Thursday, June 23, 2011

Hot off the presses! Jun 24 Mol Cell

The Jun 24 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:

  • PKM2 and the Tricky Balance of Growth and Energy in Cancer
    - Mol Cell 42(6):713-714 (2011)
    In this issue of Molecular Cell, Lv et al. (2011) identify a novel feedback mechanism in which increased glycolysis induces the acetylation and chaperone-mediated autophagic degradation of the glycolytic regulator PKM2, revealing a novel metabolic feedback loop that drives tumor growth.
  • No Country for Old Misfolded Glycoproteins
    - Mol Cell 42(6):715-717 (2011)
    Gauss et al. (2011) present evidence that an endoplasmic reticulum (ER) sugar-removing enzyme with a folding sensor subunit enables a stochastic quality control mechanism, which marks with increasing probability misfolded glycoproteins for destruction the longer they reside in the ER.
  • Sen-sing RNA Terminators
    - Mol Cell 42(6):717-718 (2011)
    In this issue of Molecular Cell, Skourti-Stathaki et al. (2011) report that human Senataxin, like its yeast homolog Sen1, promotes termination by RNA polymerase II and resolves RNA/DNA duplexes formed during transcription. Their results may help uncover a cause of motor neuron degeneration.
  • Acetylation Targets the M2 Isoform of Pyruvate Kinase for Degradation through Chaperone-Mediated Autophagy and Promotes Tumor Growth
    - Mol Cell 42(6):719-730 (2011)
    Most tumor cells take up more glucose than normal cells but metabolize glucose via glycolysis even in the presence of normal levels of oxygen, a phenomenon known as the Warburg effect. Tumor cells commonly express the embryonic M2 isoform of pyruvate kinase (PKM2) that may contribute to the metabolism shift from oxidative phosphorylation to aerobic glycolysis and tumorigenesis. Here we show that PKM2 is acetylated on lysine 305 and that this acetylation is stimulated by high glucose concentration. PKM2 K305 acetylation decreases PKM2 enzyme activity and promotes its lysosomal-dependent degradation via chaperone-mediated autophagy (CMA). Acetylation increases PKM2 interaction with HSC70, a chaperone for CMA, and association with lysosomes. Ectopic expression of an acetylation mimetic K305Q mutant accumulates glycolytic intermediates and promotes cell proliferation and tumor growth. These results reveal an acetylation regulation of pyruvate kinase and the link between ly! sine acetylation and CMA.
  • Distinct Autophagosomal-Lysosomal Fusion Mechanism Revealed by Thapsigargin-Induced Autophagy Arrest
    - Mol Cell 42(6):731-743 (2011)
    Autophagy, a catabolic pathway that delivers cellular components to lysosomes for degradation, can be activated by stressful conditions such as nutrient starvation and endoplasmic reticulum (ER) stress. We report that thapsigargin, an ER stressor widely used to induce autophagy, in fact blocks autophagy. Thapsigargin does not affect autophagosome formation but leads to accumulation of mature autophagosomes by blocking autophagosome fusion with the endocytic system. Strikingly, thapsigargin has no effect on endocytosis-mediated degradation of epidermal growth factor receptor. Molecularly, while both Rab7 and Vps16 are essential regulatory components for endocytic fusion with lysosomes, we found that Rab7 but not Vps16 is required for complete autophagy flux, and that thapsigargin blocks recruitment of Rab7 to autophagosomes. Therefore, autophagosomal-lysosomal fusion must be governed by a distinct molecular mechanism compared to general endocytic fusion.
  • Regulation of Ubiquitin Chain Initiation to Control the Timing of Substrate Degradation
    - Mol Cell 42(6):744-757 (2011)
    Processive reactions, such as transcription or translation, often proceed through distinct initiation and elongation phases. The processive formation of polymeric ubiquitin chains can accordingly be catalyzed by specialized initiating and elongating E2 enzymes, but the functional significance for this division of labor has remained unclear. Here, we have identified sequence motifs in several substrates of the anaphase-promoting complex (APC/C) that are required for efficient chain initiation by its E2 Ube2C. Differences in the quality and accessibility of these chain initiation motifs can determine the rate of a substrate's degradation without affecting its affinity for the APC/C, a mechanism used by the APC/C to control the timing of substrate proteolysis during the cell cycle. Based on our results, we propose that initiation motifs and their cognate E2s allow E3 enzymes to exert precise temporal control over substrate degradation.
  • A Ubiquitin Ligase-Associated Chaperone Holdase Maintains Polypeptides in Soluble States for Proteasome Degradation
    - Mol Cell 42(6):758-770 (2011)
    Endoplasmic reticulum-associated degradation (ERAD) employs membrane-bound ubiquitin ligases and the translocation-driving ATPase p97 to retrotranslocate misfolded proteins for proteasomal degradation. How retrotranslocated polypeptides bearing exposed hydrophobic motifs or transmembrane domains (TMDs) avoid aggregation before reaching the proteasome is unclear. Here we identify a ubiquitin ligase-associated multiprotein complex comprising Bag6, Ubl4A, and Trc35, which chaperones retrotranslocated polypeptides en route to the proteasome to improve ERAD efficiency. In vitro, Bag6, the central component of the complex, contains a chaperone-like activity capable of maintaining an aggregation-prone substrate in an unfolded yet soluble state. The physiological importance of this holdase activity is underscored by observations that ERAD substrates accumulate in detergent-insoluble aggregates in cells depleted of Bag6, or of Trc35, a cofactor that keeps Bag6 outside the nucle! us for engagement in ERAD. Our results reveal a ubiquitin ligase-associated holdase that maintains polypeptide solubility to enhance protein quality control in mammalian cells.
  • Client-Loading Conformation of the Hsp90 Molecular Chaperone Revealed in the Cryo-EM Structure of the Human Hsp90:Hop Complex
    - Mol Cell 42(6):771-781 (2011)
    Hsp90 is an essential molecular chaperone required for the folding and activation of many hundreds of cellular "client" proteins. The ATP-dependent chaperone cycle involves significant conformational rearrangements of the Hsp90 dimer and interaction with a network of cochaperone proteins. Little is known about the mechanism of client protein binding or how cochaperone interactions modulate Hsp90 conformational states. We have determined the cryo-EM structure of the human Hsp90:Hop complex that receives client proteins from the Hsp70 chaperone. Hop stabilizes an alternate Hsp90 open state, where hydrophobic client-binding surfaces have converged and the N-terminal domains have rotated and match the closed, ATP conformation. Hsp90 is thus simultaneously poised for client loading by Hsp70 and subsequent N-terminal dimerization and ATP hydrolysis. Upon binding of a single Hsp70, the Hsp90:Hop conformation remains essentially unchanged. These results identify distinct f! unctions for the Hop cochaperone, revealing an asymmetric mechanism for Hsp90 regulation and client loading.
  • A Complex of Pdi1p and the Mannosidase Htm1p Initiates Clearance of Unfolded Glycoproteins from the Endoplasmic Reticulum
    - Mol Cell 42(6):782-793 (2011)
    Endoplasmic reticulum (ER)-resident mannosidases generate asparagine-linked oligosaccharide signals that trigger ER-associated protein degradation (ERAD) of unfolded glycoproteins. In this study, we provide in vitro evidence that a complex of the yeast protein disulfide isomerase Pdi1p and the mannosidase Htm1p processes Man8GlcNAc2 carbohydrates bound to unfolded proteins, yielding Man7GlcNAc2. This glycan serves as a signal for HRD ligase-mediated glycoprotein disposal. We identified a point mutation in PDI1 that prevents complex formation of the oxidoreductase with Htm1p, diminishes mannosidase activity, and delays degradation of unfolded glycoproteins in vivo. Our results show that Pdi1p is engaged in both recognition and glycan signal processing of ERAD substrates and suggest that protein folding and breakdown are not separated but interconnected processes. We propose a stochastic model for how a given glycoprotein is partitioned into folding or degradation pathwa! ys and how the flux through these pathways is adjusted to stress conditions.
  • Human Senataxin Resolves RNA/DNA Hybrids Formed at Transcriptional Pause Sites to Promote Xrn2-Dependent Termination
    - Mol Cell 42(6):794-805 (2011)
    We present a molecular dissection of pause site-dependent transcriptional termination for mammalian RNA polymerase II (Pol II)-transcribed genes. We show that nascent transcripts form RNA/DNA hybrid structures (R-loops) behind elongating Pol II and are especially prevalent over G-rich pause sites positioned downstream of gene poly(A) signals. Senataxin, a helicase protein associated with AOA2/ALS4 neurodegenerative disorders, acts to resolve these R-loop structures and by so doing allows access of the 5′–3′ exonuclease Xrn2 at 3′ cleavage poly(A) sites. This affords 3′ transcript degradation and consequent Pol II termination. In effect, R-loops formed over G-rich pause sites, followed by their resolution by senataxin, are key steps in the termination process.
  • Recombination Hotspots and Single-Stranded DNA Binding Proteins Couple DNA Translocation to DNA Unwinding by the AddAB Helicase-Nuclease
    - Mol Cell 42(6):806-816 (2011)
    AddAB is a helicase-nuclease that processes double-stranded DNA breaks for repair by homologous recombination. This process is modulated by Chi recombination hotspots: specific DNA sequences that attenuate the nuclease activity of the translocating AddAB complex to promote downstream recombination. Using a combination of kinetic and imaging techniques, we show that AddAB translocation is not coupled to DNA unwinding in the absence of single-stranded DNA binding proteins because nascent single-stranded DNA immediately re-anneals behind the moving enzyme. However, recognition of recombination hotspot sequences during translocation activates unwinding by coupling these activities, thereby ensuring the downstream formation of single-stranded DNA that is required for RecA-mediated recombinational repair. In addition to their implications for the mechanism of double-stranded DNA break repair, these observations may affect our implementation and interpretation of helicase ass! ays and our understanding of helicase mechanisms in general.
  • NanoRNAs Prime Transcription Initiation In Vivo
    - Mol Cell 42(6):817-825 (2011)
    It is often presumed that, in vivo, the initiation of RNA synthesis by DNA-dependent RNA polymerases occurs using NTPs alone. Here, using the model Gram-negative bacterium Pseudomonas aeruginosa, we demonstrate that depletion of the small-RNA-specific exonuclease, Oligoribonuclease, causes the accumulation of oligoribonucleotides 2 to 4 nt in length, "nanoRNAs," which serve as primers for transcription initiation at a significant fraction of promoters. Widespread use of nanoRNAs to prime transcription initiation is coupled with global alterations in gene expression. Our results, obtained under conditions in which the concentration of nanoRNAs is artificially elevated, establish that small RNAs can be used to initiate transcription in vivo, challenging the idea that all cellular transcription occurs using only NTPs. Our findings further suggest that nanoRNAs could represent a distinct class of functional small RNAs that can affect gene expression through direct inco! rporation into a target RNA transcript rather than through a traditional antisense-based mechanism.
  • Integrated Approaches Reveal Determinants of Genome-wide Binding and Function of the Transcription Factor Pho4
    - Mol Cell 42(6):826-836 (2011)
    DNA sequences with high affinity for transcription factors occur more frequently in the genome than instances of genes bound or regulated by these factors. It is not clear what factors determine the genome-wide pattern of binding or regulation for a given transcription factor. We used an integrated approach to study how trans influences shape the binding and regulatory landscape of Pho4, a budding yeast transcription factor activated in response to phosphate limitation. We find that nucleosomes significantly restrict Pho4 binding. At nucleosome-depleted sites, competition from another transcription factor, Cbf1, determines Pho4 occupancy, raising the threshold for transcriptional activation in phosphate replete conditions and preventing Pho4 activation of genes outside the phosphate regulon during phosphate starvation. Pho4 binding is not sufficient for transcriptional activation—a cooperative interaction between Pho2 and Pho4 specifies genes that are activated. Comb! ining these experimental observations, we are able to globally predict Pho4 binding and its functionality.
  • The Polycomb Group Mutant esc Leads to Augmented Levels of Paused Pol II in the Drosophila Embryo
    - Mol Cell 42(6):837-844 (2011)
    Many developmental control genes contain paused RNA polymerase II (Pol II) and are thereby "poised" for rapid and synchronous activation in the early Drosophila embryo. Evidence is presented that Polycomb group (PcG) repressors can influence paused Pol II. ChIP-Seq and GRO-Seq assays were used to determine the genome-wide distributions of Pol II, H3K27me3, and H3K4me3 in extra sex combs (esc) mutant embryos. ESC is a key component of the Polycomb repressive complex 2 (PRC2), which mediates H3K27me3 modification. Enhanced Pol II occupancy is observed for thousands of genes in esc mutant embryos, including genes not directly regulated by PRC2. Thus, it would appear that silent genes lacking promoter-associated paused Pol II in wild-type embryos are converted into "poised" genes with paused Pol II in esc mutants. We suggest that this conversion of silent genes into poised genes might render differentiated cell types susceptible to switches in identity in PcG mutan! ts.

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