Hot off the presses! Oct 09 Mol Cell

The Oct 09 issue of the Mol Cell is now up on Pubget (About Mol Cell): if you're at a subscribing institution, just click the link in the latest link at the home page. (Note you'll only be able to get all the PDFs in the issue if your institution subscribes to Pubget.)

Latest Articles Include:

• Taming Supplemental Material
  - Mol Cell 36(1):1 (2009)
  
• Healing and Hurting: Molecular Mechanisms, Functions, and Pathologies of Cellular Senescence
  - Mol Cell 36(1):2-14 (2009)
  Cellular senescence is a proliferation arrest that is typically irreversible and caused by various cellular stresses, including excess rounds of cell division and cancer-causing genetic alterations. Senescence actively contributes to a tissue-level response to tissue wounding and incipient cancer, healing the tissue and suppressing tumor formation. However, in the long term, the same senescence program may hurt the tissue, thereby contributing to tissue aging. Tumor suppression, wound healing, and aging are each associated with inflammation, and here it is proposed that cellular senescence contributes to a "nonimmune cell" component of the tissue inflammatory response.
• A Small Molecule Inhibitor of Inducible Heat Shock Protein 70
  - Mol Cell 36(1):15-27 (2009)
  The multifunctional, stress-inducible molecular chaperone HSP70 has important roles in aiding protein folding and maintaining protein homeostasis. HSP70 expression is elevated in many cancers, contributing to tumor cell survival and resistance to therapy. We have determined that a small molecule called 2-phenylethynesulfonamide (PES) interacts selectively with HSP70 and leads to a disruption of the association between HSP70 and several of its cochaperones and substrate proteins. Treatment of cultured tumor cells with PES promotes cell death that is associated with protein aggregation, impaired autophagy, and inhibition of lysosomal function. Moreover, this small molecule is able to suppress tumor development and enhance survival in a mouse model of Myc-induced lymphomagenesis. The data demonstrate that PES disrupts actions of HSP70 in multiple cell signaling pathways, offering an opportunity to better understand the diverse functions of this molecular chaperone and als! o to aid in the development of new cancer therapies.
• The Otubain YOD1 Is a Deubiquitinating Enzyme that Associates with p97 to Facilitate Protein Dislocation from the ER
  - Mol Cell 36(1):28-38 (2009)
  YOD1 is a highly conserved deubiquitinating enzyme of the ovarian tumor (otubain) family, whose function has yet to be assigned in mammalian cells. YOD1 is a constituent of a multiprotein complex with p97 as its nucleus, suggesting a functional link to a pathway responsible for the dislocation of misfolded proteins from the endoplasmic reticulum. Expression of a YOD1 variant deprived of its deubiquitinating activity imposes a halt on the dislocation reaction, as judged by the stabilization of various dislocation substrates. Accordingly, we observe an increase in polyubiquitinated dislocation intermediates in association with p97 in the cytosol. This dominant-negative effect is dependent on the UBX and Zinc finger domains, appended to the N and C terminus of the catalytic otubain core domain, respectively. The assignment of a p97-associated ubiquitin processing function to YOD1 adds to our understanding of p97's role in the dislocation process.
• Structures of SPOP-Substrate Complexes: Insights into Molecular Architectures of BTB-Cul3 Ubiquitin Ligases
  - Mol Cell 36(1):39-50 (2009)
  In the largest E3 ligase subfamily, Cul3 binds a BTB domain, and an associated protein-interaction domain such as MATH recruits substrates for ubiquitination. Here, we present biochemical and structural analyses of the MATH-BTB protein, SPOP. We define a SPOP-binding consensus (SBC) and determine structures revealing recognition of SBCs from the phosphatase Puc, the transcriptional regulator Ci, and the chromatin component MacroH2A. We identify a dimeric SPOP-Cul3 assembly involving a conserved helical structure C-terminal of BTB domains, which we call "3-box" due to its facilitating Cul3 binding and its resemblance to F-/SOCS-boxes in other cullin-based E3s. Structural flexibility between the substrate-binding MATH and Cul3-binding BTB/3-box domains potentially allows a SPOP dimer to engage multiple SBCs found within a single substrate, such as Puc. These studies provide a molecular understanding of how MATH-BTB proteins recruit substrates to Cul3 and how their di! merization and conformational variability may facilitate avid interactions with diverse substrates.
• α4 Is an Essential Regulator of PP2A Phosphatase Activity
  - Mol Cell 36(1):51-60 (2009)
  The activity and specificity of serine/threonine phosphatases are governed largely by their associated proteins. α4 is an evolutionarily conserved noncatalytic subunit for PP2A-like phosphatases. Though α4 binds to only a minority of PP2A-related catalytic subunits, α4 deletion leads to progressive loss of all PP2A, PP4, and PP6 phosphatase complexes. In healthy cells, association with α4 renders catalytic (C) subunits enzymatically inactive while protecting them from proteasomal degradation until they are assembled into a functional phosphatase complex. During cellular stress, existing PP2A complexes can become unstable. Under such conditions, α4 sequesters released C subunits and is required for the adaptive increase in targeted PP2A activity that can dephosphorylate stress-induced phosphorylated substrates. Consistent with this, overexpression of α4 protects cells from a variety of stress stimuli, including DNA damage and nutrient limitation. These findings de! monstrate that α4 plays a required role in regulating the assembly and maintenance of adaptive PP2A phosphatase complexes.
• Mir-214-Dependent Regulation of the Polycomb Protein Ezh2 in Skeletal Muscle and Embryonic Stem Cells
  - Mol Cell 36(1):61-74 (2009)
  Polycomb group (PcG) proteins exert essential functions in the most disparate biological processes. The contribution of PcG proteins to cell commitment and differentiation relates to their ability to repress transcription of developmental regulators in embryonic stem (ES) cells and in committed cell lineages, including skeletal muscle cells (SMC). PcG proteins are preferentially removed from transcribed regions, but the underlying mechanisms remain unclear. Here, PcG proteins are found to occupy and repress transcription from an intronic region containing the microRNA miR-214 in undifferentiated SMC. Differentiation coincides with PcG disengagement, recruitment of the developmental regulators MyoD and myogenin, and activation of miR-214 transcription. Once transcribed, miR-214 negatively feeds back on PcG by targeting the Ezh2 3′UTR, the catalytic subunit of the PRC2 complex. miR-214-mediated Ezh2 protein reduction accelerates SMC differentiation and promotes unsched! uled transcription of developmental regulators in ES cells. Thus, miR-214 and Ezh2 establish a regulatory loop controlling PcG-dependent gene expression during differentiation.
• SKIP Interacts with c-Myc and Menin to Promote HIV-1 Tat Transactivation
  - Mol Cell 36(1):75-87 (2009)
  The Ski-interacting protein SKIP/SNW1 associates with the P-TEFb/CDK9 elongation factor and coactivates inducible genes, including HIV-1. We show here that SKIP also associates with c-Myc and Menin, a subunit of the MLL1 histone methyltransferase (H3K4me3) complex and that HIV-1 Tat transactivation requires c-Myc and Menin, but not MLL1 or H3K4me3. RNAi-ChIP experiments reveal that SKIP acts downstream of Tat:P-TEFb to recruit c-Myc and its partner TRRAP, a scaffold for histone acetyltransferases, to the HIV-1 promoter. By contrast, SKIP is recruited by the RNF20 H2B ubiquitin ligase to the basal HIV-1 promoter in a step that is bypassed by Tat and downregulated by c-Myc. Of interest, we find that SKIP and P-TEFb are dispensable for UV stress-induced HIV-1 transcription, which is strongly upregulated by treating cells with the CDK9 inhibitor flavopiridol. Thus, SKIP acts with c-Myc and Menin to promote HIV-1 Tat:P-TEFb transcription at an elongation step that is bypass! ed under stress.
• Fail-Safe Transcriptional Termination for Protein-Coding Genes in S. cerevisiae
  - Mol Cell 36(1):88-98 (2009)
  Transcription termination of RNA polymerase II (Pol II) on protein-coding genes in S. cerevisiae relies on pA site recognition by 3′ end processing factors. Here we demonstrate the existence of two alternative termination mechanisms that rescue polymerases failing to disengage from the template at pA sites. One of these fail-safe mechanisms is mediated by the NRD complex, similar to termination of short noncoding genes. The other termination mechanism is mediated by Rnt1 cleavage of the nascent transcript. Both fail-safe termination mechanisms trigger degradation of readthrough transcripts by the exosome. However, Rnt1-mediated termination can also enhance the usage of weak pA signals and thereby generate functional mRNA. We propose that these alternative Pol II termination pathways serve the dual function of avoiding transcription interference and promoting rapid removal of aberrant transcripts.
• Yeast RNase III Triggers Polyadenylation-Independent Transcription Termination
  - Mol Cell 36(1):99-109 (2009)
  Transcription termination of messenger RNA (mRNA) is normally achieved by polyadenylation followed by Rat1p-dependent 5′-3′ exoribonuleolytic degradation of the downstream transcript. Here we show that the yeast ortholog of the dsRNA-specific ribonuclease III (Rnt1p) may trigger Rat1p-dependent termination of RNA transcripts that fail to terminate near polyadenylation signals. Rnt1p cleavage sites were found downstream of several genes, and the deletion of RNT1 resulted in transcription readthrough. Inactivation of Rat1p impaired Rnt1p-dependent termination and resulted in the accumulation of 3′ end cleavage products. These results support a model for transcription termination in which cotranscriptional cleavage by Rnt1p provides access for exoribonucleases in the absence of polyadenylation signals.
• BBAP Monoubiquitylates Histone H4 at Lysine 91 and Selectively Modulates the DNA Damage Response
  - Mol Cell 36(1):110-120 (2009)
  Although the BBAP E3 ligase and its binding partner BAL are overexpressed in chemotherapy-resistant lymphomas, the role of these proteins in DNA damage responses remains undefined. Because BAL proteins modulate promoter-coupled transcription and contain structural motifs associated with chromatin remodeling and DNA repair, we reasoned that the BBAP E3 ligase might target nucleosomal proteins. Herein, we demonstrate that BBAP selectively monoubiquitylates histone H4 lysine 91 and protects cells exposed to DNA-damaging agents. Disruption of BBAP-mediated monoubiquitylation of histone H4K91 is associated with the loss of chromatin-associated H4K20 methylase, mono- and dimethyl H4K20, and a delay in the kinetics of 53BP1 foci formation at sites of DNA damage. Because 53BP1 localizes to DNA damage sites, in part, via an interaction with dimethyl H4K20, these data directly implicate BBAP in the monoubiquitylation and additional posttranslational modification of histone H4 an! d an associated DNA damage response.
• An SOS Inhibitor that Binds to Free RecA Protein: The PsiB Protein
  - Mol Cell 36(1):121-130 (2009)
  The process of bacterial conjugation involves the transfer of a conjugative plasmid as a single strand. The potentially deleterious SOS response, which is normally triggered by the appearance of single-stranded DNA, is suppressed in the recipient cell by a conjugative plasmid system centered on the product of the psiB gene. The F plasmid PsiB protein inhibits all activities of the RecA protein, including DNA binding, DNA strand exchange, and LexA protein cleavage. The proteins known to negatively regulate recombinases, such as RecA or Rad51, generally work at the level of dismantling the nucleoprotein filament. However, PsiB binds to RecA protein that is free in solution. The RecA-PsiB complex impedes formation of RecA nucleoprotein filaments on DNA.
• KEAP1 E3 Ligase-Mediated Downregulation of NF-κB Signaling by Targeting IKKβ
  - Mol Cell 36(1):131-140 (2009)
  IκB kinase β (IKKβ) is involved in tumor development and progression through activation of the nuclear factor (NF)-κB pathway. However, the molecular mechanism that regulates IKKβ degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)-based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKβ ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKβ and to upregulation of NF-κB-derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKβ degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKβ ubiquitination may contribute to tumorigenesis.
• The eIF3 Interactome Reveals the Translasome, a Supercomplex Linking Protein Synthesis and Degradation Machineries
  - Mol Cell 36(1):141-152 (2009)
  eIF3 promotes translation initiation, but relatively little is known about its full range of activities in the cell. Here, we employed affinity purification and highly sensitive LC-MS/MS to decipher the fission yeast eIF3 interactome, which was found to contain 230 proteins. eIF3 assembles into a large supercomplex, the translasome, which contains elongation factors, tRNA synthetases, 40S and 60S ribosomal proteins, chaperones, and the proteasome. eIF3 also associates with ribosome biogenesis factors and the importins-β Kap123p and Sal3p. Our genetic data indicated that the binding to both importins-β is essential for cell growth, and photobleaching experiments revealed a critical role for Sal3p in the nuclear import of one of the translasome constituents, the proteasome. Our data reveal the breadth of the eIF3 interactome and suggest that factors involved in translation initiation, ribosome biogenesis, translation elongation, quality control, and transport are physi! cally linked to facilitate efficient protein synthesis.
• A Method for Genetically Installing Site-Specific Acetylation in Recombinant Histones Defines the Effects of H3 K56 Acetylation
  - Mol Cell 36(1):153-163 (2009)
  Lysine acetylation of histones defines the epigenetic status of human embryonic stem cells and orchestrates DNA replication, chromosome condensation, transcription, telomeric silencing, and DNA repair. A detailed mechanistic explanation of these phenomena is impeded by the limited availability of homogeneously acetylated histones. We report a general method for the production of homogeneously and site-specifically acetylated recombinant histones by genetically encoding acetyl-lysine. We reconstitute histone octamers, nucleosomes, and nucleosomal arrays bearing defined acetylated lysine residues. With these designer nucleosomes, we demonstrate that, in contrast to the prevailing dogma, acetylation of H3 K56 does not directly affect the compaction of chromatin and has modest effects on remodeling by SWI/SNF and RSC. Single-molecule FRET experiments reveal that H3 K56 acetylation increases DNA breathing 7-fold. Our results provide a molecular and mechanistic underpinning ! for cellular phenomena that have been linked with K56 acetylation.