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Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2-7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45-MCM-GINS (CMG) helicases that are required for genome duplication.sup.1-4. It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes.sup.5,6. Here we show that, for daughter cells to sustain error-free DNA replication, their mother cells build up a nuclear pool of MCMs both by recycling chromatin-bound (parental) MCMs and by synthesizing new (nascent) MCMs. Although all MCMs can form pre-RCs, it is the parental pool that is inherently stable and preferentially matures into CMGs. By contrast, nascent MCM3-7 (but not MCM2) undergo rapid proteolysis in the cytoplasm, and their stabilization and nuclear translocation require interaction with minichromosome-maintenance complex-binding protein (MCMBP), a distant MCM paralogue.sup.7,8. By chaperoning nascent MCMs, MCMBP safeguards replicating genomes by increasing chromatin coverage with pre-RCs that do not participate on replication origins but adjust the pace of replisome movement to minimize errors during DNA replication. Consequently, although the paucity of pre-RCs in MCMBP-deficient cells does not alter DNA synthesis overall, it increases the speed and asymmetry of individual replisomes, which leads to DNA damage. The surplus of MCMs therefore increases the robustness of genome duplication by restraining the speed at which eukaryotic cells replicate their DNA. Alterations in physiological fork speed might thus explain why even a minor reduction in MCM levels destabilizes the genome and predisposes to increased incidence of tumour formation. Mother cells recycle parental MCMs and simultaneously synthesize nascent MCMs, both of which are inherited by daughter cells, in which the former are preferentially used to form active replisomes and the latter adjust the pace of replisome movement to minimize errors during DNA replication., Author(s): Hana Sedlackova [sup.1] , Maj-Britt Rask [sup.1] , Rajat Gupta [sup.2] , Chunaram Choudhary [sup.2] , Kumar Somyajit [sup.1] , Jiri Lukas [sup.1] Author Affiliations: (1) Protein Signaling Program, [...]

Molecular glue compounds induce protein-protein interactions that, in the context of a ubiquitin ligase, lead to protein degradation.sup.1. Unlike traditional enzyme inhibitors, these molecular glue degraders act substoichiometrically to catalyse the rapid depletion of previously inaccessible targets.sup.2. They are clinically effective and highly sought-after, but have thus far only been discovered serendipitously. Here, through systematically mining databases for correlations between the cytotoxicity of 4,518 clinical and preclinical small molecules and the expression levels of E3 ligase components across hundreds of human cancer cell lines.sup.3-5, we identify CR8--a cyclin-dependent kinase (CDK) inhibitor.sup.6--as a compound that acts as a molecular glue degrader. The CDK-bound form of CR8 has a solvent-exposed pyridyl moiety that induces the formation of a complex between CDK12-cyclin K and the CUL4 adaptor protein DDB1, bypassing the requirement for a substrate receptor and presenting cyclin K for ubiquitination and degradation. Our studies demonstrate that chemical alteration of surface-exposed moieties can confer gain-of-function glue properties to an inhibitor, and we propose this as a broader strategy through which target-binding molecules could be converted into molecular glues. The cyclin-dependent kinase inhibitor CR8 acts as a molecular glue compound by inducing the formation of a complex between CDK12-cyclin K and DDB1, which results in the ubiquitination and degradation of cyclin K., Author(s): Mikolaj Slabicki [sup.1] [sup.2] [sup.3] , Zuzanna Kozicka [sup.4] [sup.5] , Georg Petzold [sup.4] , Yen-Der Li [sup.1] [sup.2] [sup.6] , Manisha Manojkumar [sup.1] [sup.2] [sup.3] , Richard D. [...]

Reprogramming of mitochondria provides cells with the metabolic flexibility required to adapt to various developmental transitions such as stem cell activation or immune cell reprogramming, and to respond to environmental challenges such as those encountered under hypoxic conditions or during tumorigenesis.sup.1-3. Here we show that the i-AAA protease YME1L rewires the proteome of pre-existing mitochondria in response to hypoxia or nutrient starvation. Inhibition of mTORC1 induces a lipid signalling cascade via the phosphatidic acid phosphatase LIPIN1, which decreases phosphatidylethanolamine levels in mitochondrial membranes and promotes proteolysis. YME1L degrades mitochondrial protein translocases, lipid transfer proteins and metabolic enzymes to acutely limit mitochondrial biogenesis and support cell growth. YME1L-mediated mitochondrial reshaping supports the growth of pancreatic ductal adenocarcinoma (PDAC) cells as spheroids or xenografts. Similar changes to the mitochondrial proteome occur in the tumour tissues of patients with PDAC, suggesting that YME1L is relevant to the pathophysiology of these tumours. Our results identify the mTORC1-LIPIN1-YME1L axis as a post-translational regulator of mitochondrial proteostasis at the interface between metabolism and mitochondrial dynamics. Under conditions such as hypoxia or starvation, an mTORC1-lipid signalling pathway initiates mitochondrial proteolysis by YME1L., Author(s): Thomas MacVicar [sup.1] , Yohsuke Ohba [sup.1] , Hendrik Nolte [sup.1] [sup.2] , Fiona Carola Mayer [sup.1] , Takashi Tatsuta [sup.1] , Hans-Georg Sprenger [sup.1] [sup.2] , Barbara Lindner [...]

Although improvements in genetic analysis have greatly enhanced our understanding of the mechanisms behind pancreatitis, it continues to afflict many families for whom the hereditary factors remain unknown. Recent evaluation of a patient with a strong family history of pancreatitis prompted us to reexamine a large kindred originally reported over 50 years ago with an autosomal-dominant inheritance pattern of chronic pancreatitis, diabetes, and pancreatic adenocarcinoma. Whole-exome sequencing analysis identified a rare missense mutation in the gene encoding pancreas-specific protease elastase 3B (CELA3B) that cosegregates with disease. Studies of the mutant protein in vitro, in cell lines, and in CRISPR-Cas9-engineered mice indicate that this mutation causes translational upregulation of CELA3B, which, upon secretion and activation by trypsin, leads to uncontrolled proteolysis and recurrent pancreatitis. Although lesions in several other pancreatic proteases have been previously linked to hereditary pancreatitis, to our knowledge, this is the first known instance of a mutation in CELA3B and a defect in translational control contributing to this disease., Introduction Malfunction or injury to the pancreas, primarily by its own digestive enzymes, results in the inflammation and abdominal pain typical of acute pancreatitis. Repeated injury and inflammation lead to [...]

Diabetes strongly impacts protein metabolism, particularly in skeletal muscle. Insulin and IGF-1 enhance muscle protein synthesis through their receptors, but the relative roles of each in muscle proteostasis have not been fully elucidated. Using mice with muscle-specific deletion of the insulin receptor ([M-IR.sup.-/-] mice), the IGF-1 receptor (M-[IGF1R.sup.-/-] mice), or both (MIGIRKO mice), we assessed the relative contributions of IR and IGF1R signaling to muscle proteostasis. In differentiated muscle, IR expression predominated over IGF1R expression, and correspondingly, [M-IR.sup.-/-] mice displayed a moderate reduction in muscle mass whereas [M-IGF1R.sup.-/-] mice did not. However, these receptors serve complementary roles, such that double-knockout MIGIRKO mice displayed a marked reduction in muscle mass that was linked to increases in proteasomal and autophagy-lysosomal degradation, accompanied by a high- protein-turnover state. Combined muscle-specific deletion of FoxO1, FoxO3, and FoxO4 in MIGIRKO mice reversed increased autophagy and completely rescued muscle mass without changing proteasomal activity. These data indicate that signaling via IR is more important than IGF1R in controlling proteostasis in differentiated muscle. Nonetheless, the overlap of IR and IGF1R signaling is critical to the regulation of muscle protein turnover, and this regulation depends on suppression of FoxO-regulated, autophagy-mediated protein degradation., Introduction Muscle wasting and the associated decrease in muscle strength cause significant morbidity and predict risk of mortality in elderly individuals (1). A diverse range of insults including aging, immobility, [...]

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2010.07.031 Byline: Song-Hee Kim, Hyung-Jun Kim, Seonmi Kim, Jeongbin Yim Keywords: Cullin-associated and neddylation-dissociated 1 (Cand1); Cullin-RING ubiquitin ligases; Cullin3; Drosophila; Nedd8 Abbreviations: Cand1, Cullin-associated and neddylation-dissociated 1; Ci, Cubitus interruptus; CRLs, Cullin-RING ubiquitin ligases; CSN, COP9 signalosome; Cul3, Cullin3; Da-GAL4, daughterless-GAL4; DTS, dominant temperature-sensitive; HIB, hedgehog-induced BTB protein; rdx, roadkill; SCF, Skp1/Cullin1/F-box proteins Abstract: Cullin-RING ubiquitin ligases (CRLs), which comprise the largest class of E3 ligases, regulate diverse cellular processes by targeting numerous proteins. Conjugation of the ubiquitin-like protein Nedd8 with Cullin activates CRLs. Cullin-associated and neddylation-dissociated 1 (Cand1) is known to negatively regulate CRL activity by sequestering unneddylated Cullin1 (Cul1) in biochemical studies. However, genetic studies of Arabidopsis have shown that Cand1 is required for optimal CRL activity. To elucidate the regulation of CRLs by Cand1, we analyzed a Cand1 mutant in Drosophila. Loss of Cand1 causes accumulation of neddylated Cullin3 (Cul3) and stabilizes the Cul3 adaptor protein HIB. In addition, the Cand1 mutation stimulates protein degradation of Cubitus interruptus (Ci), suggesting that Cul3-RING ligase activity is enhanced by the loss of Cand1. However, the loss of Cand1 fails to repress the accumulation of Ci in Nedd8.sup.AN015 or CSN5.sup.null mutant clones. Although Cand1 is able to bind both Cul1 and Cul3, mutation of Cand1 suppresses only the accumulation of Cul3 induced by the dAPP-BP1 mutation defective in the neddylation pathway, and this effect is attenuated by inhibition of proteasome function. Furthermore, overexpression of Cand1 stabilizes the Cul3 protein when the neddylation pathway is partially suppressed. These data indicate that Cand1 stabilizes unneddylated Cul3 by preventing proteasomal degradation. Here, we propose that binding of Cand1 to unneddylated Cul3 causes a shift in the equilibrium away from the neddylation of Cul3 that is required for the degradation of substrate by CRLs, and protects unneddylated Cul3 from proteasomal degradation. Cand1 regulates Cul3-mediated E3 ligase activity not only by acting on the neddylation of Cul3, but also by controlling the stability of the adaptor protein and unneddylated Cul3. Article History: Received 11 February 2010; Revised 22 June 2010; Accepted 27 July 2010

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.09.055 Byline: Elizabeth M. Duncan (1), Tara L. Muratore-Schroeder (2), Richard G. Cook (4), Benjamin A. Garcia (2), Jeffrey Shabanowitz (2), Donald F. Hunt (2)(3), C. David Allis (1) Keywords: PROTEINS; STEMCELL; DNA Abstract: Chromatin undergoes developmentally-regulated structural and chemical changes as cells differentiate, which subsequently lead to differences in cellular function by altering patterns of gene expression. To gain insight into chromatin alterations that occur during mammalian differentiation, we turned to a mouse embryonic stem cell (ESC) model. Here we show that histone H3 is proteolytically cleaved at its N-terminus during ESC differentiation. We map the sites of H3 cleavage and identify Cathepsin L as a protease responsible for proteolytically processing the N-terminal H3 tail. In addition, our data suggest that H3 cleavage may be regulated by covalent modifications present on the histone tail itself. Our studies underscore the intriguing possibility that histone proteolysis, brought about by Cathepsin L and potentially other family members, plays a role in development and differentiation that was not previously recognized. Author Affiliation: (1) Laboratory of Chromatin Biology, The Rockefeller University, New York, NY 10065 (2) Department of Chemistry, University of Virginia, Charlottesville, VA 22904 (3) Department of Pathology, University of Virginia, Charlottesville, VA 22904 (4) Department of Immunology, Baylor College of Medicine, Houston, TX 77030 Article History: Received 19 June 2008; Revised 25 August 2008; Accepted 30 September 2008 Article Note: (miscellaneous) Published: October 16, 2008

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2008.07.032 Byline: Renate Konig (1), Yingyao Zhou (6), Daniel Elleder (2), Tracy L. Diamond (5), Ghislain M.C. Bonamy (6)(7), Jeffrey T. Irelan (6), Chih-yuan Chiang (6), Buu P. Tu (6), Paul D. De Jesus (1), Caroline E. Lilley (3), Shannon Seidel (2), Amanda M. Opaluch (1), Jeremy S. Caldwell (6), Matthew D. Weitzman (3), Kelli L. Kuhen (6), Sourav Bandyopadhyay (4), Trey Ideker (4), Anthony P. Orth (6), Loren J. Miraglia (6), Frederic D. Bushman (5), John A. Young (2), Sumit K. Chanda (1) Keywords: HUMDISEASE; CELLBIO; SYSBIO Abstract: Human Immunodeficiency Viruses (HIV-1 and HIV-2) rely upon host-encoded proteins to facilitate their replication. Here, we combined genome-wide siRNA analyses with interrogation of human interactome databases to assemble a host-pathogen biochemical network containing 213 confirmed host cellular factors and 11 HIV-1-encoded proteins. Protein complexes that regulate ubiquitin conjugation, proteolysis, DNA-damage response, and RNA splicing were identified as important modulators of early-stage HIV-1 infection. Additionally, over 40 new factors were shown to specifically influence the initiation and/or kinetics of HIV-1 DNA synthesis, including cytoskeletal regulatory proteins, modulators of posttranslational modification, and nucleic acid-binding proteins. Finally, 15 proteins with diverse functional roles, including nuclear transport, prostaglandin synthesis, ubiquitination, and transcription, were found to influence nuclear import or viral DNA integration. Taken together, the multiscale approach described here has uncovered multiprotein virus-host interactions that likely act in concert to facilitate the early steps of HIV-1 infection. Author Affiliation: (1) Infectious & Inflammatory Disease Center, Burnham Institute for Medical Research, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA (2) Infectious Disease Laboratory, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA (3) Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA (4) Department of Bioengineering, University of California, San Diego, La Jolla, CA 92037, USA (5) Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA (6) The Genomics Institute of the Novartis Research Foundation, 10675 John J. Hopkins Drive, San Diego, CA 92121, USA (7) Hudson-Alpha Institute for Biotechnology, 127 Holmes Avenue, Huntsville, AL 35801, USA Article History: Received 1 May 2008; Revised 12 June 2008; Accepted 18 July 2008 Article Note: (miscellaneous) Published: October 2, 2008