Hot off the presses! Oct 21 Mol Cell

The Oct 21 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:

• The Critical Discussion Group: Fostering Personal and Scientific Growth
  - Mol Cell 44(2):167-169 (2011)
  As scientists, we greatly benefit from discussing our work with our peers. Informal, unstructured interactions often yield highly creative feedback. With this in mind, we created a group that fosters discussion of its members' work. The group engages us in new research fields and ways of thinking, and provides us with an opportunity for co-mentoring. Three key components were essential for making this group work: the emphasis on non-hierarchical debate; the diversity of the group members; and the mutual respect existing among the participants.
• Mitochondrial SIRT3: A New Potential Therapeutic Target for Metabolic Syndrome
  - Mol Cell 44(2):170-171 (2011)
  In this issue of Molecular Cell, Hirschey et al. demonstrate that loss of the NAD+-dependent deacetylase SIRT3 and resultant mitochondrial protein hyperacetylation play a critical role in the pathogenesis of metabolic syndrome, providing new insights into the therapeutic potential of SIRT3.
• Sterols for Oxygen: The Metabolic Burden of Microbial SREBP
  - Mol Cell 44(2):172-174 (2011)
  In this issue of Molecular Cell, report a novel mechanism for oxygen-sensing in S. pombe, whereby the 2-OG-Fe(II) dioxygenase Ofd protein regulates both the DNA-binding activity and the degradation of the hypoxia regulated transcription factor, Sre1p.
• BRCA1 Forks Over New Roles in DNA-Damage Responseâ€" Before and Beyond the Breaks
  - Mol Cell 44(2):174-176 (2011)
  In this issue, Pathania et al. (2011) report the involvement of BRCA1 in ultraviolet (UV)-damage response at stalled replication forks, which extends the function of BRCA1 beyond its established role in the repair of DNA- double-strand breaks (DSBs), raising the complexity of how this tumor suppressor maintains genomic stability.
• SIRT3 Deficiency and Mitochondrial Protein Hyperacetylation Accelerate the Development of the Metabolic Syndrome
  - Mol Cell 44(2):177-190 (2011)
  Acetylation is increasingly recognized as an important metabolic regulatory posttranslational protein modification, yet the metabolic consequence of mitochondrial protein hyperacetylation is unknown. We find that high-fat diet (HFD) feeding induces hepatic mitochondrial protein hyperacetylation in mice and downregulation of the major mitochondrial protein deacetylase SIRT3. Mice lacking SIRT3 (SIRT3KO) placed on a HFD show accelerated obesity, insulin resistance, hyperlipidemia, and steatohepatitis compared to wild-type (WT) mice. The lipogenic enzyme stearoyl-CoA desaturase 1 is highly induced in SIRT3KO mice, and its deletion rescues both WT and SIRT3KO mice from HFD-induced hepatic steatosis and insulin resistance. We further identify a single nucleotide polymorphism in the human SIRT3 gene that is suggestive of a genetic association with the metabolic syndrome. This polymorphism encodes a point mutation in the SIRT3 protein, which reduces its overall enzymatic effi! ciency. Our findings show that loss of SIRT3 and dysregulation of mitochondrial protein acetylation contribute to the metabolic syndrome.
• A Pathway of Protein Translocation in Mitochondria Mediated by the AAA-ATPase Bcs1
  - Mol Cell 44(2):191-202 (2011)
  The AAA+ family in eukaryotes has many members in various cellular compartments with a role in protein unfolding and degradation. We show that the mitochondrial AAA-ATPase Bcs1 has an unusual function in protein translocation. Bcs1 mediates topogenesis of the Rieske protein, Rip1, a component of respiratory chains in bacteria, mitochondria, and chloroplasts. The oligomeric AAA-ATPase Bcs1 is involved in export of the folded Fe-S domain of Rip1 across the inner membrane and insertion of its transmembrane segment into an assembly intermediate of the cytochrome bc1 complex, thus revealing an unexpected mechanistical concept of protein translocation across membranes. Furthermore, we describe structural elements of Rip1 required for recognition and export by as well as ATP-dependent lateral release from the AAA-ATPase. In bacteria and chloroplasts Rip1 uses the Tat machinery for topogenesis; however, mitochondria have lost this machinery during evolution and a member of the! AAA-ATPase family has taken over its function.
• Peptide Switch Is Essential for Sirt1 Deacetylase Activity
  - Mol Cell 44(2):203-213 (2011)
  In mammals, the Sirtuins are composed of seven Sir2 orthologs (Sirt1–7) with a conserved deacetylase core that utilizes NAD+ as a cofactor. Interestingly, the deacetylase core of Sirt1 by itself has no catalytic activity. We found within the C-terminal domain a 25 aa sequence that is essential for Sirt1 activity (ESA). Our results indicate that the ESA region interacts with and functions as an "on switch" for the deacetylase core. The endogenous Sirt1 inhibitor DBC1, which also binds to the deacetylase core, competes with and inhibits the ESA region from interacting with the deacetylase core. We discovered an ESA mutant peptide that can bind to the deacetylase core and inhibit Sirt1 in trans. By using this mutant peptide, we were able to inhibit Sirt1 activity and to increase the chemosensitivity of androgen-refractory prostate cancer cells. Therefore, the ESA region is a potential target for development of therapies to regulate Sirt1.
• Structure and Dynamics of the Mammalian Ribosomal Pretranslocation Complex
  - Mol Cell 44(2):214-224 (2011)
  Although the structural core of the ribosome is conserved in all kingdoms of life, eukaryotic ribosomes are significantly larger and more complex than their bacterial counterparts. The extent to which these differences influence the molecular mechanism of translation remains elusive. Multiparticle cryo-electron microscopy and single-molecule FRET investigations of the mammalian pretranslocation complex reveal spontaneous, large-scale conformational changes, including an intersubunit rotation of the ribosomal subunits. Through structurally related processes, tRNA substrates oscillate between classical and at least two distinct hybrid configurations facilitated by localized changes in their L-shaped fold. Hybrid states are favored within the mammalian complex. However, classical tRNA positions can be restored by tRNA binding to the E site or by the eukaryotic-specific antibiotic and translocation inhibitor cycloheximide. These findings reveal critical distinctions in the! structural and energetic features of bacterial and mammalian ribosomes, providing a mechanistic basis for divergent translation regulation strategies and species-specific antibiotic action.
• Regulation of the Sre1 Hypoxic Transcription Factor by Oxygen-Dependent Control of DNA Binding
  - Mol Cell 44(2):225-234 (2011)
  Regulation of gene expression plays an integral role in adaptation of cells to hypoxic stress. In mammals, prolyl hydroxylases control levels of the central transcription factor hypoxia inducible factor (HIF) through regulation of HIFα subunit stability. Here, we report that the hydroxylase Ofd1 regulates the Sre1 hypoxic transcription factor in fission yeast by controlling DNA binding. Prolyl hydroxylases require oxygen as a substrate, and the activity of Ofd1 regulates Sre1-dependent transcription. In the presence of oxygen, Ofd1 binds the Sre1 N-terminal transcription factor domain (Sre1N) and inhibits Sre1-dependent transcription by blocking DNA binding. In the absence of oxygen, the inhibitor Nro1 binds Ofd1, thereby releasing Sre1N and leading to activation of genes required for hypoxic growth. In contrast to the HIF system, where proline hydroxylation is essential for regulation, Ofd1 inhibition of Sre1N does not require hydroxylation and, thus, defines a new m! echanism for hypoxic gene regulation.
• BRCA1 Is Required for Postreplication Repair after UV-Induced DNA Damage
  - Mol Cell 44(2):235-251 (2011)
  BRCA1 contributes to the response to UV irradiation. Utilizing its BRCT motifs, it is recruited during S/G2 to UV-damaged sites in a DNA replication-dependent but nucleotide excision repair (NER)-independent manner. More specifically, at UV-stalled replication forks, it promotes photoproduct excision, suppression of translesion synthesis, and the localization and activation of replication factor C complex (RFC) subunits. The last function, in turn, triggers post-UV checkpoint activation and postreplicative repair. These BRCA1 functions differ from those required for DSBR.
• The Three-Dimensional Architecture of a Bacterial Genome and Its Alteration by Genetic Perturbation
  - Mol Cell 44(2):252-264 (2011)
  We have determined the three-dimensional (3D) architecture of the Caulobacter crescentus genome by combining genome-wide chromatin interaction detection, live-cell imaging, and computational modeling. Using chromosome conformation capture carbon copy (5C), we derive ∼13 kb resolution 3D models of the Caulobacter genome. The resulting models illustrate that the genome is ellipsoidal with periodically arranged arms. The parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Repositioning these elements resulted in rotations of the chromosome that changed the subcellular positions of most genes. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Collectively, our data suggest that genome fo! lding is globally dictated by the parS sites and chromosome segregation.
• Granzyme B-Dependent Proteolysis Acts as a Switch to Enhance the Proinflammatory Activity of IL-1α
  - Mol Cell 44(2):265-278 (2011)
  Granzyme B is a cytotoxic lymphocyte-derived protease that plays a central role in promoting apoptosis of virus-infected target cells, through direct proteolysis and activation of constituents of the cell death machinery. However, previous studies have also implicated granzymes A and B in the production of proinflammatory cytokines, via a mechanism that remains undefined. Here we show that IL-1α is a substrate for granzyme B and that proteolysis potently enhanced the biological activity of this cytokine in vitro as well as in vivo. Consistent with this, compared with full-length IL-1α, granzyme B-processed IL-1α exhibited more potent activity as an immunoadjuvant in vivo. Furthermore, proteolysis of IL-1α within the same region, by proteases such as calpain and elastase, was also found to enhance its biological potency. Thus, IL-1α processing by multiple immune-related proteases, including granzyme B, acts as a switch to enhance the proinflammatory prope! rties of this cytokine.
• Serine 403 Phosphorylation of p62/SQSTM1 Regulates Selective Autophagic Clearance of Ubiquitinated Proteins
  - Mol Cell 44(2):279-289 (2011)
  Selective macroautophagy (autophagy) of ubiquitinated protein is implicated as a compensatory mechanism of the ubiquitin-proteasome system. p62/SQSTM1 is a key molecule managing autophagic clearance of polyubiquitinated proteins. However, little is known about mechanisms controlling autophagic degradation of polyubiquitinated proteins. Here, we show that the specific phosphorylation of p62 at serine 403 (S403) in its ubiquitin-associated (UBA) domain increases the affinity between UBA and polyubiquitin chain, resulting in efficiently targeting polyubiquitinated proteins in "sequestosomes" and stabilizing sequestosome structure as a cargo of ubiquitinated proteins for autophagosome entry. Casein kinase 2 (CK2) phosphorylates S403 of p62 directly. Furthermore, CK2 overexpression or phosphatase inhibition reduces the formation of inclusion bodies of the polyglutamine-expanded huntingtin exon1 fragment in a p62-dependent manner. We propose that phosphorylation of p62 a! t S403 regulates autophagic clearance of ubiquitinated proteins and protein aggregates that are poorly degraded by proteasomes.
• mTOR Drives Its Own Activation via SCFβTrCP-Dependent Degradation of the mTOR Inhibitor DEPTOR
  - Mol Cell 44(2):290-303 (2011)
  The activities of both mTORC1 and mTORC2 are negatively regulated by their endogenous inhibitor, DEPTOR. As such, the abundance of DEPTOR is a critical determinant in the activity status of the mTOR network. DEPTOR stability is governed by the 26S-proteasome through a largely unknown mechanism. Here we describe an mTOR-dependent phosphorylation-driven pathway for DEPTOR destruction via SCFβTrCP. DEPTOR phosphorylation by mTOR in response to growth signals, and in collaboration with casein kinase I (CKI), generates a phosphodegron that binds βTrCP. Failure to degrade DEPTOR through either degron mutation or βTrCP depletion leads to reduced mTOR activity, reduced S6 kinase activity, and activation of autophagy to reduce cell growth. This work expands the current understanding of mTOR regulation by revealing a positive feedback loop involving mTOR and CKI-dependent turnover of its inhibitor, DEPTOR, suggesting that misregulation of the DEPTOR destruction pathway ! might contribute to aberrant activation of mTOR in disease.
• DEPTOR, an mTOR Inhibitor, Is a Physiological Substrate of SCFβTrCP E3 Ubiquitin Ligase and Regulates Survival and Autophagy
  - Mol Cell 44(2):304-316 (2011)
  DEPTOR, an inhibitor of mTORC1 and mTORC2, is degraded via ubiquitin-proteasome pathway by an unknown E3 ubiquitin ligase. Here we report that DEPTOR is a physiological substrate of SCFβTrCP E3 ligase for targeted degradation. Upon growth factor stimulation, RSK1 and S6K1 kinases are activated to phosphorylate DEPTOR, which is then recognized by the F box protein, βTrCP, via its degron sequence for subsequent ubiquitination and degradation by SCF E3. Endogenous DEPTOR levels are negatively regulated by βTrCP. DEPTOR half-life is shortened by βTrCP but extended by a dominant-negative mutant of βTrCP, by RSK1/S6K1 inhibition, and by βTrCP degron site mutations. Biologically, DEPTOR accumulation upon βTrCP knockdown inactivates mTORC1 and activates AKT in cancer cells to confer resistance to rapamycin and paclitaxel. Furthermore, DEPTOR accumulates upon glucose deprivation and mTOR inhibition to induce autophagy. Thus, βTrCP-DEPTOR-mTOR intertwine to regulate cell! survival and autophagy.
• mTOR Generates an Auto-Amplification Loop by Triggering the βTrCP- and CK1α-Dependent Degradation of DEPTOR
  - Mol Cell 44(2):317-324 (2011)
  DEPTOR is a recently identified inhibitor of the mTOR kinase that is highly regulated at the posttranslational level. In response to mitogens, we found that DEPTOR was rapidly phosphorylated on three serines in a conserved degron, facilitating binding and ubiquitylation by the F box protein βTrCP, with consequent proteasomal degradation of DEPTOR. Phosphorylation of the βTrCP degron in DEPTOR is executed by CK1α after a priming phosphorylation event mediated by either the mTORC1 or mTORC2 complexes. Blocking the βTrCP-dependent degradation of DEPTOR via βTrCP knockdown or expression of a stable DEPTOR mutant that is unable to bind βTrCP results in mTOR inhibition. Our findings reveal that mTOR cooperates with CK1α and βTrCP to generate an auto-amplification loop to promote its own full activation. Moreover, our results suggest that pharmacologic inhibition of CK1 may be a viable therapeutic option for the treatment of cancers characterized by ac! tivation of mTOR-signaling pathways.
• Systematic and Quantitative Assessment of the Ubiquitin-Modified Proteome
  - Mol Cell 44(2):325-340 (2011)
  Despite the diverse biological pathways known to be regulated by ubiquitylation, global identification of substrates that are targeted for ubiquitylation has remained a challenge. To globally characterize the human ubiquitin-modified proteome (ubiquitinome), we utilized a monoclonal antibody that recognizes diglycine (diGly)-containing isopeptides following trypsin digestion. We identify ∼19,000 diGly-modified lysine residues within ∼5000 proteins. Using quantitative proteomics we monitored temporal changes in diGly site abundance in response to both proteasomal and translational inhibition, indicating both a dependence on ongoing translation to observe alterations in site abundance and distinct dynamics of individual modified lysines in response to proteasome inhibition. Further, we demonstrate that quantitative diGly proteomics can be utilized to identify substrates for cullin-RING ubiquitin ligases. Interrogation of the ubiquitinome allows for not only a quantit! ative assessment of alterations in protein homeostasis fidelity, but also identification of substrates for individual ubiquitin pathway enzymes.