Publications by authors named "Raymond J Deshaies"

108 Publications

In-depth proteomic analysis of proteasome inhibitors bortezomib, carfilzomib and MG132 reveals that mortality factor 4-like 1 (MORF4L1) protein ubiquitylation is negatively impacted.

J Proteomics 2021 06 10;241:104197. Epub 2021 Apr 10.

Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, 1200 E. California Blvd, Pasadena, CA 91125, USA. Electronic address:

Proteasome inhibitors are an important class of chemotherapeutic drugs. In this study, we performed a large-scale ubiquitylome analysis of the three proteasome inhibitors MG132, bortezomib and carfilzomib. Although carfilzomib is currently being used for the treatment of multiple myeloma, it has not yet been subjected to a global ubiquitylome analysis. In this study, we identified more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. We introduced stringent criteria to determine the correct ubiquitylation site ratios and used five biological replicates to achieve increased statistical power. With the vast amount of data acquired, we made proteome-wide comparisons between the proteasome inhibitors and indicate candidate proteins that will benefit from further study. We find that in addition to the expected increase in ubiquitylation in the majority of proteins, unexpectedly a select few are specifically and significantly decreased in ubiquitylation at specific sites after treatment with proteasome inhibitors. We chose to follow-up on Mortality factor 4-like 1 (MORF4L1), which was significantly decreased in ubiquitylation at lysine 187 and lysine 104 upon proteasome inhibition, but increased in protein abundance by approximately two-fold. We demonstrate that the endogenous protein level of MORF4L1 is highly regulated by the ubiquitin proteasome system. SIGNIFICANCE: This study provides a highly curated dataset of more than 14,000 unique sites of ubiquitylation in more than 4400 protein groups. For the proper quantification of ubiquitylation sites, we introduced a higher standard by quantifying only those ubiquitylation sites that are not flanked by neighboring ubiquitylation, thereby avoiding the report of incorrect ratios. The sites identified will serve to identify important targets of the ubiquitin proteasome system and aid to better understand the repertoire of proteins that are affected by inhibiting the proteasome with MG132, bortezomib, and carfilzomib. In addition, we investigated the unusual observation that ubiquitylation of the tumor suppressor Mortality factor 4-like (MORF4L1) protein decreases rather than increases upon proteasome inhibition, which may contribute to an additional anti-tumor effect of bortezomib and carfilzomib.
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http://dx.doi.org/10.1016/j.jprot.2021.104197DOI Listing
June 2021

A covalent p97/VCP ATPase inhibitor can overcome resistance to CB-5083 and NMS-873 in colorectal cancer cells.

Eur J Med Chem 2021 Mar 2;213:113148. Epub 2021 Jan 2.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA, 91125, United States; Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology, Pasadena, CA, 91125, United States. Electronic address:

Small-molecule inhibitors of p97 are useful tools to study p97 function. Human p97 is an important AAA ATPase due to its diverse cellular functions and implication in mediating the turnover of proteins involved in tumorigenesis and virus infections. Multiple p97 inhibitors identified from previous high-throughput screening studies are thiol-reactive compounds targeting Cys522 in the D2 ATP-binding domain. Thus, these findings suggest a potential strategy to develop covalent p97 inhibitors. We first used purified p97 to assay several known covalent kinase inhibitors to determine if they can inhibit ATPase activity. We evaluated their selectivity using our dual reporter cells that can distinguish p97 dependent and independent degradation. We selected a β-nitrostyrene scaffold to further study the structure-activity relationship. In addition, we used p97 structures to design and synthesize analogues of pyrazolo[3,4-d]pyrimidine (PP). We incorporated electrophiles into a PP-like compound 17 (4-amino-1-tert-butyl-3-phenyl pyrazolo[3,4-d]pyrimidine) to generate eight compounds. A selective compound 18 (N-(1-(tert-butyl)-3-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)acrylamide, PPA) exhibited excellent selectivity in an in vitro ATPase activity assay: IC of 0.6 μM, 300 μM, and 100 μM for wild type p97, yeast Cdc48, and N-ethylmaleimide sensitive factor (NSF), respectively. To further examine the importance of Cys522 on the active site pocket during PPA inhibition, C522A and C522T mutants of p97 were purified and shown to increase IC values by 100-fold, whereas replacement of Thr532 of yeast Cdc48 with Cysteine decreased the IC by 10-fold. The molecular modeling suggested the hydrogen bonds and hydrophobic interactions in addition to the covalent bonding at Cys522 between WT-p97 and PPA. Furthermore, tandem mass spectrometry confirmed formation of a covalent bond between Cys522 and PPA. An anti-proliferation assay indicated that the proliferation of HCT116, HeLa, and RPMI8226 was inhibited by PPA with IC of 2.7 μM, 6.1 μM, and 3.4 μM, respectively. In addition, PPA is able to inhibit proliferation of two HCT116 cell lines that are resistant to CB-5083 and NMS-873, respectively. Proteomic analysis of PPA-treated HCT116 revealed Gene Ontology enrichment of known p97 functional pathways such as the protein ubiquitination and the ER to Golgi transport vesicle membrane. In conclusion, we have identified and characterized PPA as a selective covalent p97 inhibitor, which will allow future exploration to improve the potency of p97 inhibitors with different mechanisms of action.
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http://dx.doi.org/10.1016/j.ejmech.2020.113148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954469PMC
March 2021

Ubiquitin-dependent proteasomal degradation of AMPK gamma subunit by Cereblon inhibits AMPK activity.

Biochim Biophys Acta Mol Cell Res 2020 08 22;1867(8):118729. Epub 2020 Apr 22.

Department of Life Science and Environmental Biochemistry, Pusan National University, Miryang 50463, Republic of Korea. Electronic address:

Cereblon (CRBN), a substrate receptor for Cullin-ring E3 ubiquitin ligase (CRL), is a major target protein of immunomodulatory drugs. An earlier study demonstrated that CRBN directly interacts with the catalytic α subunit of AMP-activated protein kinase (AMPK), a master regulator of energy homeostasis, down-regulating the enzymatic activity of AMPK. However, it is not clear how CRBN modulates AMPK activity. To investigate the mechanism of CRBN-dependent AMPK inhibition, we measured protein levels of each AMPK subunit in brains, livers, lungs, hearts, spleens, skeletal muscles, testes, kidneys, and embryonic fibroblasts from wild-type and Crbn mice. Protein levels and stability of the regulatory AMPKγ subunit were increased in Crbn mice. Increased stability of AMPKγ in Crbn MEFs was dramatically reduced by exogenous expression of Crbn. In wild-type MEFs, the proteasomal inhibitor MG132 blocked degradation of AMPKγ. We also found that CRL4 directly ubiquitinated AMPKγ. Taken together, these findings suggest that CRL4 regulates AMPK through ubiquitin-dependent proteasomal degradation of AMPKγ.
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http://dx.doi.org/10.1016/j.bbamcr.2020.118729DOI Listing
August 2020

Multispecific drugs herald a new era of biopharmaceutical innovation.

Nature 2020 04 15;580(7803):329-338. Epub 2020 Apr 15.

Amgen Research, Thousand Oaks, CA, USA.

The modern biopharmaceutical industry traces its roots to the dawn of the twentieth century, coincident with marketing of aspirin-a signature event in the history of modern drug development. Although the archetypal discovery process did not change markedly in the first seven decades of the industry, the past fifty years have seen two successive waves of transformative innovation in the development of drug molecules: the rise of 'rational drug discovery' methodology in the 1970s, followed by the invention of recombinant protein-based therapeutic agents in the 1980s. An incipient fourth wave is the advent of multispecific drugs. The successful development of prospectively designed multispecific drugs has the potential to reconfigure our ideas of how target-based therapeutic molecules can work, and what it is possible to achieve with them. Here I review the two major classes of multispecific drugs: those that enrich a therapeutic agent at a particular site of action and those that link a therapeutic target to a biological effector. The latter class-being freed from the constraint of having to directly modulate the target upon binding-may enable access to components of the proteome that currently cannot be targeted by drugs.
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http://dx.doi.org/10.1038/s41586-020-2168-1DOI Listing
April 2020

Harnessing the Power of Proteolysis for Targeted Protein Inactivation.

Mol Cell 2020 02 30;77(3):446-460. Epub 2020 Jan 30.

AMGEN Research, One Amgen Center Drive, Thousand Oaks, CA 91320, USA.

Two decades into the twenty-first century, a confluence of breakthrough technologies wielded at the molecular level is presenting biologists with unique opportunities to unravel the complexities of the cellular world. CRISPR/Cas9 allows gene knock-outs, knock-ins, and single-base editing at chromosomal loci. RNA-based tools such as siRNA, antisense oligos, and morpholinos can be used to silence expression of specific genes. Meanwhile, protein knockdown tools that draw inspiration from natural regulatory mechanisms and facilitate elimination of native or degron-tagged proteins from cells are rapidly emerging. The acute and reversible reduction in protein levels enabled by these methods allows for precise determination of loss-of-function phenotypes free from secondary effects or compensatory adaptation that can confound nucleic-acid-based methods that involve slow depletion or permanent loss of a protein. In this Review, we summarize the ingenious ways biologists have exploited natural mechanisms for protein degradation to direct the elimination of specific proteins at will. This has led to advancements not only in basic research but also in the therapeutic space with the introduction of PROTACs into clinical trials for cancer patients.
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http://dx.doi.org/10.1016/j.molcel.2020.01.010DOI Listing
February 2020

PIKES Analysis Reveals Response to Degraders and Key Regulatory Mechanisms of the CRL4 Network.

Mol Cell 2020 03 20;77(5):1092-1106.e9. Epub 2020 Jan 20.

Genentech, 1 DNA Way, South San Francisco, 94080 CA, USA. Electronic address:

Co-opting Cullin4 RING ubiquitin ligases (CRL4s) to inducibly degrade pathogenic proteins is emerging as a promising therapeutic strategy. Despite intense efforts to rationally design degrader molecules that co-opt CRL4s, much about the organization and regulation of these ligases remains elusive. Here, we establish protein interaction kinetics and estimation of stoichiometries (PIKES) analysis, a systematic proteomic profiling platform that integrates cellular engineering, affinity purification, chemical stabilization, and quantitative mass spectrometry to investigate the dynamics of interchangeable multiprotein complexes. Using PIKES, we show that ligase assemblies of Cullin4 with individual substrate receptors differ in abundance by up to 200-fold and that Cand1/2 act as substrate receptor exchange factors. Furthermore, degrader molecules can induce the assembly of their cognate CRL4, and higher expression of the associated substrate receptor enhances degrader potency. Beyond the CRL4 network, we show how PIKES can reveal systems level biochemistry for cellular protein networks important to drug development.
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http://dx.doi.org/10.1016/j.molcel.2019.12.013DOI Listing
March 2020

Assembly and Regulation of CRL Ubiquitin Ligases.

Adv Exp Med Biol 2020 ;1217:33-46

Department of Biochemistry, Purdue University, West Lafayette, IN, USA.

Cullin-RING ubiquitin ligases (CRLs) determine the substrate specificity of ubiquitination reactions, and substrates are recruited to the cullin core through binding to their cognate substrate receptor modules. Because a family of substrate receptors compete for the same cullin core, the assembly and activity of CRLs are dynamically regulated to fulfill the needs of the cell to adapt to the changing pool of proteins demanding ubiquitination. Cullins are modified by NEDD8, a ubiquitin-like protein. This process, referred to as neddylation, promotes the E3 activity of CRLs by inducing conformational rearrangement in the Cullin-RING catalytic core. Cand1 is a cullin-associated protein whose binding is excluded by cullin neddylation. Although early biochemical studies suggested that Cand1 inhibits CRL activity, genetic studies revealed its positive role in ubiquitination. Emerging evidence from kinetic and quantitative proteomic studies demonstrated that Cand1 stimulates assembly of new Skp1-Cul1-F-box protein (SCF) complexes by exchanging the Skp1-F-box protein substrate receptor modules. Furthermore, aided by refined experimental design as well as computational simulation, an attractive model has been developed in which substrate, neddylation cycle and Cand1-mediated "adaptive exchange" collaborate to maintain the dynamics of the cellular SCF repertoire. Here, we review and discuss recent advances that have deepened our understanding of CRL regulation.
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http://dx.doi.org/10.1007/978-981-15-1025-0_3DOI Listing
February 2020

Multisystem Proteinopathy Mutations in VCP/p97 Increase NPLOC4·UFD1L Binding and Substrate Processing.

Structure 2019 12 14;27(12):1820-1829.e4. Epub 2019 Oct 14.

Department of Molecular & Cell Biology, University of California, Berkeley, CA 94720, USA; Howard Hughes Medical Institute, University of California, Berkeley, CA 94720, USA; California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, CA 94720, USA. Electronic address:

Valosin-containing protein (VCP)/p97 is an essential ATP-dependent protein unfoldase. Dominant mutations in p97 cause multisystem proteinopathy (MSP), a disease affecting the brain, muscle, and bone. Despite the identification of numerous pathways that are perturbed in MSP, the molecular-level defects of these p97 mutants are not completely understood. Here, we use biochemistry and cryoelectron microscopy to explore the effects of MSP mutations on the unfoldase activity of p97 in complex with its substrate adaptor NPLOC4⋅UFD1L (UN). We show that all seven analyzed MSP mutants unfold substrates faster. Mutant homo- and heterohexamers exhibit tighter UN binding and faster substrate processing. Our structural studies suggest that the increased UN affinity originates from a decoupling of p97's nucleotide state and the positioning of its N-terminal domains. Together, our data support a gain-of-function model for p97-UN-dependent processes in MSP and underscore the importance of N-terminal domain movements for adaptor recruitment and substrate processing by p97.
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http://dx.doi.org/10.1016/j.str.2019.09.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6929323PMC
December 2019

Epidithiodiketopiperazines Inhibit Protein Degradation by Targeting Proteasome Deubiquitinase Rpn11.

Cell Chem Biol 2018 11 23;25(11):1350-1358.e9. Epub 2018 Aug 23.

Division of Biology and Biological Engineering, California Institute of Technology, Box 114-96, Pasadena, CA 91125, USA; Howard Hughes Medical Institute, Chevy Chase, MD 26335, USA; Amgen Discovery Research, One Amgen Center Drive MS 29-M-B, Thousand Oaks, CA 91320, USA. Electronic address:

The 26S proteasome is the major proteolytic machine for breaking down cytosolic and nuclear proteins in eukaryotes. Due to the lack of a suitable assay, it is difficult to measure routinely and quantitatively the breakdown of proteins by the 26S proteasome in vitro. In the present study, we developed an assay to monitor proteasome-mediated protein degradation. Using this assay, we discovered that epidithiodiketopiperazine (ETPs) blocked the degradation of our model substrate in vitro. Further characterization revealed that ETPs inhibited proteasome function by targeting the essential proteasomal deubiquitinase Rpn11 (POH1/PSMD14). ETPs also inhibited other JAMM (JAB1/MPN/Mov34 metalloenzyme) proteases such as Csn5 and AMSH. An improved ETP with fewer non-specific effects, SOP11, stabilized a subset of proteasome substrates in cells, induced the unfolded protein response, and led to cell death. SOP11 represents a class of Rpn11 inhibitor and provides an alternative route to develop proteasome inhibitors.
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http://dx.doi.org/10.1016/j.chembiol.2018.07.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6309308PMC
November 2018

Increased proteasomal activity supports photoreceptor survival in inherited retinal degeneration.

Nat Commun 2018 04 30;9(1):1738. Epub 2018 Apr 30.

Department of Ophthalmology, Duke University School of Medicine, Durham, NC, 27510, USA.

Inherited retinal degenerations, affecting more than 2 million people worldwide, are caused by mutations in over 200 genes. This suggests that the most efficient therapeutic strategies would be mutation independent, i.e., targeting common pathological conditions arising from many disease-causing mutations. Previous studies revealed that one such condition is an insufficiency of the ubiquitin-proteasome system to process misfolded or mistargeted proteins in affected photoreceptor cells. We now report that retinal degeneration in mice can be significantly delayed by increasing photoreceptor proteasomal activity. The largest effect is observed upon overexpression of the 11S proteasome cap subunit, PA28α, which enhanced ubiquitin-independent protein degradation in photoreceptors. Applying this strategy to mice bearing one copy of the P23H rhodopsin mutant, a mutation frequently encountered in human patients, quadruples the number of surviving photoreceptors in the inferior retina of 6-month-old mice. This striking therapeutic effect demonstrates that proteasomes are an attractive target for fighting inherited blindness.
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http://dx.doi.org/10.1038/s41467-018-04117-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5928105PMC
April 2018

Vms1 and ANKZF1 peptidyl-tRNA hydrolases release nascent chains from stalled ribosomes.

Nature 2018 05 9;557(7705):446-451. Epub 2018 Apr 9.

Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, USA.

Ribosomal surveillance pathways scan for ribosomes that are transiently paused or terminally stalled owing to structural elements in mRNAs or nascent chain sequences. Some stalls in budding yeast are sensed by the GTPase Hbs1, which loads Dom34, a catalytically inactive member of the archaeo-eukaryotic release factor 1 superfamily. Hbs1-Dom34 and the ATPase Rli1 dissociate stalled ribosomes into 40S and 60S subunits. However, the 60S subunits retain the peptidyl-tRNA nascent chains, which recruit the ribosome quality control complex that consists of Rqc1-Rqc2-Ltn1-Cdc48-Ufd1-Npl4. Nascent chains ubiquitylated by the E3 ubiquitin ligase Ltn1 are extracted from the 60S subunit by the ATPase Cdc48-Ufd1-Npl4 and presented to the 26S proteasome for degradation. Failure to degrade the nascent chains leads to protein aggregation and proteotoxic stress in yeast and neurodegeneration in mice. Despite intensive investigations on the ribosome quality control pathway, it is not known how the tRNA is hydrolysed from the ubiquitylated nascent chain before its degradation. Here we show that the Cdc48 adaptor Vms1 is a peptidyl-tRNA hydrolase. Similar to classical eukaryotic release factor 1, Vms1 activity is dependent on a conserved catalytic glutamine. Evolutionary analysis indicates that yeast Vms1 is the founding member of a clade of eukaryotic release factor 1 homologues that we designate the Vms1-like release factor 1 clade.
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http://dx.doi.org/10.1038/s41586-018-0022-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226276PMC
May 2018

Cand1-Mediated Adaptive Exchange Mechanism Enables Variation in F-Box Protein Expression.

Mol Cell 2018 03;69(5):773-786.e6

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA; Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA; Amgen, One Amgen Center Way, Thousand Oaks, CA 91320, USA. Electronic address:

Skp1⋅Cul1⋅F-box (SCF) ubiquitin ligase assembly is regulated by the interplay of substrate binding, reversible Nedd8 conjugation on Cul1, and the F-box protein (FBP) exchange factors Cand1 and Cand2. Detailed investigations into SCF assembly and function in reconstituted systems and Cand1/2 knockout cells informed the development of a mathematical model for how dynamical assembly of SCF complexes is controlled and how this cycle is coupled to degradation of an SCF substrate. Simulations predicted an unanticipated hypersensitivity of Cand1/2-deficient cells to FBP expression levels, which was experimentally validated. Together, these and prior observations lead us to propose the adaptive exchange hypothesis, which posits that regulation of the k of an FBP from SCF by the actions of substrate, Nedd8, and Cand1 molds the cellular repertoire of SCF complexes and that the plasticity afforded by this exchange mechanism may enable large variations in FBP expression during development and in FBP gene number during evolution.
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http://dx.doi.org/10.1016/j.molcel.2018.01.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5836512PMC
March 2018

Alcohol-abuse drug disulfiram targets cancer via p97 segregase adaptor NPL4.

Nature 2017 12 6;552(7684):194-199. Epub 2017 Dec 6.

Danish Cancer Society Research Center, DK-2100 Copenhagen, Denmark.

Cancer incidence is rising and this global challenge is further exacerbated by tumour resistance to available medicines. A promising approach to meet the need for improved cancer treatment is drug repurposing. Here we highlight the potential for repurposing disulfiram (also known by the trade name Antabuse), an old alcohol-aversion drug that has been shown to be effective against diverse cancer types in preclinical studies. Our nationwide epidemiological study reveals that patients who continuously used disulfiram have a lower risk of death from cancer compared to those who stopped using the drug at their diagnosis. Moreover, we identify the ditiocarb-copper complex as the metabolite of disulfiram that is responsible for its anti-cancer effects, and provide methods to detect preferential accumulation of the complex in tumours and candidate biomarkers to analyse its effect on cells and tissues. Finally, our functional and biophysical analyses reveal the molecular target of disulfiram's tumour-suppressing effects as NPL4, an adaptor of p97 (also known as VCP) segregase, which is essential for the turnover of proteins involved in multiple regulatory and stress-response pathways in cells.
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http://dx.doi.org/10.1038/nature25016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5730499PMC
December 2017

Structural Basis for the Inhibitory Effects of Ubistatins in the Ubiquitin-Proteasome Pathway.

Structure 2017 12 16;25(12):1839-1855.e11. Epub 2017 Nov 16.

Department of Chemistry and Biochemistry, Center for Biomolecular Structure and Organization, University of Maryland, College Park, MD 20742, USA. Electronic address:

The discovery of ubistatins, small molecules that impair proteasomal degradation of proteins by directly binding to polyubiquitin, makes ubiquitin itself a potential therapeutic target. Although ubistatins have the potential for drug development and clinical applications, the lack of structural details of ubiquitin-ubistatin interactions has impeded their development. Here, we characterized a panel of new ubistatin derivatives using functional and binding assays. The structures of ubiquitin complexes with ubistatin B and hemi-ubistatin revealed direct interactions with ubiquitin's hydrophobic surface patch and the basic/polar residues surrounding it. Ubistatin B binds ubiquitin and diubiquitin tighter than a high-affinity ubiquitin receptor and shows strong preference for K48 linkages over K11 and K63. Furthermore, ubistatin B shields ubiquitin conjugates from disassembly by a range of deubiquitinases and by the 26S proteasome. Finally, ubistatin B penetrates cancer cells and alters the cellular ubiquitin landscape. These findings highlight versatile properties of ubistatins and have implications for their future development and use in targeting ubiquitin-signaling pathways.
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http://dx.doi.org/10.1016/j.str.2017.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5731780PMC
December 2017

Composition and Regulation of the Cellular Repertoire of SCF Ubiquitin Ligases.

Cell 2017 Nov 2;171(6):1326-1339.e14. Epub 2017 Nov 2.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA; Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125, USA. Electronic address:

SCF (Skp1-Cullin-F-box) ubiquitin ligases comprise several dozen modular enzymes that have diverse roles in biological regulation. SCF enzymes share a common catalytic core containing Cul1⋅Rbx1, which is directed toward different substrates by a variable substrate receptor (SR) module comprising 1 of 69 F-box proteins bound to Skp1. Despite the broad cellular impact of SCF enzymes, important questions remain about the architecture and regulation of the SCF repertoire, including whether SRs compete for Cul1 and, if so, how this competition is managed. Here, we devise methods that preserve the in vivo assemblages of SCF complexes and apply quantitative mass spectrometry to perform a census of these complexes (the "SCFome") in various states. We show that Nedd8 conjugation and the SR exchange factor Cand1 have a profound effect on shaping the SCFome. Together, these factors enable rapid remodeling of SCF complexes to promote biased assembly of SR modules bound to substrate.
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http://dx.doi.org/10.1016/j.cell.2017.10.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5711595PMC
November 2017

Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP•NPLOC4•UFD1L is enhanced by a mutation that causes multisystem proteinopathy.

Proc Natl Acad Sci U S A 2017 05 16;114(22):E4380-E4388. Epub 2017 May 16.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125;

p97 is a "segregase" that plays a key role in numerous ubiquitin (Ub)-dependent pathways such as ER-associated degradation. It has been hypothesized that p97 extracts proteins from membranes or macromolecular complexes to enable their proteasomal degradation; however, the complex nature of p97 substrates has made it difficult to directly observe the fundamental basis for this activity. To address this issue, we developed a soluble p97 substrate-Ub-GFP modified with K48-linked ubiquitin chains-for in vitro p97 activity assays. We demonstrate that WT p97 can unfold proteins and that this activity is dependent on the p97 adaptor NPLOC4-UFD1L, ATP hydrolysis, and substrate ubiquitination, with branched chains providing maximal stimulation. Furthermore, we show that a p97 mutant that causes inclusion body myopathy, Paget's disease of bone, and frontotemporal dementia in humans unfolds substrate faster, suggesting that excess activity may underlie pathogenesis. This work overcomes a significant barrier in the study of p97 and will allow the future dissection of p97 mechanism at a level of detail previously unattainable.
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http://dx.doi.org/10.1073/pnas.1706205114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5465906PMC
May 2017

Thiolutin is a zinc chelator that inhibits the Rpn11 and other JAMM metalloproteases.

Nat Chem Biol 2017 Jul 1;13(7):709-714. Epub 2017 May 1.

Heidelberg University Biochemistry Center, Heidelberg, Germany.

Thiolutin is a disulfide-containing antibiotic and anti-angiogenic compound produced by Streptomyces. Its biological targets are not known. We show that reduced thiolutin is a zinc chelator that inhibits the JAB1/MPN/Mov34 (JAMM) domain-containing metalloprotease Rpn11, a deubiquitinating enzyme of the 19S proteasome. Thiolutin also inhibits the JAMM metalloproteases Csn5, the deneddylase of the COP9 signalosome; AMSH, which regulates ubiquitin-dependent sorting of cell-surface receptors; and BRCC36, a K63-specific deubiquitinase of the BRCC36-containing isopeptidase complex and the BRCA1-BRCA2-containing complex. We provide evidence that other dithiolopyrrolones also function as inhibitors of JAMM metalloproteases.
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http://dx.doi.org/10.1038/nchembio.2370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792653PMC
July 2017

p97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates.

Proc Natl Acad Sci U S A 2017 04 20;114(14):3565-3571. Epub 2017 Mar 20.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125;

Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4 and p97 pathways.
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http://dx.doi.org/10.1073/pnas.1700949114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389304PMC
April 2017

Capzimin is a potent and specific inhibitor of proteasome isopeptidase Rpn11.

Nat Chem Biol 2017 05 28;13(5):486-493. Epub 2017 Feb 28.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California, USA.

The proteasome is a vital cellular machine that maintains protein homeostasis, which is of particular importance in multiple myeloma and possibly other cancers. Targeting of proteasome 20S peptidase activity with bortezomib and carfilzomib has been widely used to treat myeloma. However, not all patients respond to these compounds, and those who do eventually suffer relapse. Therefore, there is an urgent and unmet need to develop new drugs that target proteostasis through different mechanisms. We identified quinoline-8-thiol (8TQ) as a first-in-class inhibitor of the proteasome 19S subunit Rpn11. A derivative of 8TQ, capzimin, shows >5-fold selectivity for Rpn11 over the related JAMM proteases and >2 logs selectivity over several other metalloenzymes. Capzimin stabilized proteasome substrates, induced an unfolded protein response, and blocked proliferation of cancer cells, including those resistant to bortezomib. Proteomic analysis revealed that capzimin stabilized a subset of polyubiquitinated substrates. Identification of capzimin offers an alternative path to develop proteasome inhibitors for cancer therapy.
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http://dx.doi.org/10.1038/nchembio.2326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570473PMC
May 2017

Discovery of an Inhibitor of the Proteasome Subunit Rpn11.

J Med Chem 2017 02 13;60(4):1343-1361. Epub 2017 Feb 13.

Department of Chemistry and Biochemistry, University of California San Diego , La Jolla, California 92093, United States.

The proteasome plays a crucial role in degradation of normal proteins that happen to be constitutively or inducibly unstable, and in this capacity it plays a regulatory role. Additionally, it degrades abnormal/damaged/mutant/misfolded proteins, which serves a quality-control function. Inhibitors of the proteasome have been validated in the treatment of multiple myeloma, with several FDA-approved therapeutics. Rpn11 is a Zn-dependent metalloisopeptidase that hydrolyzes ubiquitin from tagged proteins that are trafficked to the proteasome for degradation. A fragment-based drug discovery (FBDD) approach was utilized to identify fragments with activity against Rpn11. Screening of a library of metal-binding pharmacophores (MBPs) revealed that 8-thioquinoline (8TQ, IC value ∼2.5 μM) displayed strong inhibition of Rpn11. Further synthetic elaboration of 8TQ yielded a small molecule compound (35, IC value ∼400 nM) that is a potent and selective inhibitor of Rpn11 that blocks proliferation of tumor cells in culture.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01379DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5761724PMC
February 2017

The pseudophosphatase STYX targets the F-box of FBXW7 and inhibits SCFFBXW7 function.

EMBO J 2017 02 22;36(3):260-273. Epub 2016 Dec 22.

Institute of Basic Medical Sciences, University of Oslo, Oslo, Norway

The F-box protein FBXW7 is the substrate-recruiting subunit of an SCF ubiquitin ligase and a major tumor-suppressor protein that is altered in several human malignancies. Loss of function of FBXW7 results in the stabilization of numerous proteins that orchestrate cell proliferation and survival. Little is known about proteins that directly regulate the function of this protein. In the current work, we have mapped the interactome of the enigmatic pseudophosphatase STYX We reasoned that a catalytically inactive phosphatase might have adopted novel mechanisms of action. The STYX interactome contained several F-box proteins, including FBXW7. We show that STYX binds to the F-box domain of FBXW7 and disables its recruitment into the SCF complex. Therefore, STYX acts as a direct inhibitor of FBXW7, affecting the cellular levels of its substrates. Furthermore, we find that levels of STYX and FBXW7 are anti-correlated in breast cancer patients, which affects disease prognosis. We propose the STYX-FBXW7 interaction as a promising drug target for future investigations.
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http://dx.doi.org/10.15252/embj.201694795DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286380PMC
February 2017

Nrf1 can be processed and activated in a proteasome-independent manner.

Curr Biol 2016 09;26(18):R834-R835

Department of Pathology, Virginia Commonwealth University, Richmond, VA 23298, USA. Electronic address:

In response to proteasome inhibition, the transcription factor Nrf1 facilitates de novo synthesis of proteasomes by inducing proteasome subunit (PSM) genes [1,2]. Previously, we showed that activation of the p120 form of Nrf1, a membrane-bound protein in the endoplasmic reticulum (ER) with the bulk of its polypeptide in the lumen, involves its retrotranslocation into the cytosol in a manner that depends on the AAA-ATPase p97/VCP [3]. This is followed by proteolytic processing and mobilization of the transcriptionally active p110 form of Nrf1 to the nucleus. A subsequent study suggested that site-specific proteolytic processing of Nrf1 by the proteasome yields an active 75 kDa fragment [4]. We show here that under conditions where all three active sites of the proteasome are completely blocked, p120 Nrf1 can still be proteolytically cleaved to the p110 form, which is translocated to the nucleus to activate transcription of PSM genes. Thus, our results indicate that a proteasome-independent pathway can promote the release of active p110 Nrf1 from the ER membrane.
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http://dx.doi.org/10.1016/j.cub.2016.08.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6156719PMC
September 2016

A conserved quality-control pathway that mediates degradation of unassembled ribosomal proteins.

Elife 2016 08 23;5. Epub 2016 Aug 23.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, United States.

Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes.
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http://dx.doi.org/10.7554/eLife.19105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026473PMC
August 2016

Valosin-containing protein (VCP)-Adaptor Interactions are Exceptionally Dynamic and Subject to Differential Modulation by a VCP Inhibitor.

Mol Cell Proteomics 2016 09 12;15(9):2970-86. Epub 2016 Jul 12.

From the ‡Division of Biology and Biological Engineering, California Institute of Technology, 1200 E. California Blvd, Pasadena, California 91125; ‡‡Howard Hughes Medical Institute, Pasadena, California 91125

Protein quality control (PQC) plays an important role in stemming neurodegenerative diseases and is essential for the growth of some cancers. Valosin-containing protein (VCP)/p97 plays a pivotal role in multiple PQC pathways by interacting with numerous adaptors that link VCP to specific PQC pathways and substrates and influence the post-translational modification state of substrates. However, our poor understanding of the specificity and architecture of the adaptors, and the dynamic properties of their interactions with VCP hinders our understanding of fundamental features of PQC and how modulation of VCP activity can best be exploited therapeutically. In this study we use multiple mass spectrometry-based proteomic approaches combined with biophysical studies to characterize the interaction of adaptors with VCP. Our results reveal that most VCP-adaptor interactions are characterized by rapid dynamics that in some cases are modulated by the VCP inhibitor NMS873. These findings have significant implications for both the regulation of VCP function and the impact of VCP inhibition on different VCP-adaptor complexes.
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http://dx.doi.org/10.1074/mcp.M116.061036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5013311PMC
September 2016

Ribosomal proteins produced in excess are degraded by the ubiquitin-proteasome system.

Mol Biol Cell 2016 09 6;27(17):2642-52. Epub 2016 Jul 6.

Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125 Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA 91125

Ribosome assembly is an essential process that consumes prodigious quantities of cellular resources. Ribosomal proteins cannot be overproduced in Saccharomyces cerevisiae because the excess proteins are rapidly degraded. However, the responsible quality control (QC) mechanisms remain poorly characterized. Here we demonstrate that overexpression of multiple proteins of the small and large yeast ribosomal subunits is suppressed. Rpl26 overexpressed from a plasmid can be detected in the nucleolus and nucleoplasm, but it largely fails to assemble into ribosomes and is rapidly degraded. However, if the endogenous RPL26 loci are deleted, plasmid-encoded Rpl26 assembles into ribosomes and localizes to the cytosol. Chemical and genetic perturbation studies indicate that overexpressed ribosomal proteins are degraded by the ubiquitin-proteasome system and not by autophagy. Inhibition of the proteasome led to accumulation of multiple endogenous ribosomal proteins in insoluble aggregates, consistent with the operation of this QC mechanism in the absence of ribosomal protein overexpression. Our studies reveal that ribosomal proteins that fail to assemble into ribosomes are rapidly distinguished from their assembled counterparts and ubiquitinated and degraded within the nuclear compartment.
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http://dx.doi.org/10.1091/mbc.E16-05-0290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007085PMC
September 2016

Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle.

Elife 2016 03 31;5. Epub 2016 Mar 31.

Department of Biology, Institute of Biochemistry, Swiss Federal Institute of Technology, Zurich, Switzerland.

The COP9-Signalosome (CSN) regulates cullin-RING ubiquitin ligase (CRL) activity and assembly by cleaving Nedd8 from cullins. Free CSN is autoinhibited, and it remains unclear how it becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation. Both CSN and neddylated substrate undergo large conformational changes upon binding, with important roles played by the N-terminal domains of Csn2 and Csn4 and the RING domain of Rbx1 in enabling formation of a high affinity, fully active complex. The RING domain is crucial for deneddylation, and works in part through conformational changes involving insert-2 of Csn6. Nedd8 deconjugation and re-engagement of the active site zinc by the autoinhibitory Csn5 glutamate-104 diminish affinity for Cul1/Rbx1 by ~100-fold, resulting in its rapid ejection from the active site. Together, these mechanisms enable a dynamic deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network.
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http://dx.doi.org/10.7554/eLife.12102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4878873PMC
March 2016

Glutamine Triggers Acetylation-Dependent Degradation of Glutamine Synthetase via the Thalidomide Receptor Cereblon.

Mol Cell 2016 Mar;61(6):809-20

Division of Biology and Biological Engineering, California Institute of Technology, Box 114-96, Pasadena, CA 91125, USA; Howard Hughes Medical Institute. Electronic address:

Cereblon (CRBN), a substrate receptor for the cullin-RING ubiquitin ligase 4 (CRL4) complex, is a direct protein target for thalidomide teratogenicity and antitumor activity of immunomodulatory drugs (IMiDs). Here we report that glutamine synthetase (GS) is an endogenous substrate of CRL4(CRBN). Upon exposing cells to high glutamine concentration, GS is acetylated at lysines 11 and 14, yielding a degron that is necessary and sufficient for binding and ubiquitylation by CRL4(CRBN) and degradation by the proteasome. Binding of acetylated degron peptides to CRBN depends on an intact thalidomide-binding pocket but is not competitive with IMiDs. These findings reveal a feedback loop involving CRL4(CRBN) that adjusts GS protein levels in response to glutamine and uncover a new function for lysine acetylation.
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http://dx.doi.org/10.1016/j.molcel.2016.02.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4889030PMC
March 2016
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