Contact Reinhard Jahn Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (446K) Supplementary Figures 1–5 Additional data Entities in this article * Synaptotagmin-1 Syt1 Rattus norvegicus * View in UniProt * View in Entrez Gene * Syntaxin-4 Stx4 Rattus norvegicus * View in UniProt * View in Entrez Gene * Syntaxin-1A Stx1a Rattus norvegicus * View in UniProt * View in Entrez Gene * Vesicle-associated membrane protein 2 Vamp2 Rattus norvegicus * View in UniProt * View in Entrez Gene * Synaptosomal-associated protein 23 Snap23 Rattus norvegicus * View in UniProt * View in Entrez Gene * Synaptosomal-associated protein 25 Snap25 Rattus norvegicus * View in UniProt * View in Entrez Gene
• Mechanism and function of synaptotagmin-mediated membrane apposition
  - Nat Struct Mol Biol 18(7):813-821 (2011)
  Nature Structural & Molecular Biology | Article Mechanism and function of synaptotagmin-mediated membrane apposition * Enfu Hui1, 2, 3 * Jon D Gaffaney1, 3 * Zhao Wang1 * Colin P Johnson1 * Chantell S Evans1 * Edwin R Chapman1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:813–821Year published:(2011)DOI:doi:10.1038/nsmb.2075Received01 June 2010Accepted28 April 2011Published online05 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Synaptotagmin-1 is a Ca2+ sensor that triggers synchronous neurotransmitter release. The first documented biochemical property of synaptotagmin-1 was its ability to aggregate membranes in response to Ca2+. However, the mechanism and function of this process were poorly understood. Here we show that synaptotagmin-1–mediated vesicle aggregation is driven by trans interactions between synaptotagmin-1 molecules bound to different membranes. We found a strong correlation between the ability of Ca2+-bound synaptotagmin-1 to aggregate vesicles and to stimulate SNARE-mediated membrane fusion. Moreover, artificial aggregation of membranes—using non-synaptotagmin proteins—also efficiently promoted fusion of SNARE-bearing liposomes. Finally, using a modified fusion assay, we observed that synaptotagmin-1 drove the assembly of otherwise non-fusogenic individual t-SNARE proteins into fusion-competent heterodimers, independently of aggregation. Thus, membrane aggregation and t-SNARE! assembly appear to be two key aspects of fusion reactions that are regulated by Ca2+-bound synaptotagmin-1 and catalyzed by SNAREs. View full text Figures at a glance * Figure 1: The cytoplasmic domain of synaptotagmin-1 aggregates phosphatidylserine-harboring vesicles in a Ca2+-dependent manner. () Kinetics of turbidity changes (absorbance at 400 nm, A400nm) of the protein-free liposome suspension (pf) in response to sequential addition of 1 μM C2AB, 1 mM Ca2+ and 2 mM EGTA. (–) The same experiment as in but using t-SNARE–bearing liposomes (designated as Tr, ) v-SNARE-bearing liposomes (designated as Vr, ) or a mixture of Tr and Vr at a 9:1 molar ratio (). In all panels, the total lipid concentration was ~113 μM. The Ca2+C2AB-dependent increases in liposome turbidity were fit with a double exponential function, and the time constant (τ) of the fast component is reported in the text. The triangle indicates the irreversible A400nm signal that probably resulted from vesicle fusion. The representative data shown were reproducible in two independent trials. * Figure 2: C2AB aggregates liposomes through a mechanism distinct from those of avidin-biotin and poly-D-lysine. () C2AB-mediated liposome aggregation (A400nm) was assayed as a function of C2AB concentration. The turbidity was measured 10 min after mixing C2AB and liposomes (113 μM total lipid) in the presence of 1 mM Ca2+ (closed circles). As controls, liposomes were omitted from the mixture and the turbidity measured (open circles). () The protein-alone aggregation signals were subtracted from the aggregation signals for the protein-lipid mixtures, to obtain corrected signals for C2AB-mediated liposome aggregation. () Liposome aggregation was assayed as a function of poly-D-lysine concentration. () Avidin-induced aggregation of biotinylated liposomes (2% biotin-phosphatidylethanolamine) was assayed as function of avidin concentration. () A model summarizing C2AB-mediated liposome aggregation at different protein-to-lipid ratios. () 0.3 μM C2AB was mixed with increasing lipid concentrations, in the presence of either 0.2 mM EGTA or 1 mM Ca2+, and the turbidity was measured 10 min af! ter mixing. At relatively high lipid concentrations, the background turbidity became substantial, leading to an upward deflection in the signal. () Ca2+-C2AB–triggered increases in turbidity were corrected from () Poly-D-lysine (1 μg ml−1) was mixed with increasing lipid concentrations, and the turbidity of the mixture was measured. () Avidin (1 μM) was mixed with increasing concentrations of biotinylated liposomes, and the turbidity of the mixture was plotted as a function of lipid concentration. Data are represented as mean ± s.e.m., n = 3. * Figure 3: Mutational analysis of synaptotagmin-1–mediated membrane aggregation. () Liposome suspensions containing 113 μM total lipid were mixed with increasing concentrations of protein in the presence of 1 mM Ca2+, incubated for 10 min at room temperature, and the turbidity was plotted as a function of protein concentration for each of the indicated synaptotagmin-1 mutants. () The threshold protein concentration for aggregation was defined as the lowest concentration tested that gave rise to ≥50% increase in turbidity. The representative data shown were reproducible in two or three independent trials. * Figure 4: Mapping the membrane-binding interface of C2B using NBD fluorescence reporters. () Structural model46 depicting individual amino acid residues of C2B, in the context of C2AB, that were replaced with cysteines; for simplicity only C2B is shown. The environmentally sensitive fluorophore NBD was used to label each of these sole cysteine residues. () Membrane penetration activity of each residue depicted in . The NBD fluorescence spectrum of each C2AB mutant (0.3 μM) was obtained in the presence of liposomes plus either 0.2 mM EGTA (red) or 1 mM Ca2+ (black). For each mutant, fluorescence was normalized to the peak intensity of the spectrum in EGTA. Ca2+ triggered a marked fluorescence increase when NBD was placed on the tip of the Ca2+-binding loops, but not when NBD was placed on other surfaces of C2B. * Figure 5: FRET between NBD-labeled C2AB and rhodamine-labeled liposomes. () Each NBD-labeled cysteine mutant of C2AB (0.3 μM) shown in Figure 4a was mixed with liposomes that harbored 0.3% rhodamine-phosphatidylethanolamine. Fluorescence spectra were obtained before (blue) and after (green) the addition of 1 mM Ca2+. Mixtures of NBD-labeled C2AB and rhodamine-free liposomes were also tested in parallel as references (EGTA, red; Ca2+, black). For each mutant, all four spectra were normalized to the maximum intensity of samples obtained using rhodamine-free liposomes in EGTA. * Figure 6: Synaptotagmin-1–mediated vesicle aggregation occurs through trans interactions. () Pre-pinning of C2AB to liposomes. Phosphatidylserine-harboring liposomes (1 mM total lipid) were mixed with 2 μM wild-type C2AB or C2AB (R398, 399Q) in the presence of 1 mM Ca2+, and subjected to co-sedimentation assays (see Online Methods). Representative gel shows that both wild-type and mutant C2AB were completely depleted from the supernatant. () Model depicting the experiment. C2AB (2 μM) was pre-pinned to liposomes (1 mM total lipid) that harbored 2% of either biotin-phosphatidylethanolamine or dansyl-phosphatidylethanolamine. Immediately after preparation, C2AB-coated liposomes were mixed with naked liposomes or the other set of C2AB-coated liposomes in the presence of 1 mM Ca2+ or 2 mM EGTA. Avidin beads (30 μl, 50% slurry) were then added to precipitate biotin-phosphatidylethanolamine vesicles (along with any bound dansyl-labeled vesicles) by low-speed centrifugation (2,300g, 1 min). Beads were washed three times with 500 ml of buffer containing either 1 mM Ca! 2+ or 2 mM EGTA and incubated with 150 μl of n-dodecylmaltoside, and the supernatant was collected and the fluorescence intensity of dansyl determined. () Dansyl fluorescence of the avidin beads under the conditions indicated. Replacement of wild-type C2AB with C2AB (R398, 399Q) completely abolished the dansyl signal to background levels. Errors bars represent s.e.m., n = 3. * Figure 7: The ability of synaptotagmin-1 to aggregate vesicles is correlated with its ability to stimulate fusion of v-SNARE vesicles with t-SNARE heterodimer vesicles. () Representative NBD de-quenching signals corresponding to fusion between SNARE-bearing vesicles (Tr and Vr) regulated by the indicated concentrations of C2AB. () The final extent of fusion (closed circles) as shown in and the degree of aggregation (open circles) as shown in Figure 3 were plotted against C2AB concentration in the same plot. In most cases, vesicle aggregation activity and the fusion-promoting activity of C2AB were closely correlated. () The protein dose-responses for vesicle aggregation and the stimulation of fusion, mediated by each of the indicated synaptotagmin-1 constructs, were compared. The representative data shown were reproducible in two or three independent trials. * Figure 8: Stimulation of SNARE-catalyzed fusion by other vesicle-aggregating proteins. () The final extent (at 10 min) of Ca2+-dependent, cPLA2-C2–mediated vesicle aggregation was plotted as a function of cPLA2-C2 concentration. The turbidity (A400nm) of the vesicle suspension in the presence of EGTA was subtracted from signals obtained in the presence of Ca2+. Data were fit with a sigmoidal dose-response function (variable slope) using GraphPad Prism 5.0 software. () Representative NBD de-quenching signal corresponding to Ca2+-regulated fusion between SNARE-bearing vesicles (Tr and Vr) in the presence of indicated concentrations of cPLA2-C2. () The final extent of fusion at 60 min, as shown in , was plotted against cPLA2-C2 concentration. () The final extent of aggregation and fusion were both plotted against cPLA2-C2 concentration. () The final extent of avidin-mediated aggregation of biotinylated vesicles was plotted as a function of avidin concentration. () Representative NBD de-quenching signals corresponding to fusion between SNARE-bearing, biotinylate! d vesicles at increasing avidin concentrations. () The extent of fusion at 60 min plotted against avidin concentration. () The final extent of aggregation and fusion plotted against avidin concentration. Representative data are shown from two or three independent trials that yielded similar results. * Figure 9: Ca2+-C2AB, but not other aggregating agents, accelerates fusion between syntaxin-bearing vesicles (SYXr) and Vr in the presence of soluble SNAP-25. () SYXr, Vr and other reactants were added to the reaction at the time points indicated by arrows. In response to Ca2+, C2AB efficiently stimulated fusion of SYXr and Vr in the presence of soluble SNAP-25 (purple trace). Omission of either soluble SNAP-25 or C2AB abrogated the Ca2+-dependent stimulatory effect (green trace and red trace, respectively). Replacing C2AB with avidin or cPLA2-C2 did not restore regulated fusion. () Models depicting how Ca2+–synaptotagmin-1 stimulates fusion of SNARE-bearing SUVs by both aggregating membranes and assembling t-SNAREs into fusion competent heterodimers. Shown are representative fusion traces from three independent trials. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Enfu Hui & * Jon D Gaffaney Affiliations * Howard Hughes Medical Institute and Department of Neuroscience, University of Wisconsin, Madison, Wisconsin, USA. * Enfu Hui, * Jon D Gaffaney, * Zhao Wang, * Colin P Johnson, * Chantell S Evans & * Edwin R Chapman * Present address: Department of Molecular and Cellular Pharmacology, University of California, San Francisco, San Francisco, California, USA. * Enfu Hui Contributions E.R.C. conceived of and supervised the project; E.H., J.D.G. and E.R.C. designed the experiments and wrote the manuscript; E.H. conducted all the aggregation assays on synaptotagmin-1 proteins and cPLA2-C2, membrane penetration assays and FRET-based membrane binding assays; J.D.G. conducted all the fusion assays on synaptotagmin-1 proteins and cPLA2-C2 and performed experiments for Figure 6 with Z.W.; Z.W. carried out experiments for Figure 9; C.P.J. conducted avidin-biotin–mediated aggregation and fusion assays in Figure 2d,i and Figure 8e–h; C.S.E. carried out experiments in Supplementary Figure 8. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Edwin R Chapman Author Details * Enfu Hui Search for this author in: * NPG journals * PubMed * Google Scholar * Jon D Gaffaney Search for this author in: * NPG journals * PubMed * Google Scholar * Zhao Wang Search for this author in: * NPG journals * PubMed * Google Scholar * Colin P Johnson Search for this author in: * NPG journals * PubMed * Google Scholar * Chantell S Evans Search for this author in: * NPG journals * PubMed * Google Scholar * Edwin R Chapman Contact Edwin R Chapman Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–10 and Supplementary Methods Additional data Entities in this article * Synaptotagmin-1 Syt1 Rattus norvegicus * View in UniProt * View in Entrez Gene * Annexin A4 ANXA4 Bos taurus * View in UniProt * View in Entrez Gene * Syntaxin-1A Stx1a Rattus norvegicus * View in UniProt * View in Entrez Gene * Vesicle-associated membrane protein 2 Vamp2 Mus musculus * View in UniProt * View in Entrez Gene * Cytosolic phospholipase A2 PLA2G4A Homo sapiens * View in UniProt * View in Entrez Gene * Synaptosomal-associated protein 25 Snap25 Mus musculus * View in UniProt * View in Entrez Gene
• An ALS-associated mutation affecting TDP-43 enhances protein aggregation, fibril formation and neurotoxicity
  - Nat Struct Mol Biol 18(7):822-830 (2011)
  Nature Structural & Molecular Biology | Article An ALS-associated mutation affecting TDP-43 enhances protein aggregation, fibril formation and neurotoxicity * Weirui Guo1, 2, 8 * Yanbo Chen1, 3, 8 * Xiaohong Zhou1, 2, 8 * Amar Kar2, 7 * Payal Ray2 * Xiaoping Chen2 * Elizabeth J Rao4 * Mengxue Yang1 * Haihong Ye1 * Li Zhu1 * Jianghong Liu1 * Meng Xu5 * Yanlian Yang5 * Chen Wang5 * David Zhang2 * Eileen H Bigio6 * Marsel Mesulam6 * Yan Shen3 * Qi Xu3 * Kazuo Fushimi2 * Jane Y Wu1, 2 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:822–830Year published:(2011)DOI:doi:10.1038/nsmb.2053Received07 August 2010Accepted04 April 2011Published online12 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Mutations in TARDBP, encoding TAR DNA-binding protein-43 (TDP-43), are associated with TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We compared wild-type TDP-43 and an ALS-associated mutant TDP-43in vitro and in vivo. The A315T mutant enhances neurotoxicity and the formation of aberrant TDP-43 species, including protease-resistant fragments. The C terminus of TDP-43 shows sequence similarity to prion proteins. Synthetic peptides flanking residue 315 form amyloid fibrils in vitro and cause neuronal death in primary cultures. These data provide evidence for biochemical similarities between TDP-43 and prion proteins, raising the possibility that TDP-43 derivatives may cause spreading of the disease phenotype among neighboring neurons. Our work also suggests that decreasing the abundance of neurotoxic TDP-43 species, enhancing degradation or clearance of such TDP-43 derivatives and blocking the spread of the ! disease phenotype may have therapeutic potential for TDP-43 proteinopathies. View full text Figures at a glance * Figure 1: Expression of A315T mutant hTDP-43 in motor neurons (MNs) leads to enhanced axonal damage and more severe impairment of locomotive function. (–) MNs expressing either wild-type hTDP-43 (WT; ) or the A315T mutant () showed marked axon swelling (indicated by arrows) and disruption of axonal integrity, whereas MNs in vector control flies () showed normal axonal morphology. Fluorescence microscopic images are shown, including membrane GFP (mGFP, green; panel 1), RFP signal (red; panel 2) and overlay of images in different channels (panel 3) (scale bars, 50 μm). Axon swelling is marked by white arrows, whereas a loss of axonal integrity is marked by the purple arrow. Images of the third-instar larvae of corresponding groups are shown in panel 4, with rulers in orange (scale bar, 1 mm). () Flies expressing hTDP-43 in MNs show functional deficits. Two and three independent lines of flies expressing WT TDP-43 or the A315T mutant, respectively, were scored and compared with the vector control group (n = 20 in each group). Data were compared using one-way ANOVA with a Bonferroni post hoc test. A315T mutant flies showed ! more severe movement deficits. ***P < 0.001. Fly genotypes: control, OK371-Gal4/UAS-mGFP/UAS-RFP; WT, OK371-Gal4/UAS-mGFP/UAS-Wt-hTDP43-RFP; A315T, OK371-Gal4/UAS-mGFP/UAS-A315T-hTDP43-RFP. * Figure 2: Expression of the A315T mutant causes more severe motor neuron (MN) damage. () Control flies have normal MNs with well-organized clusters in the ventral nerve cord (VNC). mGFP, membrane GFP; red fluorescent protein, RFP; Nu, Hoechst dye nuclear staining. (,) MNs in third-instar larvae of transgenic flies expressing hTDP-43 show cell death and morphological abnormality in MN clusters, especially in the last three VNC segments. MN damage is much more prominent in flies expressing the A315T mutant. Arrowheads mark swollen neurons with the mGFP area enlarged. Arrows mark MNs with fragmented or condensed nuclei and reduced mGFP signals. Quantification of MNs in the last three VNC segments indicates that 79 ± 5% of MNs expressing the A315T mutant, as compared to 32 ± 3% of those expressing wild-(WT) type TDP, show cell body swelling or condensed nuclei (with six flies in each group scored in three independent experiments). Fly genotypes: , OK371-Gal4/UAS-mGFP/UAS-RFP; , OK371-Gal4/UAS-mGFP/UAS-TDP-43-RFP; , OK371-Gal4/UAS-mGFP/UAS-A315T TDP-43-RFP. Scal! e bars, 20 μm. * Figure 3: FTLD-TDP brain samples show abnormal TDP-43–immunoreactive species. () RIPA-soluble protein lysates were prepared from postmortem brain tissues from seven control subjects and seven subjects with TDP-43–immunoreactive FTLD (see Online Methods and Supplementary Methods). Control samples were from non–cognitively impaired subjects with minimal Alzheimer's disease pathology containing Braak & Braak tangle stages II–III, except for one with mild cognitive impairment and pathological diagnosis of early Alzheimer's disease (lane 7). The samples were analyzed by western blotting using specific anti–TDP-43 antibodies. Several TDP-43–positive bands were detected, including the predicted band migrating at 43 kDa (*), bands migrating at ~74 kDa (arrow) and a band migrating faster than 37 kDa (arrowhead). The 74-kDa species was prominent in samples from seven of the subjects with FTLD-TDP (lanes 8–14) but was detectable at only a low level in samples from three (lanes 5–7) out of seven of the control subjects with Alzheimer's disease (lane! s 1–7). () Actin was used as a control showing that similar amounts of total proteins were loaded. * Figure 4: Biochemical characterization of TDP-43–immunoreactive species. () Stable HEK293 cells expressing HA-tagged wild-type or A315T hTDP-43 were lysed in RIPA buffer. The RIPA-insoluble fraction was extracted in RIPA buffer containing 2% (w/v) Sarkosyl and 500 mM NaCl. Sarkosyl-soluble (S) and Sarkosyl-insoluble pellet (P) fractions were separated by centrifugation and analyzed by western blotting using anti-HA. In addition to the expected 43-kDa band (*), a prominent band migrating at approximately 75 kDa was detected in the Sarkosyl-insoluble pellet from cells expressing A315T TDP-43 (lane 4). This 75-kDa was detected only at a low level in the Sarkosyl-soluble fraction (lane 2) and was not detectable in the cell lysates expressing wild-type TDP-43. () SDS-resistant aberrant TDP-43 species were detected in the Sarkosyl-insoluble pellet of lysates from cells expressing TDP-43. The Sarkosyl-soluble fractions (lanes 1 and 3) or Sarkosyl-insoluble pellets (lanes 2 and 4) from either the wild-type (lanes 1 and 2) or A315T mutant (lanes 3-4) TDP-! 43 cells were analyzed by semidenaturing agarose gel electrophoresis (SDD-AGE) as described47 followed by western blotting using a specific anti–TDP-43 antibody. Sarkosyl-insoluble fractions were extracted using 3% SDS buffer before being loaded on SDD-AGE. SDS-resistant oligomeric species (migrating slower than the monomer species) were substantially more abundant in cells expressing the A315T mutant than in those expressing the wild-type TDP-43. (,) RIPA-soluble fractions of cell lysates expressing either wild-type TDP-43-HA () or A315T TDP-43-HA () were loaded onto a gel filtration column with different fractions examined by western blotting using anti-HA or anti-TPX antibodies. IN, input cell lysates. The arrow and arrowheads mark the 75-kDa and 60-kDa high-molecular-weight species, respectively. The asterisk marks the expected 43-kDa monomer TDP-43 band. Some gel lanes have been omitted for reasons of space; the complete gel image is provided in Supplementary Figure ! 3. () Western blot signals in and were plotted for the 43-kDa ! species (green), 75-kDa species (red) of A315T TDP-43 and TPX band (black) for different fractions, with 440-kDa and 67-kDa size markers indicated. The 23-kDa TPX protein was detected in fractions 32–35. The aberrant 75-kDa A315T TDP-43 species was detected in fractions 24–29, corresponding to molecular weight range from 440 kDa to 67 kDa. * Figure 5: Cells expressing A315T TDP-43 show high-molecular-weight phosphorylated protein species that are resistant to heat, DTT and urea and produce fragments partially resistant to protease K (PK) treatment. () High-molecular-weight bands are detected in lysates from cells expressing A315T TDP-43. Stable cells expressing HA-tagged wild-type or A315T TDP-43 (marked by WT or A, respectively) were lysed and subjected to western blotting using anti-HA antibody. In addition to the 43-kDa band (*), both higher-molecular-weight (75 kDa, arrows) and lower-molecular-weight bands (arrowheads) were detected. The 75-kDa species were resolved into 75–76-kDa doublets on some gels. () The abundance of the 75-kDa species (arrow) increased when lysates from cells expressing A315T TDP-43 were treated with okadaic acid (OA) and decreased when lysates were treated with alkaline phosphatase (AP). Cells expressing HA-tagged wild-type (lanes 1 and 2) or A315T mutant (lanes 3 and 4) TDP-43 were treated with the control vehicle or OA, and cell lysates were examined by western blotting using anti-HA antibody (lanes 1–4). Lanes 5 and 6 show western blots using anti–TDP-43 of reaction products treate! d with control or AP, after immunoprecipitation of cell lysates from cells expressing the A315T mutant using anti-HA (a-HA IP). The 43-kDa TDP-43 (*), higher-molecular-weight species (**) and lower-molecular-weight products (arrowhead) are also shown. () The 75-kDa species detected in cells expressing A315T TDP-43 was not affected by treatment with heat (20 °C, lane 2; 100 °C, lane 4), 200 mM DTT (lane 6) or 6 M urea (lane 8) in the presence of protease inhibitors. () Increased amounts of protease K–resistant TDP-43 derivatives were detected in cells expressing the A315T mutant TDP-43. Cell lysates from the wild-type or A315T TDP-43-HA cells were treated with protease K at different concentrations, separated on SDS-PAGE and western blotted using either anti-HA (lanes 18) or anti–TDP-43 (lanes 9 and 10) antibodies. Although the 75-kDa and 43-kDa TDP-43 species are sensitive to protease K treatment, a number of lower-molecular-weight bands, especially TDP-43–reactive ! species (5–10 kDa), are resistant to protease K (lanes 9–1! 0). Representative bands partially resistant to protease K including 2524 kDa, 1514 kDa and 1312 kDa are marked by arrowheads; a cluster of TDP-43 reactive bands ~510 kDa are marked by #. () Western blotting of cell lysates using anti-HA after treatment with protease K at 1 μg ml−1, with lanes 1 and 2, and lanes 3 and 4, containing duplicates of reactions. () Quantification of western blotting signals of protease K–resistant bands in . Shown is the ratio of the corresponding band to the total amounts of signals including the full-length 43-kDa band, demonstrating that partially protease K–resistant bands (2524 kDa or 1110 kDa) were more abundant in cells expressing the A315T TDP-43 (black bars, A) than in those expressing wild-type TDP-43 (white bars, wild type). Error bars, s.e.m. * Figure 6: Sequence features and structural prediction of the C-terminal fragments of TDP-43. Molecular dynamics (MD) simulation of TDP-43 synthetic peptides corresponding to residues 286331 suggests that the A315T mutation increases the tendency of the protein to form β-sheet structures and to stay in extended conformation(s). () The alignment of peptide sequences of the C-terminal domain of TDP-43 (Ser233Met414) with the prion proteins (PRNP) from Homo sapiens (Hs) and Pan troglodytes (Pt) reveals a moderate level of sequence similarity. The identical amino acid residues are in red and underlined; similar residues are in green and underlined. Proteinopathy-associated mutations of TDP-43 are shown as yellow highlighted residues above the corresponding region. (–) The peptide properties as predicted using the Protscale server of the Swiss Institute of Bioinformatics (SIB)24. The synthetic 46-mer peptides of either wild-type (blue) or the A315T mutant (pink) TDP-43 were analyzed for the flexibility scale, as described by Bhaskaran and Ponnuswamy25, and for predicte! d β-sheet and β-turn, using the Deleage-Roux scale26. The peptide profiles were smoothened using an equal-weight sliding window of nine amino acids. () The β-sheet probability of amino acid residues in TDP-43 peptides as further analyzed using a Ramachandran plot27. A residue is defined as having a β-sheet structure when −150 < φ < −60 and 100 < ϕ < 170 on the Ramachandran plot27. The probability of 1 means that the residue is 100% in β-sheet conformation during the course of MD simulation. The blue, pink and green lines represent statistics from simulations of the wild type, the A315T mutant and the A315E mutant, respectively. The 46-mer peptides were analyzed in their entirety; data are shown for amino acid positions 20–45, corresponding to amino acid 306–330 (note that amino acid position 30 corresponds to A315T in TDP-43 protein, as marked by '*315' with an arrow). () The probability distribution of the radius of gyration of the TDP-43 peptides by MD simu! lation. The radius of gyration is defined as the root-mean-squ! are distance of the collection of atoms from their common center of gravity. Radii of gyration in the range of 9–11 Å and 12–20 Å correspond to collapsed and extended conformations, respectively. The radii of gyration were calculated using the 46-mer peptide. The A315T mutant shows higher probability at 14–16 Å of radius of gyration, suggesting its higher tendency to stay extended. * Figure 7: Synthetic TDP-43 peptides (wild-type or A315T mutant, Gln286–Gln331) form fibrils in vitro. () Time course of ThT binding of synthetic TDP-43 peptides. The wild-type (blue circles) or A315T mutant (red triangles) TDP-43 peptides or the control peptide (black crosses) is presented with dynamic changes in ThT binding at 37 °C. () EM images of fibrils formed with the wild-type or A315T TDP-43 peptides. Higher magnification is shown below. Scale bars, 200 nm. () Time-lapse AFM reveals dynamic process of protofibril and fibril formation of TDP-43 peptides. Time-lapse AFM images of wild-type TDP-43 (–) and A315T mutant TDP-43 synthetic peptide (–) during the formation of protofibrils and fibrils after incubation of peptides in aqueous solution at a concentration of 20 mM for 0, 7 h, 13 h and 17 h. All the images were obtained on a mica surface. (,) Corresponding cross-sectional profiles for the wild-type () and A315T mutant () TDP-43 peptides along the white lines, respectively. Z scale bars for the AFM images are as marked on the right. Red crosses in and mark the ! locations for the height measurements, which correspond to the red lines in and . * Figure 8: The A315T mutant synthetic TDP-43 peptide causes enhanced neurotoxicity as compared to the wild-type peptide in primary cortical neuronal cultures. () Fluorescence microscopic images of primary cortical neurons treated with the control peptide, wild-type TPD-43 peptide, phosphorylated A315T TDP-43 peptide or amyloid1–42 peptide (Ctrl, WT, A315T and Aβ42, respectively). Microscopic images corresponding to phase-contrast images (PC), Tuj1 immunostaining, Hoechst dye nuclear staining (Nu), TUNEL staining and the superimpositions of the corresponding groups of images are shown. The large arrowheads mark TUNEL-positive neurons undergoing cell death, whereas the small arrowhead in the A315T group marks an abnormal nucleus that is TUNEL negative. Neurites with normal morphology are marked by arrows. Most neurites in the A315T group were damaged, showing either abnormal varicosities or disruption of normal integrity. Scale bar, 20 μm. (,) Quantification of neuronal death as the percentage of TUNEL-positive neurons in the corresponding groups shown in . The graph shows average values ± s.e.m. of data collected from three in! dependent experiments. Statistical analysis in was performed with one-way ANOVA with Tukey post hoc test, and that in with two-way ANOVA with Bonferroni post hoc test. ***P < 0.001; *P < 0.05. Author information * Abstract * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Weirui Guo, * Yanbo Chen & * Xiaohong Zhou Affiliations * State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Science, Beijing, China. * Weirui Guo, * Yanbo Chen, * Xiaohong Zhou, * Mengxue Yang, * Haihong Ye, * Li Zhu, * Jianghong Liu & * Jane Y Wu * Department of Neurology, Center for Genetic Medicine, Lurie Cancer Center, Northwestern University Feinberg School of Medicine, Chicago, USA. * Weirui Guo, * Xiaohong Zhou, * Amar Kar, * Payal Ray, * Xiaoping Chen, * David Zhang, * Kazuo Fushimi & * Jane Y Wu * National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Science and Peking Union Medical College, Tsinghua University, Beijing, China. * Yanbo Chen, * Yan Shen & * Qi Xu * Trumbull College, Yale University, New Haven, Connecticut, USA. * Elizabeth J Rao * National Center for Nanoscience and Technology, Beijing, China. * Meng Xu, * Yanlian Yang & * Chen Wang * The Cognitive Neurology & Alzheimer's Disease Center, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA. * Eileen H Bigio & * Marsel Mesulam * Present address: Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland, USA. * Amar Kar Contributions J.Y.W., W.G., X.Z., K.F. and E.J.R. designed the study; W.G., Y.C., X.Z., A.K., P.R., X.C., E.J.R., M.Y., L.Z., J.L., M.X., Y.Y., C.W., D.Z., K.F., E.J.R. and J.Y.W. performed the experiments and analyzed the data; H.Y., L.Z., J.L., Y.S., K.F., Q.X. and J.Y.W. supervised the experiments and discussed and analyzed the data; E.H.B. and M.M. provided crucial tissue samples and revised the manuscript; W.G., E.J.R. and J.Y.W. wrote the paper. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Jane Y Wu or * Qi Xu Author Details * Weirui Guo Search for this author in: * NPG journals * PubMed * Google Scholar * Yanbo Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaohong Zhou Search for this author in: * NPG journals * PubMed * Google Scholar * Amar Kar Search for this author in: * NPG journals * PubMed * Google Scholar * Payal Ray Search for this author in: * NPG journals * PubMed * Google Scholar * Xiaoping Chen Search for this author in: * NPG journals * PubMed * Google Scholar * Elizabeth J Rao Search for this author in: * NPG journals * PubMed * Google Scholar * Mengxue Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Haihong Ye Search for this author in: * NPG journals * PubMed * Google Scholar * Li Zhu Search for this author in: * NPG journals * PubMed * Google Scholar * Jianghong Liu Search for this author in: * NPG journals * PubMed * Google Scholar * Meng Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Yanlian Yang Search for this author in: * NPG journals * PubMed * Google Scholar * Chen Wang Search for this author in: * NPG journals * PubMed * Google Scholar * David Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Eileen H Bigio Search for this author in: * NPG journals * PubMed * Google Scholar * Marsel Mesulam Search for this author in: * NPG journals * PubMed * Google Scholar * Yan Shen Search for this author in: * NPG journals * PubMed * Google Scholar * Qi Xu Contact Qi Xu Search for this author in: * NPG journals * PubMed * Google Scholar * Kazuo Fushimi Search for this author in: * NPG journals * PubMed * Google Scholar * Jane Y Wu Contact Jane Y Wu Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Movies * Supplementary Movie 1 (442K) Molecular dynamics simulation suggests that the 46mer TDP-43 peptides adopt multiple conformations including collapsed globular conformation at the N-terminal half and extended β-sheet conformation at the C-terminal region, in agreement with the Protscale analyses (Fig. 7). Molecular dynamics simulations were carried out on Wt and A315T mutant TDP-43 synthetic peptide (Q286-Q331) using TINKER, a software tool for molecular design3. The force field used in this molecular dynamics simulation was CHARMM-19, in combination with a statistical potential of mean force DOPE (discrete optimized protein energy)4. Non-bonded forces (electrostatic and van der Waals) were truncated at 5Å. After the TDP peptide was energy minimized and equilibrated for 100-ps with 1-fs time step, ten (peptide dimer) or thirty (monomer) independent 100-ps trajectories were produced using different random initial conditions. The canonical ensemble simulations were kept at a constant temperature of 300K. T! he amino-termini of both peptides are at the lower right corner, with the carboxyl termini at the upper left corner. The backbones of the wild type and A315T mutant peptides are shown in green and blue ribbons respectively with corresponding Ala315 or Thr315 residues marked in red. PDF files * Supplementary Text and Figures (4M) Supplementary Methods and Supplementary Figures 1–7 Additional data Entities in this article * TAR DNA-binding protein 43 TARDBP Homo sapiens * View in UniProt * View in Entrez Gene * TAR DNA-binding protein 43 Tardbp Mus musculus * View in UniProt * View in Entrez Gene * TAR-binding protein TBPH Drosophila melanogaster * View in UniProt * View in Entrez Gene * Major prion protein PRNP Homo sapiens * View in UniProt * View in Entrez Gene * RNA-binding protein FUS FUS Homo sapiens * View in UniProt * View in Entrez Gene * Microtubule-associated protein tau MAPT Homo sapiens * View in UniProt * View in Entrez Gene * Cystic fibrosis transmembrane conductance regulator CFTR Homo sapiens * View in UniProt * View in Entrez Gene * Glyceraldehyde-3-phosphate dehydrogenase GAPDH Homo sapiens * View in UniProt * View in Entrez Gene
• KAP-1 phosphorylation regulates CHD3 nucleosome remodeling during the DNA double-strand break response
  - Nat Struct Mol Biol 18(7):831-839 (2011)
  Nature Structural & Molecular Biology | Article KAP-1 phosphorylation regulates CHD3 nucleosome remodeling during the DNA double-strand break response * Aaron A Goodarzi1 * Thomas Kurka1 * Penelope A Jeggo1 * Affiliations * Contributions * Corresponding authorsJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:831–839Year published:(2011)DOI:doi:10.1038/nsmb.2077Received08 October 2010Accepted30 March 2011Published online05 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg KAP-1 poses a substantial barrier to DNA double-strand break (DSB) repair within heterochromatin that is alleviated by ATM-dependent KAP-1 phosphorylation (pKAP-1). Here we address the mechanistic consequences of pKAP-1 that promote heterochromatic DSB repair and chromatin relaxation. KAP-1 function involves autoSUMOylation and recruitment of nucleosome deacetylation, methylation and remodeling activities. Although heterochromatin acetylation or methylation changes were not detected, radiation-induced pKAP-1 dispersed the nucleosome remodeler CHD3 from DSBs and triggered concomitant chromatin relaxation; pKAP-1 loss reversed these effects. Depletion or inactivation of CHD3, or ablation of its interaction with KAP-1SUMO1, bypassed pKAP-1's role in repair. Though KAP-1 SUMOylation was unaffected after irradiation, CHD3 dissociated from KAP-1SUMO1 in a pKAP-1–dependent manner. We demonstrate that KAP-1Ser824 phosphorylation generates a motif that directly perturbs interaction! s between CHD3′s SUMO-interacting motif and SUMO1, dispersing CHD3 from heterochromatin DSBs and enabling repair. View full text Figures at a glance * Figure 1: Heterochromatic DSB repair and DSB-induced nucleosome spacing alterations require sustained ATM activity and KAP-1 phosphorylation. () Confluence-arrested G0- and G1-phase NIH3T3 cells were irradiated (2 Gy) and harvested over 2 d. ATMi was added or removed at the indicated times, before immunostaining for γH2AX, H3TrimethylK9 and DAPI. Total IRIF and those overlapping with heterochromatin (= dense DAPI + H3TrimethylK9) were enumerated. Euchromatin fraction = heterochromatin subtracted from total. Repair kinetics = % of initial IRIF within euchromatin or heterochromatin over time. () IBR3 cells were irradiated (8 Gy) and left for 24 h (t = 0 min with ATMi). Cells were then pulsed with ATMi as indicated and immunostained for γH2AX (green) and pKAP-1 (red). pKAP-1 overlapping with γH2AX or throughout the rest of the nucleus was quantified. () HeLa cells were treated with 200 ng ml−1 NCS for 0.5 h before the addition or removal of DMSO or ATMi. Assays were carried out with DNA-PKi to prolong window of chromatin relaxation. Nuclei were processed and 2.5 μg DNA was visualized as described in Online Meth! ods. Lower panels show quantified signal as percent of total (for each lane) across distance from the well to end of gel. Calibrated kilobase pair (kbp) sizes are indicated. () 1BR3 cells were treated as in and immunostained for γH2AX and either pKAP-1, H3Di/TriMeK9, H3AcetylK9 or H4TriMeK20. Signal intensities at IRIF were quantified for 200–300 foci per experiment. All data (–) are mean of ≥3 experiments; error bars, s.d. * Figure 2: Ionizing radiation reduces CHD3 retention at sites of damage after ionizing radiation in an ATM-dependent, rapid and reversible manner. () Confluence-arrested primary human fibroblasts were irradiated (as indicated), detergent extracted 1 h later and immunostained for γH2AX (green), DAPI (blue) and either endogenous CHD3 (red) or pKAP-1 (not shown). Graph shows quantified pKAP-1 and CHD3 signal; 20–40 nuclei were scored per experiment. () HeLa cells transfected with CHD3Flag were treated as indicated, harvested 1 h after irradiation and extracted and lysed as descibed in Online Methods. Equal volumes of each sample were immunoblotted for indicated proteins. Red arrows highlight KAP-1– and chromatin-enriched fractions in which CHD3 shows dynamic behavior with or without ionizing radiation and/or ATMi. () Confluence-arrested primary human fibroblasts were irradiated, pulsed with ATMi (as in Fig. 1b,d) and then extracted and stained as in . CHD3 signal intensity at γH2AX foci (green line, diamonds) or away from γH2AX foci (red line, squares) was quantified for 200–300 foci or 20–30 nuclei per experim! ent. () Representative images from , showing the differential impact of a 1 h DMSO or ATMi pulse (24 h after 8-Gy irradiation) on CHD3 levels overlapping with persisting γH2AX. (,) HeLa cells transfected with scrambled siRNA (siMock) or siRNA directed against KAP-1, CHD3 or CHD4 were treated ± 200 ng ml−1 NCS and harvested, processed and quantified as described in Figure 1c. All data (–) are mean of ≥3 experiments; error bars, s.d. * Figure 3: Depletion or catalytic mutation of CHD3 alleviates the need for ATM activity or KAP-1 phosphorylation in DSB repair. () HeLa cells were transfected with scrambled siRNA (siMock) or siRNA directed against KAP-1, CHD3 or CHD4 and immunostained for indicated proteins. () Cells were transfected as in , treated with DMSO or ATMi, irradiated (3 Gy), harvested 0.5 h or 24 h later and immunostained for γH2AX and either KAP-1, CHD3 or CHD4. Average γH2AX foci numbers per nuclei were quantified in cells with confirmed knockdown. () HeLa cells were transfected with siRNA as in , transfected with indicated siRNA-resistant constructs and treated, stained and quantified as in . () CHD3 schematic. () HeLa cells transfected with indicated Flag-tagged CHD3 (wild type (WT), K767Q, D883N, P1966X or A1642X) or wild-type HA-tagged KAP-1 were made into extracts and incubated with anti-HA agarose beads. Washed immunoprecipitates were immunoblotted (alongside 50 μg input) for anti-Flag, anti-HA or anti-Ku70. () HeLa cells, transfected with CHD3 siRNA as in , were transfected with the indicated constructs and t! reated (or not) with ATMi, irradiated, immunostained and quantified as in . () HeLa cells depleted for the indicated targets (by siRNA) were stained with H3Di/TriMeK9 or H3AcetylK9. Total nuclear levels of each histone modification were measured in cells (scoring 50–100 per experiment) with confirmed knockdown. All data (–) are mean of ≥3 experiments; error bars, s.d. * Figure 4: Ablating KAP-1 SUMOylation alleviates the need for pKAP-1 in DSB repair, and CHD3 interactions with KAP-1SUMO, but not overall KAP-1 SUMOylation, are altered after ionizing radiation. (,) KAP-1–depleted HeLa cells transfected with indicated siRNA-resistant, HA-tagged KAP-1 constructs were treated (or not) with ATMi, irradiated, harvested as indicated and immunostained for γH2AX and KAP-1 or hemagglutinin (as appropriate). Average γH2AX foci numbers per nuclei were quantified in cells with confirmed KAP-1 knockdown or hemagglutinin expression. () HeLa cells expressing indicated KAP-1HA and SUMO1His constructs were irradiated and treated with ATMi where indicated. Extracts were prepared and enriched for SUMOHis-tagged proteins with TALON beads before being immunoblotted (alongside 50-μg input) for indicated proteins. () Upper panel, HeLa cells expressing KAP-1HA and SUMO1His were irradiated and harvested as indicated. Lower panel, quantified, normalized SUMOylated KAP-1 signal (n = 3) in cells expressing indicated constructs. (,) HeLa cells expressing Flag-tagged CHD3 (wild-type) or KAP-1HA constructs were irradiated and/or treated with ATMi, made into! extracts and incubated with anti-HA beads. Washed immunoprecipitates (with 50-μg input) were immunoblotted for indicated proteins. () HeLa cells were transfected with CHD3Flag, KAP-1HA and SUMO1His constructs as indicated. 24 h later, cells were irradiated and/or treated with ATMi, made into extracts and incubated with anti-Flag beads. Washed immunoprecipitates (with 50-μg input) were immunoblotted for indicated proteins. All data (–) are mean of ≥3 experiments; error bars, s.d. * Figure 5: pKAP-1 peptides disrupt interactions between SUMO1 and the SIM domain of CHD3 in vitro. () Coomassie-stained PAGE gels of >99% purity SUMO1. () Anti-SUMO1 immunoblots of purified SUMO1. () Increasing SUMO1 amounts were added to biotin-CHDSIM bound to Streptavidin Fe-particles. Bound SUMO1 was eluted and immunoblotted as in . () Experiment described in was conducted with the extra step of adding purified KAP-1 phospho (pSer824) or dephospho (Ser824) peptides (upper panel) or Artemis phospho (pS645) or dephospho (S645) peptide (lower panel) after SUMO1 was added to Biotin-CHDSIM particles. () Quantified data shown in , representing mean and s.d. of three to six independent experiments. () Structural model of human KAP-1's PHD + bromodomain (residues 624–812, gray) with SUMO1 (pale blue) with a bound SIM (green). Models were generated at http://www.rcsb.org/ (structures 2RO1 and 2ASQ ). SUMO1 + SIM structures are positioned (arbitrarily) relative to two known SUMO-conjugation sites (Lys779, Lys804) in KAP-1. KAP-1 residues 813–835 are predicted to be unstructu! red. Given the known SUMOylation anchor points and the established SUMO1 dimensions, KAP-1's tail is in sufficient proximity to reach SUMO1's SIM-binding groove, suggesting that it might displace SIM domains from SUMO1. () In resting cells, nucleosome remodeling activity (promoting compaction) is recruited to heterochromatin by means of interactions between KAP-1SUMO1 and CHD3's SIM-domain. Following DSB-induced ATM activation, KAP-1 is phosphorylated at Ser824, which confers increased negativity, enabling it to interfere with SUMO1–CHD3 interactions and causing release of CHD3 from KAP-1–enriched heterochromatin. Author information * Abstract * Author information * Supplementary information Affiliations * Genome Damage and Stability Centre, University of Sussex, East Sussex, UK. * Aaron A Goodarzi, * Thomas Kurka & * Penelope A Jeggo Contributions A.A.G. and T. K. conducted the experiments. A.A.G. and P.A.J. coauthored the manuscript and conceived of and designed the study. Competing financial interests The authors declare no competing financial interests. Corresponding authors Correspondence to: * Aaron A Goodarzi or * Penelope A Jeggo Author Details * Aaron A Goodarzi Contact Aaron A Goodarzi Search for this author in: * NPG journals * PubMed * Google Scholar * Thomas Kurka Search for this author in: * NPG journals * PubMed * Google Scholar * Penelope A Jeggo Contact Penelope A Jeggo Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information PDF files * Supplementary Text and Figures (2M) Supplementary Figures 1–5 and Supplementary Discussion Additional data Entities in this article * Histone-lysine N-methyltransferase SETDB1 SETDB1 Homo sapiens * View in UniProt * View in Entrez Gene * Histone deacetylase 2 HDAC2 Homo sapiens * View in UniProt * View in Entrez Gene * Double-strand break repair protein MRE11A MRE11A Homo sapiens * View in UniProt * View in Entrez Gene * Chromodomain-helicase-DNA-binding protein 4 CHD4 Homo sapiens * View in UniProt * View in Entrez Gene * Serine-protein kinase ATM ATM Homo sapiens * View in UniProt * View in Entrez Gene * Chromodomain-helicase-DNA-binding protein 3 CHD3 Homo sapiens * View in UniProt * View in Entrez Gene * Histone deacetylase 1 HDAC1 Homo sapiens * View in UniProt * View in Entrez Gene * Small ubiquitin-related modifier 1 SUMO1 Homo sapiens * View in UniProt * View in Entrez Gene * E3 SUMO-protein ligase PIAS1 PIAS1 Homo sapiens * View in UniProt * View in Entrez Gene * Mediator of DNA damage checkpoint protein 1 MDC1 Homo sapiens * View in UniProt * View in Entrez Gene * Protein artemis DCLRE1C Homo sapiens * View in UniProt * View in Entrez Gene * Nibrin NBN Homo sapiens * View in UniProt * View in Entrez Gene * DNA repair protein RAD50 RAD50 Homo sapiens * View in UniProt * View in Entrez Gene * X-ray repair cross-complementing protein 6 XRCC6 Homo sapiens * View in UniProt * View in Entrez Gene * Transcription intermediary factor 1-beta TRIM28 Homo sapiens * View in UniProt * View in Entrez Gene * Metastasis-associated protein MTA2 MTA2 Homo sapiens * View in UniProt * View in Entrez Gene * E3 ubiquitin-protein ligase RNF168 RNF168 Homo sapiens * View in UniProt * View in Entrez Gene * DNA-dependent protein kinase catalytic subunit PRKDC Homo sapiens * View in UniProt * View in Entrez Gene * Tumor suppressor p53-binding protein 1 TP53BP1 Homo sapiens * View in UniProt * View in Entrez Gene * SUMO-conjugating enzyme UBC9 UBE2I Homo sapiens * View in UniProt * View in Entrez Gene * SUMO-activating enzyme subunit 2 UBA2 Homo sapiens * View in UniProt * View in Entrez Gene * SUMO-activating enzyme subunit 1 SAE1 Homo sapiens * View in UniProt * View in Entrez Gene
• Misregulation of miR-1 processing is associated with heart defects in myotonic dystrophy
  - Nat Struct Mol Biol 18(7):840-845 (2011)
  Nature Structural & Molecular Biology | Article Misregulation of miR-1 processing is associated with heart defects in myotonic dystrophy * Frédérique Rau1, 2, 3, 4 * Fernande Freyermuth1, 2, 3, 4 * Charlotte Fugier1, 2, 3, 4 * Jean-Philippe Villemin5 * Marie-Christine Fischer1, 2, 3, 4 * Bernard Jost1, 2, 3, 4 * Doulaye Dembele1, 2, 3, 4 * Geneviève Gourdon6, 7, 8 * Annie Nicole6, 7, 8 * Denis Duboc8, 9 * Karim Wahbi10, 11, 12, 13 * John W Day14 * Harutoshi Fujimura15 * Masanori P Takahashi16 * Didier Auboeuf10 * Natacha Dreumont1, 2, 3, 4 * Denis Furling10, 11, 12, 13 * Nicolas Charlet-Berguerand1, 2, 3, 4 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:840–845Year published:(2011)DOI:doi:10.1038/nsmb.2067Received14 September 2010Accepted07 April 2011Published online19 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Myotonic dystrophy is an RNA gain-of-function disease caused by expanded CUG or CCUG repeats, which sequester the RNA binding protein MBNL1. Here we describe a newly discovered function for MBNL1 as a regulator of pre-miR-1 biogenesis and find that miR-1 processing is altered in heart samples from people with myotonic dystrophy. MBNL1 binds to a UGC motif located within the loop of pre-miR-1 and competes for the binding of LIN28, which promotes pre-miR-1 uridylation by ZCCHC11 (TUT4) and blocks Dicer processing. As a consequence of miR-1 loss, expression of GJA1 (connexin 43) and CACNA1C (Cav1.2), which are targets of miR-1, is increased in both DM1- and DM2-affected hearts. CACNA1C and GJA1 encode the main calcium- and gap-junction channels in heart, respectively, and we propose that their misregulation may contribute to the cardiac dysfunctions observed in affected persons. View full text Figures at a glance * Figure 1: The processing of miR-1 is altered in myotonic dystrophies. () Quantitative real-time PCR (qRT-PCR) analysis of the expression of mature miR-1, miR-126, miR-138, miR-199 and miR-499 relative to the U6 small nuclear (sn) RNA in heart samples of adults with DM1 or DM2 and in control (CTL) heart samples from 2 unaffected persons, 1 person with dilated cardiomyopathy and 5 people with ALS. ***P < 0.001. () qRT-PCR analysis of the expression of pre-miR-1 and mature miR-1 relative to the U6 snRNA in heart samples of control (2 unaffected, 1 with dilated cardiomyopathy and 5 with ALS), DM1-affected and DM2-affected subjects. *P < 0.05, ***P < 0.001. () qRT-PCR analysis of the expression of pre-miR-1 and mature miR-1 in H9C2 rat cardiomyocytes differentiated for 6 days and infected with recombinant adenovirus (MOI 100) expressing GFP (CTL) or 960 CUG repeats (CUG). The mean of at least three independent infections is depicted as the percentage of mature or pre-miR-1 relative to the U6 snRNA. Error bars indicate s.d. ***P < 0.001. () 5 μg of! total RNA extracted from HeLa cells co-transfected with ectopic pri-miR-1-1 or pri-miR-1-2 minigenes and a plasmid expressing either no (CTL) or 960 CUG repeats (CUG) were analyzed by northern blot analysis using an antisense miR-1 [γ-32P]–labeled probe. The mean of at least three independent transfections is depicted as the percentage of mature miR-1 relative to pre-miR-1. Ethidium bromide staining demonstrates equal loading. Error bars indicate s.d. * Figure 2: MBNL1 regulates the processing of miR-1. () Northern blot analysis of RNA extracted from HeLa cells co-transfected with ectopic pri-miR-1-1 minigene and a plasmid expressing either no CTG (CTL), 960 CUG repeats (CUG), MBNL1, CUGBP1 or shRNA directed against MBNL1 (shMBNL1 #1) or against CUGBP1 (shCUGBP1). The mean of at least three independent transfections is depicted as the percentage of mature miR-1 relative to pre-miR-1. Error bars indicate s.d., and ethidium bromide staining demonstrates equal loading. () Top, qRT-PCR analysis of the expression of endogenous pre-miR-1 and mature miR-1 in H9C2 rat cardiomyocytes infected with recombinant adenovirus expressing an shRNA against LacZ (shCTL) or against Mbnl1 (shMBNL1). The mean of at least three independent infections is depicted as the percentage of mature or pre-miR-1 relative to the U6 snRNA. Error bars indicate s.d. **P < 0.01. Bottom, Mbnl1 depletion was confirmed by western blotting. () Sequence of the wild-type human pre-miR-1-1. Mature miR-1 is indicated i! n italic. The UGC motif mutated in UAC is indicated in upper case. () UV-cross-linking analysis of pre-miR-1-1, pre-miR-1-2 and mutated pre-miR-1-1 (UGC MUT) using purified bacterial recombinant GST-MBNL1ΔCter and uniformly [α-32P]CTP-labeled RNAs. () UV-cross-linking binding of GST-MBNL1ΔCter to uniformly [α-32P]CTP-labeled pre-miR-1-1 RNA is competed by increasing amounts of unlabeled RNA composed of 10 CUG repeats. The mean of at least three independent experiments is depicted as the binding of MBNL1 to pre-miR-1. Error bars indicate s.d. * Figure 3: LIN28 regulates the processing of miR-1. () Northern blot analysis of RNA extracted from HeLa cells co-transfected with ectopic pri-miR-1-1 or pri-miR-1-2 and a plasmid expressing either GFP (CTL) or Flag-LIN28. The mean of at least three independent transfections is depicted as the percentage of mature miR-1 relative to pre-miR-1. Error bars indicate s.d. () Top, qRT-PCR analysis of the expression of endogenous pre-miR-1 and mature miR-1 in undifferentiated H9C2 rat cardiomyoblastes infected with recombinant lentivirus expressing an shRNA against Gapdh (shCTL) or against Lin28B (shLIN28B). The mean of at least three independent infections is depicted as the percentage of mature or pre-miR-1 relative to the U6 snRNA. Error bars indicate s.d. **P < 0.01. Bottom, Lin28B depletion was confirmed by western blotting. () Immunoprecipitated Flag-TUT4 uridylates pre-miR-1-1 in presence of [α-32P]UTP and 100 nM of His-LIN28. Pre-miR-16 and pre-Let-7-A1 are negative and positive controls, respectively. () Immunoprecipitated! Myc-Dicer processes [γ-32P]–labeled pre-miR-1 into mature miR-1 but does not cleave pre-miR-1 that was previously uridylated by TUT4 and LIN28. The asterisk (*) indicates a nonspecific degradation product. () Top, sequence of human pre-miR-1-1. Mature miR-1 is indicated in italics. Mutated UGC and AAG motifs are indicated in upper case (MUT). Bottom, UV-cross-linking analysis of pre-miR-1-1, pre-miR-1-2 and mutated pre-miR-1-1 (MUT) was conducted using purified His-LIN28 and uniformly [α-32P]CTP-labeled RNAs. () UV-cross-linking binding of GST-MBNL1ΔCter to uniformly [α-32P]CTP-labeled pre-miR-1-1 RNA is competed by the indicated increasing amounts of His-LIN28. * Figure 4: Targets of miR-1 are upregulated in myotonic dystrophies. () Sequence alignments between miR-1 and the 3′ UTR of human CACNA1C. () Luciferase activity of HeLa cells co-transfected with either no (CTL) or 40 nM of miR-1 mimic and a plasmid expressing no, CACNA1C or GJA1 miR-1 binding sites (3×) cloned within the Renilla luciferase 3′ UTR. The mean of at least three independent transfections is depicted as the luciferase activity. Error bars indicate s.d. **P < 0.01, ***P < 0.001. () Western blot analysis of the expression of endogenous Cacna1c, Gja1 and calnexin in H9C2 rat cardiomyoblasts transfected with 50 nM of miR-1 mimic or 50 nM of anti-miR-1. () qRT-PCR analysis of the expression of CACNA1C and GJA1 relative to RPLP0 mRNA in heart samples of control (2 unaffected, 1 with dilated cardiomyopathy and 5 with ALS), DM1- and DM2-affected subjects. Error bars indicate s.d. () Western blot analysis of the expression of CACNA1C, GJA1 and calnexin in membrane extracts from heart samples of control (2 unaffected, 1 with dilated ca! rdiomyopathy and 5 with ALS), DM1- and DM2-affected subjects. * Figure 5: Model of miR-1 alteration in myotonic dystrophies. () MBNL1 and LIN28 compete for binding to pre-miR-1 loop. In presence of MBNL1, the processing of pre-miR-1 in mature miR-1 is favored and results in regulated expression of miR-1 targets. () In people with myotonic dystrophy, the sequestration of MBNL1 by expanded CUG or CCUG repeats allows LIN28 or LIN28B to bind to pre-miR-1, which promotes its consequent uridylation by TUT4. Uridylated pre-miR-1 is resistant to Dicer cleavage, which results in lower amounts of miR-1 and increased levels of its targets, GJA1 and CACNA1C. Author information * Abstract * Author information * Supplementary information Affiliations * Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France. * Frédérique Rau, * Fernande Freyermuth, * Charlotte Fugier, * Marie-Christine Fischer, * Bernard Jost, * Doulaye Dembele, * Natacha Dreumont & * Nicolas Charlet-Berguerand * Institut National de la Santé et de la Recherche Médicale (INSERM) U964, Illkirch, France. * Frédérique Rau, * Fernande Freyermuth, * Charlotte Fugier, * Marie-Christine Fischer, * Bernard Jost, * Doulaye Dembele, * Natacha Dreumont & * Nicolas Charlet-Berguerand * Centre National de la Recherche Scientifique (CNRS) UMR7104, Illkirch, France. * Frédérique Rau, * Fernande Freyermuth, * Charlotte Fugier, * Marie-Christine Fischer, * Bernard Jost, * Doulaye Dembele, * Natacha Dreumont & * Nicolas Charlet-Berguerand * Strasbourg University, Illkirch, France. * Frédérique Rau, * Fernande Freyermuth, * Charlotte Fugier, * Marie-Christine Fischer, * Bernard Jost, * Doulaye Dembele, * Natacha Dreumont & * Nicolas Charlet-Berguerand * Institut National de la Santé et de la Recherche Médicale U590, Lyon, France. * Jean-Philippe Villemin * Necker Hospital, Paris, France. * Geneviève Gourdon & * Annie Nicole * Institut National de la Santé et de la Recherche Médicale U781, Paris, France. * Geneviève Gourdon & * Annie Nicole * Université Paris 5, Paris, France. * Geneviève Gourdon, * Annie Nicole & * Denis Duboc * Cochin Hospital, Paris, France. * Denis Duboc * Université Pierre et Marie Curie (UM76), Paris, France. * Karim Wahbi, * Didier Auboeuf & * Denis Furling * Institut de Myologie, Paris, France. * Karim Wahbi & * Denis Furling * Institut National de la Santé et de la Recherche Médicale U974, Paris, France. * Karim Wahbi & * Denis Furling * Centre National de la Recherche Scientifique UMR7215, Paris, France. * Karim Wahbi & * Denis Furling * University of Minnesota, Minneapolis, Minnesota, USA. * John W Day * Department of Neurology, Toneyama Hospital, Osaka, Japan. * Harutoshi Fujimura * Department of Neurology, Osaka University Graduate School of Medicine, Osaka, Japan. * Masanori P Takahashi Contributions Experiments were conducted by F.R., F.F., C.F., J.-P.V., D.D., N.D., M.-C.F., A.N., D.A. and B.J. Clinical samples and patient data were obtained from J.W.D., D.D., K.W., D.F., G.G., H.F., D.D., M.P.T. and from the Research Resource Network supported by the Research Grant for Nervous and Mental Disorders from the Ministry of Health, Labour and Welfare, Japan. The study was designed and coordinated by N.D., D.F. and N.C.-B. The paper was written by N.C.-B. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Nicolas Charlet-Berguerand Author Details * Frédérique Rau Search for this author in: * NPG journals * PubMed * Google Scholar * Fernande Freyermuth Search for this author in: * NPG journals * PubMed * Google Scholar * Charlotte Fugier Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-Philippe Villemin Search for this author in: * NPG journals * PubMed * Google Scholar * Marie-Christine Fischer Search for this author in: * NPG journals * PubMed * Google Scholar * Bernard Jost Search for this author in: * NPG journals * PubMed * Google Scholar * Doulaye Dembele Search for this author in: * NPG journals * PubMed * Google Scholar * Geneviève Gourdon Search for this author in: * NPG journals * PubMed * Google Scholar * Annie Nicole Search for this author in: * NPG journals * PubMed * Google Scholar * Denis Duboc Search for this author in: * NPG journals * PubMed * Google Scholar * Karim Wahbi Search for this author in: * NPG journals * PubMed * Google Scholar * John W Day Search for this author in: * NPG journals * PubMed * Google Scholar * Harutoshi Fujimura Search for this author in: * NPG journals * PubMed * Google Scholar * Masanori P Takahashi Search for this author in: * NPG journals * PubMed * Google Scholar * Didier Auboeuf Search for this author in: * NPG journals * PubMed * Google Scholar * Natacha Dreumont Search for this author in: * NPG journals * PubMed * Google Scholar * Denis Furling Search for this author in: * NPG journals * PubMed * Google Scholar * Nicolas Charlet-Berguerand Contact Nicolas Charlet-Berguerand Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Author information * Supplementary information Excel files * Supplementary Data (4M) () Analysis using the Agilent GeneSpring GX Software of miRNA microarray performed on RNA extracted from three independent cultures of primary muscle cultures (differentiated 10 days) derived from controls and age-matched DM1 patients. Among the various miRNA with a mis-regulation of fold change >2 only, miR-1, miR-126, miR-138, miR-146, miR-199 and miR-499 had a corrected p value < 0.05. () Raw data and crude analysis using excel. Data are accessible through NCBI GEO GSE24109. PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–3, Supplementary Table 1 and Supplementary Methods Additional data Entities in this article * Insulin-like growth factor I IGF1 Homo sapiens * View in UniProt * View in Entrez Gene * Muscleblind-like protein BLES04 Mbnl1 Rattus norvegicus * View in UniProt * View in Entrez Gene * Exportin-5 XPO5 Homo sapiens * View in UniProt * View in Entrez Gene * MicroRNA let-7f-2 Mirlet7f-2 Rattus norvegicus * View in Entrez Gene * MicroRNA mir-126 Mir126 Rattus norvegicus * View in Entrez Gene * Lin-28 homolog B Lin28b Rattus norvegicus * View in UniProt * View in Entrez Gene * Protein lin-28 homolog B LIN28B Homo sapiens * View in UniProt * View in Entrez Gene * Voltage-dependent L-type calcium channel subunit alpha-1C Cacna1c Rattus norvegicus * View in UniProt * View in Entrez Gene * MicroRNA let-7a-1 MIRLET7A1 Homo sapiens * View in Entrez Gene * Myocyte-specific enhancer factor 2A MEF2A Homo sapiens * View in UniProt * View in Entrez Gene * Calmodulin Homo sapiens * View in UniProt * Myotonin-protein kinase DMPK Homo sapiens * View in UniProt * View in Entrez Gene * Voltage-dependent L-type calcium channel subunit alpha-1C CACNA1C Homo sapiens * View in UniProt * View in Entrez Gene * CUGBP Elav-like family member 1 CELF1 Homo sapiens * View in UniProt * View in Entrez Gene * Cellular nucleic acid-binding protein CNBP Homo sapiens * View in UniProt * View in Entrez Gene * Terminal uridylyltransferase 4 ZCCHC11 Homo sapiens * View in UniProt * View in Entrez Gene * Protein lin-28 homolog A LIN28A Homo sapiens * View in UniProt * View in Entrez Gene * Gap junction alpha-1 protein GJA1 Homo sapiens * View in UniProt * View in Entrez Gene * Endoribonuclease Dicer DICER1 Homo sapiens * View in UniProt * View in Entrez Gene * MicroRNA 1-1 MIR1-1 Homo sapiens * View in Entrez Gene * Insulin receptor INSR Homo sapiens * View in UniProt * View in Entrez Gene * Chloride channel protein 1 CLCN1 Homo sapiens * View in UniProt * View in Entrez Gene * Muscleblind-like protein 1 MBNL1 Homo sapiens * View in UniProt * View in Entrez Gene * MicroRNA 499 MIR499 Homo sapiens * View in Entrez Gene * MicroRNA 126 MIR126 Homo sapiens * View in Entrez Gene * MicroRNA 1a-2 Mir1a-2 Mus musculus * View in Entrez Gene * MicroRNA 1-2 MIR1-2 Homo sapiens * View in Entrez Gene * Gap junction alpha-1 protein Gja1 Rattus norvegicus * View in UniProt * View in Entrez Gene * Calnexin Canx Rattus norvegicus * View in UniProt * View in Entrez Gene * Microprocessor complex subunit DGCR8 DGCR8 Homo sapiens * View in UniProt * View in Entrez Gene * 60S acidic ribosomal protein P0 RPLP0 Homo sapiens * View in UniProt * View in Entrez Gene * Ribonuclease 3 DROSHA Homo sapiens * View in UniProt * View in Entrez Gene * Calnexin CANX Homo sapiens * View in UniProt * View in Entrez Gene * MicroRNA let-7f-2 MIRLET7F2 Homo sapiens * View in Entrez Gene * Triadin TRDN Homo sapiens * View in UniProt * View in Entrez Gene * CUGBP Elav-like family member 2 CELF2 Homo sapiens * View in UniProt * View in Entrez Gene * Twinfilin-1 TWF1 Homo sapiens * View in UniProt * View in Entrez Gene * Potassium voltage-gated channel subfamily A member 5 KCNA5 Homo sapiens * View in UniProt * View in Entrez Gene
• Integrating energy calculations with functional assays to decipher the specificity of G protein–RGS protein interactions
  - Nat Struct Mol Biol 18(7):846-853 (2011)
  Nature Structural & Molecular Biology | Article Integrating energy calculations with functional assays to decipher the specificity of G protein–RGS protein interactions * Mickey Kosloff1 * Amanda M Travis1 * Dustin E Bosch2 * David P Siderovski2 * Vadim Y Arshavsky1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:846–853Year published:(2011)DOI:doi:10.1038/nsmb.2068Received05 November 2010Accepted07 April 2011Published online19 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Abstract * Abstract * Accession codes * Author information * Supplementary information Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg The diverse Regulator of G protein Signaling (RGS) family sets the timing of G protein signaling. To understand how the structure of RGS proteins determines their common ability to inactivate G proteins and their selective G protein recognition, we combined structure-based energy calculations with biochemical measurements of RGS activity. We found a previously unidentified group of variable 'Modulatory' residues that reside at the periphery of the RGS domain–G protein interface and fine-tune G protein recognition. Mutations of Modulatory residues in high-activity RGS proteins impaired RGS function, whereas redesign of low-activity RGS proteins in critical Modulatory positions yielded complete gain of function. Therefore, RGS proteins combine a conserved core interface with peripheral Modulatory residues to selectively optimize G protein recognition and inactivation. Finally, we show that our approach can be extended to analyze interaction specificity across other large pro! tein families. View full text Figures at a glance * Figure 1: The GAP activities of ten representative RGS domains are not correlated with their subfamily classification. () The kgap constant for each domain was calculated as described in Supplementary Methods from single-exponential fits to the time course of GTP hydrolyzed by Gαo (400 nM) with or without added RGS protein (20 nM). Data are mean ± s.e.m.; n ≥ 4. High-activity, low-activity and no-activity RGS proteins are green, magenta and red (RGS2 only), respectively. () Phylogenetic tree of 19 human RGS domains. RGS proteins whose activity was tested in this study are colored as in . * Figure 2: Positions of Significant & Conserved and Modulatory residues in multiple RGS proteins. () Residue-level sequence map summarizing structure analysis and energy calculations of eight RGS–Gα crystal structures with PDB codes 1AGR (RGS4); 2IK8 (RGS16h, human RGS16); 3C7K (RGS16m, mouse RGS16); 2IHB (RGS10); 2GTP (RGS1); 2ODE (RGS8); 1FQJ (RGS9); 2V4Z (RGS2*, gain-of-function RGS2 triple mutant; see Online Methods). The sequences in the multiple sequence alignment are taken from the crystal structures. RGS protein residues that contribute substantially to the interaction with the Gα subunit are color-coded in the panel according to the type of their energetic contribution (see key). Putative Significant & Conserved and Modulatory positions are marked above the alignment by red asterisks and purple triangles, respectively. () 3D visualization of the different types of energetic contributions by individual RGS protein residues (spheres, colored as in ). The eight superimposed RGS domain structures are viewed through the semitransparent surface of Gα. () Signific! ant & Conserved and Modulatory residues in the eight superimposed RGS domain structures are red and purple spheres, respectively. Orientation is the same as in . () 3D visualization as in , rotated 90° about the y axis. * Figure 3: Mutations in Modulatory positions impair the GAP activities of RGS4 and RGS16 in an additive manner. () Sequences of RGS4 mutations in Modulatory positions (RGS4a–RGS4d) and a Significant & Conserved position (RGS4e). () Sequences of RGS16 mutations in Modulatory positions (RGS16a–RGS16d) and a Significant & Conserved position (RGS16e). The annotated sequences of wild-type RGS4 and RGS16 in and are from Figure 2a. () GAP activities of RGS4 mutants determined by single-turnover GTPase assays. GTP-loaded Gαo (400 nM) was incubated with or without RGS4 (40 nM) for 1 min. GAP activities are expressed as a percentage of wild-type RGS4 activity. Values are mean ± s.e.m. (n ≥ 4). () GAP activities of the RGS16 mutants, determined as in . Experiments were conducted in triplicate. * Figure 4: Redesign of RGS17 gain-of-function mutants. () Sequences of RGS16 (annotated as in Fig. 2a), RGS17 and its mutants. RGS17 residues in Significant & Conserved or Modulatory positions that are different from RSG16 are marked as follows: those predicted to interfere with high GAP activity are orange, and those appearing in other high-activity RGS proteins are bold black. Residues in RGS17 mutants that were replaced by RGS16 residues are blue. () Positions of the four RGS17 sites mutated in the redesign experiments. The RGS16 residues used as the template for the redesign are visualized on the superimposed structures of Gαi1–RGS16 (PDB 2IK8) and Gαo–RGS16 (PDB 3CK7), viewed through the semitransparent surface of the Gα subunit. Corresponding RGS17 residue numbers are in parentheses. () GAP activities of the redesigned RGS17 mutants compared to activities of wild-type proteins. kgap values were determined as in Figure 1 and are mean ± s.e.m. (n ≥ 4). * Figure 5: Redesign of RGS18 gain-of-function mutants. () Sequences of RGS16, RGS18 and its mutants, color-coded as in Figure 4a. () Positions of the three RGS18 sites mutated in the redesign experiments, visualized on the superimposed Gα–RGS16 structures as in Figure 4b. Corresponding RGS18 residue numbers are in parentheses. () GAP activities of the redesigned RGS18 mutants compared to activities of wild-type proteins. kgap values were determined as in Figure 4c and are mean ± s.e.m. (n ≥ 4). * Figure 6: Residues contributing substantially to colicin E7–immunity protein interactions. Energy calculations were carried out on the following structures: wild-type E7–Im7 complexes (wt1–wt5, PDB 7CEI, 2JAZ, 2JB0, 2JBG and 1ZNV); computationally redesigned E7–Im7 (cr1 and cr2, PDB 1UJZ and 2ERH); and E7 bound to Im9 proteins evolved in vitro to bind E7 with high affinity (ie1 and ie2, PDB 3GJN and 3GKL). () Residue-level sequence map of wild-type and engineered immunity proteins. Sequences in the multiple sequence alignment are taken from the crystal structures. Residues that contribute substantially to the interaction are color-coded according to type of energy contribution (see key). Consensus analysis was applied to the five wild-type proteins and Significant & Conserved and Modulatory positions were determined for all nine structures as in Figure 2. () The nine E7–Im structures, superimposed via the Im proteins. () Visualization of the energy-contribution types for wild-type E7–Im7 residues (spheres, colored as in ). The E7 and Im7 structures are i! n an 'open book' view (rotated 90° relative to about the x axis in opposite directions). () Energy contributions of residues in the computationally redesigned E7–Im7, shown as in . () Energy contributions of residues in E7 and the in vitro–evolved Im9 proteins, shown as in . * Figure 7: Positions of Significant & Conserved and Modulatory residues in the Gα subunits interacting with RGS domains. () Significant & Conserved RGS residues (red) interact with all three Gα switch regions (SW I–SW III). () Modulatory RGS residues (purple) interact with switch II and III and the helical domain of the Gα subunits. In both and , Significant & Conserved positions within the Gα subunits are orange and Modulatory positions are blue. () The different types of energetic contributions by individual Gα residues (spheres, colored as in Fig. 2). The eight superimposed Gα subunits are viewed through the semitransparent surface of the RGS domain and are rotated 90° about the y axis and 30° about the x axis relative to . () Significant & Conserved and Modulatory residues in the Gα structures (red and purple spheres, respectively). () Same as in , rotated 90° about the y axis. Accession codes * Abstract * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 1AGR * 2IK8 * 3C7K * 2IHB * 2GTP * 2ODE * 1FQJ * 2V4Z * 7CEI * 2JAZ * 2JB0 * 2JBG * 1ZNV * 1UJZ * 2ERH * 3GJN * 3GKL * 3CK7 * 1AGR * 2IK8 * 3C7K * 2IHB * 2GTP * 2ODE * 1FQJ * 2V4Z * 7CEI * 2JAZ * 2JB0 * 2JBG * 1ZNV * 1UJZ * 2ERH * 3GJN * 3GKL * 3CK7 Author information * Abstract * Accession codes * Author information * Supplementary information Affiliations * Duke Eye Center, Duke University Medical Center, Durham, North Carolina, USA. * Mickey Kosloff, * Amanda M Travis & * Vadim Y Arshavsky * Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA. * Dustin E Bosch & * David P Siderovski Contributions M.K. designed and carried out computational analysis and biochemical experiments, analyzed data and prepared the manuscript, A.M.T. carried out experiments and prepared the manuscript, D.E.B. carried out experiments and prepared the manuscript, D.P.S. supervised the project and prepared the manuscript and V.Y.A. supervised the project and analysis and prepared the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Vadim Y Arshavsky Author Details * Mickey Kosloff Search for this author in: * NPG journals * PubMed * Google Scholar * Amanda M Travis Search for this author in: * NPG journals * PubMed * Google Scholar * Dustin E Bosch Search for this author in: * NPG journals * PubMed * Google Scholar * David P Siderovski Search for this author in: * NPG journals * PubMed * Google Scholar * Vadim Y Arshavsky Contact Vadim Y Arshavsky Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Abstract * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (639K) Supplementary Figures 1–4, Supplementary Tables 1 and 2, and Supplementary Methods Additional data Entities in this article * Colicin-E7 Escherichia coli * View in UniProt * Guanine nucleotide-binding protein G(o) subunit alpha GNAO1 Homo sapiens * View in UniProt * View in Entrez Gene * Regulator of G-protein signaling 7 RGS7 Homo sapiens * View in UniProt * View in Entrez Gene * Regulator of G-protein signaling 1 RGS1 Homo sapiens * View in UniProt * View in Entrez Gene * Regulator of G-protein signaling 2 RGS2 Homo sapiens * View in UniProt * View in Entrez Gene * Retinal rod rhodopsin-sensitive cGMP 3',5'-cyclic phosphodiesterase subunit gamma PDE6G Homo sapiens * View in UniProt * View in Entrez Gene * Guanine nucleotide-binding protein G(i) subunit alpha-1 GNAI1 Homo sapiens * View in UniProt * View in Entrez Gene * Regulator of G-protein signaling 4 RGS4 Homo sapiens * View in UniProt * View in Entrez Gene * Regulator of G-protein signaling 16 RGS16 Homo sapiens * View in UniProt * View in Entrez Gene * Regulator of G-protein signaling 10 RGS10 Homo sapiens * View in UniProt * View in Entrez Gene * Regulator of G-protein signaling 8 RGS8 Homo sapiens * View in UniProt * View in Entrez Gene * Regulator of G-protein signaling 9 RGS9 Homo sapiens * View in UniProt * View in Entrez Gene * Colicin-E7 immunity protein Escherichia coli * View in UniProt * Regulator of G-protein signaling 18 RGS18 Homo sapiens * View in UniProt * View in Entrez Gene * Regulator of G-protein signaling 17 RGS17 Homo sapiens * View in UniProt * View in Entrez Gene * Regulator of G-protein signaling 14 RGS14 Homo sapiens * View in UniProt * View in Entrez Gene * Regulator of G-protein signaling 16 Rgs16 Mus musculus * View in UniProt * View in Entrez Gene * Guanine nucleotide-binding protein G(k) subunit alpha GNAI3 Homo sapiens * View in UniProt * View in Entrez Gene * Colicin-E9 immunity protein ceiI Escherichia coli * View in UniProt * View in Entrez Gene
• Eisosome-driven plasma membrane organization is mediated by BAR domains
  - Nat Struct Mol Biol 18(7):854-856 (2011)
  Nature Structural & Molecular Biology | Brief Communication Eisosome-driven plasma membrane organization is mediated by BAR domains * Natasza E Ziółkowska1 * Lena Karotki1 * Michael Rehman1 * Juha T Huiskonen2 * Tobias C Walther3 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:854–856Year published:(2011)DOI:doi:10.1038/nsmb.2080Received21 November 2010Accepted27 April 2011Published online19 June 2011 Highlighting tool Genes and ProteinsUpdate Highlighting Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Plasma membranes are organized into domains of different protein and lipid composition. Eisosomes are key complexes for yeast plasma membrane organization, containing primarily Pil1 and Lsp1. Here we show that both proteins consist mostly of a banana-shaped BAR domain common to membrane sculpting proteins, most similar to the ones of amphiphysin, arfaptin 2 and endophilin 2. Our data reveal a previously unrecognized family of BAR-domain proteins involved in plasma membrane organization. View full text Figures at a glance * Figure 1: Molecular structure of Lsp1 and Pil1. () X-ray structure of Lsp1 ASIA in a ribbon representation of the dimer (green and gray monomers). () Lsp1 sequence diagram showing the residues included in Lsp1 ASIA (green and yellow) and the residues not visible in the electron density map (yellow). () Structure alignment of A, B and C chains in the asymmetric unit of the Lsp1 ASIA X-ray structure. The gray portion was used to align the chains. The flexible parts of the structure are shown in blue, green and red (chains A, B and C). () Theoretical model of a Pil1 homodimer. Amino acids of low sequence similarity to Lsp1 are indicated in orange. * Figure 2: Lsp1 and Pil1 belong to the superfamily of BAR domain–containing proteins. () Alignment of S. cerevisiaeLsp1 ASIA dimer (PDB 3PLT; green) and the D. melanogasteramphiphysin dimer (PDB 1URU; brown). () Phylogenetic tree generated using a structure-based sequence alignment of Lsp1 ASIA with BAR domain–containing proteins. Dm, D. melanogaster; Hs. H. sapiens; Sc, S. cerevisiae; Gs, Galdieria sulphuraria. Scale bar in amino acid substitutions per site. () Lsp1 ASIA dimer surface colored according to evolutionary conservation (top panels) and electrostatic potential (bottom panels) on the concave surface and a close-up view of the positively charged surface patch. * Figure 3: The positively charged patch on the Pil1 concave surface is required for normal Pil1 localization and function. () Representative confocal midsections of cells expressing Pil1-GFP wild-type (WT) and Pil1-GFP mutants (K130E R133E, R133E or K63A K66A K130A R133A (KKKR-A)) in pil1Δ and pil1Δ lsp1Δ cells. Scale bar, 2.5 μm. () Quantification of foci per cell, normalized to WT. () Quantification of GFP fluorescence of foci. () Quantification of cytosolic GFP fluorescence. () Yeast strains with plasmids harboring a WT copy of PIL1 on a URA3 plasmid and a WT or mutant copy of PIL1 on a LEU2 plasmid were tested for growth on 5-fluoroorotic acid (5-FOA) plates at 24 °C, which indicates the ability of the mutant copy of PIL1 to complement pil1Δ function. An agar plate with synthetic complete medium lacking uracil (– URA) is shown as a control for growth. pil1 mutants are indicated with pil1*. The yeast strains used in this work are listed in Supplementary Table 2. Error bars, s.d. Accession codes * Accession codes * Author information * Supplementary information Primary accessions Protein Data Bank * 3PLT * 3PLT Referenced accessions Protein Data Bank * 1URU * 1I49 * 1URU * 1I49 Author information * Accession codes * Author information * Supplementary information Affiliations * Max Planck Institute of Biochemistry, Organelle Architecture and Dynamics, Martinsried, Germany. * Natasza E Ziółkowska, * Lena Karotki & * Michael Rehman * Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK. * Juha T Huiskonen * Yale University School of Medicine, Department of Cell Biology, New Haven, Connecticut, USA. * Tobias C Walther Contributions All authors contributed to design and execution of experiments. N.E.Z. produced the protein, grew crystals, solved the structure of Lsp1 and performed the confocal microscopy. N.E.Z. and T.C.W. wrote the manuscript. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Tobias C Walther Author Details * Natasza E Ziółkowska Search for this author in: * NPG journals * PubMed * Google Scholar * Lena Karotki Search for this author in: * NPG journals * PubMed * Google Scholar * Michael Rehman Search for this author in: * NPG journals * PubMed * Google Scholar * Juha T Huiskonen Search for this author in: * NPG journals * PubMed * Google Scholar * Tobias C Walther Contact Tobias C Walther Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information Movies * Supplementary Video 1 (94K) Pil1-GFP R126E in the pil1Δ lsp1Δ strain forms long rods traversing the cytoplasm. Ylr413w-RFPmars is used as a membrane staining marker. z-stack images collected at 0.2-μm distances. PDF files * Supplementary Text and Figures (1M) Supplementary Figures 1–8, Supplementary Tables 1–2 and Supplementary Methods Additional data Entities in this article * 3-isopropylmalate dehydrogenase LEU2 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Sphingolipid long chain base-responsive protein PIL1 PIL1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Protein SUR7 SUR7 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Arginine permease CAN1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Orotidine 5'-phosphate decarboxylase URA3 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Actin cytoskeleton-regulatory complex protein PAN1 PAN1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Amphiphysin Amph Drosophila melanogaster * View in UniProt * View in Entrez Gene * Sphingolipid long chain base-responsive protein LSP1 LSP1 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) * View in UniProt * View in Entrez Gene * Endophilin-A2 SH3GL1 Homo sapiens * View in UniProt * View in Entrez Gene * Arfaptin-2 ARFIP2 Homo sapiens * View in UniProt * View in Entrez Gene
• Crystal structure of the trithorax group protein ASH2L reveals a forkhead-like DNA binding domain
  - Nat Struct Mol Biol 18(7):857-859 (2011)
  Nature Structural & Molecular Biology | Brief Communication Crystal structure of the trithorax group protein ASH2L reveals a forkhead-like DNA binding domain * Sabina Sarvan1, 5 * Vanja Avdic1, 5 * Véronique Tremblay1, 5 * Chandra-Prakash Chaturvedi2, 3 * Pamela Zhang1 * Sylvain Lanouette1 * Alexandre Blais1, 2 * Joseph S Brunzelle4 * Marjorie Brand2, 3 * Jean-François Couture1 * Affiliations * Contributions * Corresponding authorJournal name:Nature Structural & Molecular BiologyVolume: 18,Pages:857–859Year published:(2011)DOI:doi:10.1038/nsmb.2093Received17 August 2010Accepted30 May 2011Published online05 June 2011 Article tools * Full text * Print * Email * Download PDF * Download citation * Order reprints * Rights and permissions * Share/bookmark * Connotea * CiteULike * Facebook * Twitter * Delicious * Digg Absent, small or homeotic discs–like 2 (ASH2L) is a trithorax group (TrxG) protein and a regulatory subunit of the SET1 family of lysine methyltransferases. Here we report that ASH2L binds DNA using a forkhead-like helix-wing-helix (HWH) domain. In vivo, the ASH2L HWH domain is required for binding to the β-globin locus control region, histone H3 Lys4 (H3K4) trimethylation and maximal expression of the β-globin gene (Hbb-1), validating the functional importance of the ASH2L DNA binding domain. View full text Figures at a glance * Figure 1: ASH2L is composed of a C4 zinc finger and a structurally conserved helix-wing-helix domain. () Overall structure of ASH2L N-terminal domain. β-strands and α-helices are rendered in blue and orange, respectively; the zinc atom is depicted in green. () ASH2L harbors a structurally conserved helix-wing-helix domain. Evolutionarily conserved surface residues are colored according to the sequence alignment (Supplementary Fig. 1). () Superimposition of ASH2L and FOXO4 HWH domains. ASH2L is rendered as in , and FOXO4 is colored in gray. () Electrostatic potential surface of FOXO4 () and ASH2L (). DNA carbon atoms are rendered in yellow. Electrostatic potentials are contoured from +10 kBTe−1 (blue) to −10 kBTe−1 (red), where e is the electron, T is temperature and kB is the Boltzmann constant. Arrows indicate the canonical and putative DNA binding α-helix of FOXO4 and ASH2L. * Figure 2: The N-terminal domain of ASH2L binds DNA. () ASH2LN directly binds HS2. EMSA of biotinylated HS2 using 4μM of ASH2LN. Specificity of the binding is confirmed using 10×, 25× and 50× molar excess of non-biotinylated probe (Competitor) or with 10× and 25× molar excess of an AGGCT–CTGGG substituted HS2 element (HS2mut). () ASH2L binds GC-rich DNA motifs. Gene ontology analysis of the human genes containing a good match for the SELEX Motif #1 (inset) in their proximal promoters (green histogram bar). Gray bars represent five control sets of randomly selected genes. (Enrichment of ASH2L to selected target genes is presented in Supplementary Fig. 6, Supplementary Methods and Supplementary Table 2). () ASH2LN is important for the recruitment of ASH2L to HS2. Flag-tagged ASH2L constructs were ectopically expressed in differentiated mouse erythroleukemia (MEL) cells, and ChIP was done with an anti-Flag antibody. Relative binding of Flag-ASH2LFL, Flag-ASH2LN and Flag-ASH2LC was measured by ChIP at the HS2 site of the �! �-globin LCR. Immunopurified DNA was quantified as previously described5. Average values of duplicate quantitative PCR (qPCR) reactions are shown; error bars represent s.d. Each experiment was done twice with independent chromatin samples. * Figure 3: Role of ASH2L HWH domain in the regulation of β-globin gene expression. () Mutation of ASH2L α5-helix impairs binding of HS2 in EMSA. ASH2L α5-helix is essential for ASH2L binding to HS2 (), maximal expression of β-globin () and H3K4 trimethylation (). Binding of ASH2L and enrichment of H3K4me3 was measured by ChIP using Dox-induced differentiated MEL cells transfected with either the empty vector (CMV), ASH2L-WT, ASH2L-K225E or ASH2L-K229E. The inset shows the western blot analysis of Dox-inducible knockdown (Kd) of endogenous ASH2L rescued with shRNA-resistant flag-tagged ASH2L-WT, ASH2L-K225E and ASH2L-K229E in differentiated MEL cells. TFIIH p89 is used as a loading control. β-major globin gene (βmaj-globin) transcription relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was assessed by real-time quantitative PCR (qRT-PCR) as previously described5. Values were averaged as in Figure 2c. Accession codes * Accession codes * Author information * Supplementary information Referenced accessions Protein Data Bank * 3S32 * 3S32 Author information * Accession codes * Author information * Supplementary information Primary authors * These authors contributed equally to this work. * Sabina Sarvan, * Vanja Avdic & * Véronique Tremblay Affiliations * Ottawa Institute of Systems Biology, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada. * Sabina Sarvan, * Vanja Avdic, * Véronique Tremblay, * Pamela Zhang, * Sylvain Lanouette, * Alexandre Blais & * Jean-François Couture * The Sprott Center for Stem Cell Research, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Ontario, Canada. * Chandra-Prakash Chaturvedi, * Alexandre Blais & * Marjorie Brand * Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada. * Chandra-Prakash Chaturvedi & * Marjorie Brand * Feinberg School of Medicine, Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, Chicago, Illinois, USA. * Joseph S Brunzelle Contributions J.-F.C. and M.B. designed the experiments. S.S. and V.A were responsible for doing the EMSA and analyzing the crystal structure. V.T. did the SELEX and validated the ASH2L target genes. C.-P.C. carried out the ChIP and qRT-PCR studies. P.Z. and S.L. did the methyltransferase assays and sequence alignment, respectively. J.S.B. collected the data and calculated the initial phases of the model. A.B. analyzed the SELEX data. J.-F.C. provided scientific direction for the project and wrote the manuscript. J.S.B., A.B. and M.B commented on the paper. Competing financial interests The authors declare no competing financial interests. Corresponding author Correspondence to: * Jean-François Couture Author Details * Sabina Sarvan Search for this author in: * NPG journals * PubMed * Google Scholar * Vanja Avdic Search for this author in: * NPG journals * PubMed * Google Scholar * Véronique Tremblay Search for this author in: * NPG journals * PubMed * Google Scholar * Chandra-Prakash Chaturvedi Search for this author in: * NPG journals * PubMed * Google Scholar * Pamela Zhang Search for this author in: * NPG journals * PubMed * Google Scholar * Sylvain Lanouette Search for this author in: * NPG journals * PubMed * Google Scholar * Alexandre Blais Search for this author in: * NPG journals * PubMed * Google Scholar * Joseph S Brunzelle Search for this author in: * NPG journals * PubMed * Google Scholar * Marjorie Brand Search for this author in: * NPG journals * PubMed * Google Scholar * Jean-François Couture Contact Jean-François Couture Search for this author in: * NPG journals * PubMed * Google Scholar Supplementary information * Accession codes * Author information * Supplementary information PDF files * Supplementary Text and Figures (3.4M) Supplementary Figures 1–6, Supplementary Tables 1 and 2, and Supplementary Methods Additional data