Latest Articles Include:
- The Proteasome's Crown for Destruction
Sakata E Saeki Y Tanaka K - Mol Cell 34(5):519-520 (2009)
Three recent Molecular Cell papers from (Zhang et al., 2009a) and (Zhang et al., 2009b) and Djuranovic et al. (2009) provide new insights into how proteasomal ATPases recognize, unfold, and translocate their substrates, thus enhancing our understanding of regulated proteolysis. - RVB1/RVB2: Running Rings around Molecular Biology
Jha S Dutta A - Mol Cell 34(5):521-533 (2009)
RVB1/RVB2 (also known as Pontin/Reptin, TIP49/TIP48, RuvbL1/RuvbL2, ECP54/ECP51, INO80H/INO80J, TIH1/TIH2, and TIP49A/TIP49B) are two highly conserved members of the AAA+ family that are present in different protein and nucleoprotein complexes. Recent studies implicate the RVB-containing complexes in many cellular processes such as transcription, DNA damage response, snoRNP assembly, cellular transformation, and cancer metastasis. In this review, we discuss recent advances in our understanding of RVB-containing complexes and their role in these pathways. - H3K4me3 Stimulates the V(D)J RAG Complex for Both Nicking and Hairpinning in trans in Addition to Tethering in cis: Implications for Translocations
Shimazaki N Tsai AG Lieber MR - Mol Cell 34(5):535-544 (2009)
The PHD finger of the RAG2 polypeptide of the RAG1/RAG2 complex binds to the histone H3 modification, trimethylated lysine 4 (H3K4me3), and in some manner increases V(D)J recombination. In the absence of biochemical studies of H3K4me3 on purified RAG enzyme activity, the precise role of H3K4me3 remains unclear. Here, we find that H3K4me3 stimulates purified RAG enzymatic activity at both the nicking (2- to 5-fold) and hairpinning (3- to 11-fold) steps of V(D)J recombination. Remarkably, this stimulation can be achieved with free H3K4me3 peptide (in trans), indicating that H3K4me3 functions via two distinct mechanisms. It not only tethers the RAG enzyme complex to a region of DNA, but it also induces a substantial increase in the catalytic turnover number (kcat) of the RAG complex. The H3K4me3 catalytic stimulation applies to suboptimal cryptic RSS sites located at H3K4me3 peaks that are critical in the inception of human T cell acute lymphoblastic lymphomas. - Potent Transcriptional Interference by Pausing of RNA Polymerases over a Downstream Promoter
Palmer AC Ahlgren-Berg A Egan JB Dodd IB Shearwin KE - Mol Cell 34(5):545-555 (2009)
Elongating RNA polymerases (RNAPs) can interfere with transcription from downstream promoters by inhibiting DNA binding by RNAP and activators. However, combining quantitative measurement with mathematical modeling, we show that simple RNAP elongation cannot produce the strong asymmetric interference observed between a natural face-to-face promoter pair in bacteriophage lambda. Pausing of elongating polymerases over the RNAP-binding site of the downstream promoter is demonstrated in vivo and is shown by modeling to account for the increased interference. The model successfully predicts the effects on interference of treatments increasing or reducing pausing. Gene regulation by pausing-enhanced occlusion provides a general and potentially widespread mechanism by which even weak converging or tandem transcription, either coding or noncoding, can bring about strong in cis repression. - Polypyrimidine Tract Binding Protein Stabilizes the Encephalomyocarditis Virus IRES Structure via Binding Multiple Sites in a Unique Orientation
Kafasla P Morgner N Pöyry TA Curry S Robinson CV Jackson RJ - Mol Cell 34(5):556-568 (2009)
Polypyrimidine tract binding (PTB) protein is a regulator of alternative pre-mRNA splicing, and also stimulates the initiation of translation dependent on many viral internal ribosome entry segments/sites (IRESs). It has four RNA-binding domains (RBDs), but although the contacts with many IRESs have been mapped, the orientation of binding (i.e., which RBD binds to which site in the IRES) is unknown. To answer this question, 16 derivatives of PTB1, each with a single cysteine flanking the RNA-binding surface in an RBD, were constructed and used in directed hydroxyl radical probing with the encephalomyocarditis virus IRES. The results, together with mass spectrometry data on the stoichiometry of PTB binding to different IRES derivatives, show that the minimal IRES binds a single PTB in a unique orientation, with RBD1 and RBD2 binding near the 3′ end, and RBD3 contacting the 5′ end, thereby constraining and stabilizing the three-dimensional structural fold of the IRES. - An Unfolded CH1 Domain Controls the Assembly and Secretion of IgG Antibodies
Feige MJ Groscurth S Marcinowski M Shimizu Y Kessler H Hendershot LM Buchner J - Mol Cell 34(5):569-579 (2009)
A prerequisite for antibody secretion and function is their assembly into a defined quaternary structure, composed of two heavy and two light chains for IgG. Unassembled heavy chains are actively retained in the endoplasmic reticulum (ER). Here, we show that the CH1 domain of the heavy chain is intrinsically disordered in vitro, which sets it apart from other antibody domains. It folds only upon interaction with the light-chain CL domain. Structure formation proceeds via a trapped intermediate and can be accelerated by the ER-specific peptidyl-prolyl isomerase cyclophilin B. The molecular chaperone BiP recognizes incompletely folded states of the CH1 domain and competes for binding to the CL domain. In vivo experiments demonstrate that requirements identified for folding the CH1 domain in vitro, including association with a folded CL domain and isomerization of a conserved proline residue, are essential for antibody assembly and secretion in the cell. - Structure and Activity of the N-Terminal Substrate Recognition Domains in Proteasomal ATPases
Djuranovic S Hartmann MD Habeck M Ursinus A Zwickl P Martin J Lupas AN Zeth K - Mol Cell 34(5):580-590 (2009)
The proteasome forms the core of the protein quality control system in archaea and eukaryotes and also occurs in one bacterial lineage, the Actinobacteria. Access to its proteolytic compartment is controlled by AAA ATPases, whose N-terminal domains (N domains) are thought to mediate substrate recognition. The N domains of an archaeal proteasomal ATPase, Archaeoglobus fulgidus PAN, and of its actinobacterial homolog, Rhodococcus erythropolis ARC, form hexameric rings, whose subunits consist of an N-terminal coiled coil and a C-terminal OB domain. In ARC-N, the OB domains are duplicated and form separate rings. PAN-N and ARC-N can act as chaperones, preventing the aggregation of heterologous proteins in vitro, and this activity is preserved in various chimeras, even when these include coiled coils and OB domains from unrelated proteins. The structures suggest a molecular mechanism for substrate processing based on concerted radial motions of the coiled coils relative to ! the OB rings. - The Nfkb1 and Nfkb2 Proteins p105 and p100 Function as the Core of High-Molecular-Weight Heterogeneous Complexes
Savinova OV Hoffmann A Ghosh G - Mol Cell 34(5):591-602 (2009)
Nfkb1 and Nfkb2 proteins p105 and p100 serve both as NF-κB precursors and inhibitors of NF-κB dimers. In a biochemical characterization of endogenous cytoplasmic and purified recombinant proteins, we found that p105 and p100 assemble into high-molecular-weight complexes that contribute to the regulation of all NF-κB isoforms. Unlike the classical inhibitors IκBα, -β, and -, high-molecular-weight complexes of p105 and p100 proteins bind NF-κB subunits in two modes: through direct dimerization of Rel homology domain-containing NF-κB polypeptides and through interactions of the p105 and p100 ankyrin repeats with preformed NF-κB dimers, thereby mediating the bona fide IκB activities, IκBγ and IκBδ. Our biochemical evidence suggests an assembly pathway in which kinetic mechanisms control NF-κB dimer formation via processing and assembly of large complexes that contain IκB activities. - LysRS Serves as a Key Signaling Molecule in the Immune Response by Regulating Gene Expression
Yannay-Cohen N Carmi-Levy I Kay G Yang CM Han JM Kemeny DM Kim S Nechushtan H Razin E - Mol Cell 34(5):603-611 (2009)
Lysyl-tRNA synthetase (LysRS) was found to produce diadenosine tetraphosphate (Ap4A) in vitro more than two decades ago. Here, we used LysRS silencing in mast cells in combination with transfected normal and mutated LysRS to demonstrate in vivo the critical role played by LysRS in the production of Ap4A in response to immunological challenge. Upon such challenge, LysRS was phosphorylated on serine 207 in a MAPK-dependent manner, released from the multisynthetase complex, and translocated into the nucleus. We previously demonstrated that LysRS forms a complex with MITF and its repressor Hint-1, which is released from the complex by its binding to Ap4A, enabling MITF to transcribe its target genes. Here, silencing LysRS led to reduced Ap4A production in immunologically activated cells, which resulted in a lower level of MITF inducible genes. Our data demonstrate that specific LysRS serine 207 phosphorylation regulates Ap4A production in immunologically stimulated mast ce! lls, thus implying that LysRS is a key mediator in gene regulation. - Resetting the Site: Redirecting Integration of an Insertion Sequence in a Predictable Way
Guynet C Achard A Hoang BT Barabas O Hickman AB Dyda F Chandler M - Mol Cell 34(5):612-619 (2009)
Target site choice is a complex and poorly understood aspect of DNA transposition despite its importance in rational transposon-mediated gene delivery. Though most transposons choose target sites essentially randomly or with some slight sequence or structural preferences, insertion sequence IS608 from Helicobacter pylori, which transposes using single-stranded DNA, always inserts just 3′ of a TTAC tetranucleotide. Our results from studies on the IS608 transposition mechanism demonstrated that the transposase recognizes its target site by co-opting an internal segment of transposon DNA and utilizes it for specific recognition of the target sites through base-pairing. This suggested a way to redirect IS608 transposition to novel target sites. As we demonstrate here, we can now direct insertions in a predictable way into a variety of different chosen target sequences, both in vitro and in vivo. - HMGN Proteins Act in Opposition to ATP-Dependent Chromatin Remodeling Factors to Restrict Nucleosome Mobility
Rattner BP Yusufzai T Kadonaga JT - Mol Cell 34(5):620-626 (2009)
The high-mobility group N (HMGN) proteins are abundant nonhistone chromosomal proteins that bind specifically to nucleosomes at two high-affinity sites. Here we report that purified recombinant human HMGN1 (HMG14) and HMGN2 (HMG17) potently repress ATP-dependent chromatin remodeling by four different molecular motor proteins. In contrast, mutant HMGN proteins with double Ser-to-Glu mutations in their nucleosome-binding domains are unable to inhibit chromatin remodeling. The HMGN-mediated repression of chromatin remodeling is reversible and dynamic. With the ACF chromatin remodeling factor, HMGN2 does not directly inhibit the ATPase activity but rather appears to reduce the affinity of the factor to chromatin. These findings suggest that HMGN proteins serve as a counterbalance to the action of the many ATP-dependent chromatin remodeling activities in the nucleus. - EDEM1 Recognition and Delivery of Misfolded Proteins to the SEL1L-Containing ERAD Complex
Cormier JH Tamura T Sunryd JC Hebert DN - Mol Cell 34(5):627-633 (2009)
Terminally misfolded or unassembled secretory proteins are retained in the endoplasmic reticulum (ER) and subsequently cleared by the ER-associated degradation (ERAD) pathway. The degradation of ERAD substrates involves mannose trimming of N-linked glycans; however, the mechanisms of substrate recognition and sorting to the ERAD pathway are poorly defined. EDEM1 (ER degradation-enhancing α-mannosidase-like 1 protein) has been proposed to play a role in ERAD substrate signaling or recognition. We show that EDEM1 specifically binds nonnative proteins in a glycan-independent manner. Inhibition of mannosidase activity with kifunensine or disruption of the EDEM1 mannosidase-like domain by mutation had no effect on EDEM1 substrate binding but diminished its association with the ER membrane adaptor protein SEL1L. These results support a model whereby EDEM1 binds nonnative proteins and uses its mannosidase-like domain to target aberrant proteins to the ER membrane dislocation! and ubiquitination complex containing SEL1L.
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