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The cJun NH2-terminal kinase (JNK) signaling pathway in the liver promotes systemic changes in metabolism by regulating peroxisome proliferator-activated receptor α (PPARα)-dependent expression of the hepatokine fibroblast growth factor 21 (FGF21). Hepatocyte-specific gene ablation studies demonstrated that theMapk9gene (encoding JNK2) plays a key mechanistic role. Mutually exclusive inclusion of exons 7a and 7b yields expression of the isoforms JNK2α and JNK2β. Here we demonstrate thatFgf21gene expression and metabolic regulation are primarily regulated by the JNK2α isoform. To identify relevant substrates of JNK2α, we performed a quantitative phosphoproteomic study of livers isolated from control mice, mice with JNK deficiency in hepatocytes, and mice that express only JNK2α or JNK2β in hepatocytes. We identified the JNK substrate retinoid X receptor α (RXRα) as a protein that exhibited JNK2α-promoted phosphorylation in vivo. RXRα functions as a heterodimeric partner of PPARα and may therefore mediate the effects of JNK2α signaling onFgf21expression. To test this hypothesis, we established mice with hepatocyte-specific expression of wild-type or mutated RXRα proteins. We found that the RXRα phosphorylation site Ser260was required for suppression ofFgf21gene expression. Collectively, these data establish a JNK-mediated signaling pathway that regulates hepaticFgf21expression.

Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin-proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.

Introduction: The chromatin factor BRD9 is a genetic dependency in some cancers, often referred to as SMARCB1-perturbed cancers. Two types of genetic alterations result in SMARCB1 perturbation: SS18-SSX gene fusion and SMARCB1 loss-of-function mutations. In synovial sarcoma, a rare and aggressive soft tissue malignancy comprising approximately 10% of all soft tissue sarcomas, the presence of the SS18-SSX fusion gene drives the disruption of SMARCB1 function and leads to a synthetic lethal dependence on BRD9. In SMARCB1-null solid tumors, for example malignant rhabdoid tumors (MRT), poorly differentiated chordomas, and epithelioid sarcomas, the absence of SMARCB1 protein results in a similar BRD9 dependence. Thus, in SMARCB1-perturbed cancers, including synovial sarcoma and SMARCB1-null cancers, degradation of BRD9 is hypothesized to result in an anticancer effect. CFT8634 is an orally bioavailable heterobifunctional degrader that induces ternary complex formation with BRD9 and an E3 ligase, leading to the ubiquitination of BRD9 and its subsequent degradation by the proteasome. Results: Here we describe the chemical structure of CFT8634 and an overview of the medicinal chemistry path leading to its discovery. In vitro, CFT8634 promotes rapid, potent, deep, and selective degradation of BRD9 with a half-maximal degradation concentration (DC50) of 2 nM in a synovial sarcoma cell line. In long-term growth assays, CFT8634 is effective at impairing cell growth in a concentration-dependent manner specifically in SMARCB1-perturbed contexts. In vivo, oral dosing of CFT8634 in xenograft models of SMARCB1-perturbed cancers leads to robust and dose-dependent degradation of BRD9, which translates to significant and dose-dependent inhibition of tumor growth in preclinical xenograft models. Conclusion: The preclinical data presented herein support the clinical development of CFT8634 for the treatment of synovial sarcoma and SMARCB1-null tumors. Citation Format: Katrina L. Jackson, Roman V. Agafonov, Mark W. Carlson, Prasoon Chaturvedi, David Cocozziello, Kyle Cole, Richard Deibler, Scott J. Eron, Andrew Good, Ashley A. Hart, Minsheng He, Christina S. Henderson, Hongwei Huang, Marta Isasa, R. Jason Kirby, Linda Lee, Michelle Mahler, Moses Moustakim, Christopher G. Nasveschuk, Michael Palmer, Laura L. Poling, Roy M. Pollock, Matthew Schnaderbeck, Stan Spence, Gesine K. Veits, Jeremy L. Yap, Ning Yin, Rhamy Zeid, Adam S. Crystal, Andrew J. Phillips, Stewart L. Fisher. The discovery and characterization of CFT8634: A potent and selective degrader of BRD9 for the treatment of SMARCB1-perturbed cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND09.

Introduction: Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 inhibits the growth of multiple myeloma (MM) cells, confirming their dependency on IKZF1/3. IMiDs (lenalidomide, pomalidomide) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. However, most patients treated with lenalidomide or pomalidomide eventually develop progressive disease due to acquired resistance, underscoring the unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high binding affinity to cereblon (CRBN), rapid and deep IKZF1/3 degradation, and potent dose-dependent efficacy in vivo. Results: A series of novel benzoimidazolone-based CRBN ligands with potent binding affinity were discovered and their binding modes were informed by CRBN co-crystal structures. Although the benzoimidazolone-based CRBN binders did not exhibit IKZF1/3 degradation activity, structural insights into their unique binding modes and knowledge of the IKZF1/3 degradation pharmacophore were combined to enable identification of a novel benzoisoindolone-based ligand that exhibited a 10-fold potency increase in biochemical CRBN binding and a 30-fold potency increase in H929 MM cell growth inhibition when compared to lenalidomide. Additional rounds of structure-based drug design, degradation and phenotypic profiling led to the discovery of CFT7455, a highly potent, selective and orally bioavailable degrader of IKZF1/3. CFT7455 demonstrated an 800 and 1600-fold improvement in CRBN binding compared to pomalidomide in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hrs. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines, as well as H929 cells made resistant to IMiDs. In vivo, CFT7455 catalyzed deep and durable degradation of IKZF3, translating into potent antitumor activity in multiple myeloma xenograft models. CFT7455 also retained its activity in models resistant or insensitive to clinically approved IMiDs as single agent or in combination with standard of care agent dexamethasone. Conclusion: Overall, CFT7455 is a next generation IKZF1/3 degrader, with improved potency and anticancer efficacy in preclinical models compared to existing IMiDs. These features make CFT7455 an exciting drug candidate, as a single agent or for use in combination. CFT7455 is currently being studied in a Ph1 clinical trial. Citation Format: James A. Henderson, Scott J. Eron, Andrew Good, R Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Ashley A. Hart, Christina S. Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Adam S. Crystal, Roy M. Pollock, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. The discovery and characterization of CFT7455: A potent and selective degrader of IKZF1/3 for the treatment of relapsed/refractory multiple myeloma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr ND13.

Introduction Ikaros family zinc finger protein 1 and 3 (IKZF1/3) are essential transcription factors (TF) for terminal differentiation of B and T cells. Depletion of IKZF1/3 in MM cells inhibits growth, confirming their dependency on IKZF1/3. IMiDs (lenalidomide[len], pomalidomide[pom]) are effective therapies for treatment of MM and promote degradation of IKZF1/3 via their interaction with CRL4-CRBN E3 ligase. Most patients treated with len or pom eventually develop progressive disease due to acquired resistance, underscoring an unmet medical need. CFT7455 is a novel IKZF1/3 degrader optimized for high affinity cereblon (CRBN) binding, rapid IKZF1/3 degradation, and potent in vivo efficacy. Results CFT7455 demonstrated an 800 to 1600-fold improvement in CRBN binding compared to pom in biochemical and cellular NanoBRET assays, respectively. In H929 MM cells expressing HiBiT-tagged IKZF1, CFT7455 induced >75% degradation of IKZF1 within 1.5 hr. The high binding affinity and degradation catalysis shown with CFT7455 enabled potent antiproliferative activity across a panel of MM cell lines as well as H929 cells made resistant to IMiDs. In RPMI-8226 MM mice xenografts, CFT7455 (0.1 mg/kg/day) resulted in deep, durable degradation of IKZF3 (21% and 9.5% of vehicle levels at 4 and 24 hr, respectively). Protein levels for IRF4, an essential TF in MM, declined over 7 days with daily CFT7455 treatment to 8% of vehicle levels. Dose dependent efficacy ranged from 0.003-0.1 mg/kg/day, with tumor regression evident at doses ≥0.01 mg/kg/day. Pom was inactive in this model at a human equivalent dose (3 mg/kg/day), with no observed tumor shrinkage in these mice following 17 days of dosing. Switching pom treatment to CFT7455 (0.1 mg/kg/day) on Day 18 led to tumor regression in 67% of animals on Day 28 and 100% tumor regression on Day 35, demonstrating that CFT7455 penetrates large tissues and is efficacious in rapidly growing, IMiD resistant tumors. In the H929 tumor xenograft model, administration of CFT7455 (0.1 mg/kg/day) promoted tumor regression (95% tumor growth inhibition by 7 days); dosing was stopped after 21 days. On Day 63, half the tumors remained below their starting tumor volume. Additionally, CFT7455 demonstrated durable tumor regression in the aggressive MM1.S systemic MM tumor model. In mice bearing RPMI-8226 xenograft tumors, the combination of CFT7455 (QD) and dexamethasone (QW) was more active, and demonstrated a significant survival improvement, compared to either agent alone. Conclusions CFT7455 is a highly potent, catalytic degrader of IKZF1/3, with marked antitumor activity as a single agent and in combination with dexamethasone. Importantly, CFT7455 retains activity in models resistant or insensitive to IMiDs. These results warrant investigation of CFT7455 as a therapeutic approach for MM and a clinical study is planned. Citation Format: James A. Henderson, R. Jason Kirby, Samantha Perino, Roman V. Agafonov, Prasoon Chaturvedi, Bradley Class, David Cocozziello, Scott J. Eron, Andrew Good, Ashley A. Hart, Christina Henderson, Marta Isasa, Brendon Ladd, Matt Schnaderbeck, Michelle Mahler, Roy M. Pollock, Adam S. Crystal, Christopher G. Nasveschuk, Andrew J. Phillips, Stewart L. Fisher, David A. Proia. CFT7455: A novel, IKZF1/3 degrader that demonstrates potent tumor regression in IMiD-resistant multiple myeloma (MM) xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB007.

Methionine is an essential sulfur amino acid that is engaged in key cellular functions such as protein synthesis and is a precursor for critical metabolites involved in maintaining cellular homeostasis. In mammals, in response to nutrient conditions, the liver plays a significant role in regulating methionine concentrations by altering its flux through the transmethylation, transsulfuration, and transamination metabolic pathways. A comprehensive understanding of how hepatic methionine metabolism intersects with other regulatory nutrient signaling and transcriptional events is, however, lacking. Here, we show that methionine and derived-sulfur metabolites in the transamination pathway activate the GCN5 acetyltransferase promoting acetylation of the transcriptional coactivator PGC-1α to control hepatic gluconeogenesis. Methionine was the only essential amino acid that rapidly induced PGC-1α acetylation through activating the GCN5 acetyltransferase. Experiments employing metabolic pathway intermediates revealed that methionine transamination, and not the transmethylation or transsulfuration pathways, contributed to methionine-induced PGC-1α acetylation. Moreover, aminooxyacetic acid, a transaminase inhibitor, was able to potently suppress PGC-1α acetylation stimulated by methionine, which was accompanied by predicted alterations in PGC-1α-mediated gluconeogenic gene expression and glucose production in primary murine hepatocytes. Methionine administration in mice likewise induced hepatic PGC-1α acetylation, suppressed the gluconeogenic gene program, and lowered glycemia, indicating that a similar phenomenon occurs in vivo. These results highlight a communication between methionine metabolism and PGC-1α-mediated hepatic gluconeogenesis, suggesting that influencing methionine metabolic flux has the potential to be therapeutically exploited for diabetes treatment.

The abundance of cell surface glucose transporters must be precisely regulated to ensure optimal growth under constantly changing environmental conditions. We recently conducted a proteomic analysis of the cellular response to trivalent arsenic, a ubiquitous environmental toxin and carcinogen. A surprising finding was that a subset of glucose transporters was among the most downregulated proteins in the cell upon arsenic exposure. Here we show that this downregulation reflects targeted arsenic-dependent degradation of glucose transporters. Degradation occurs in the vacuole and requires the E2 ubiquitin ligase Ubc4, the E3 ubiquitin ligase Rsp5, and K63-linked ubiquitin chains. We used quantitative proteomic approaches to determine the ubiquitinated proteome after arsenic exposure, which helped us to identify the ubiquitination sites within these glucose transporters. A mutant lacking all seven major glucose transporters was highly resistant to arsenic, and expression of a degradation-resistant transporter restored arsenic sensitivity to this strain, suggesting that this pathway represents a protective cellular response. Previous work suggests that glucose transporters are major mediators of arsenic import, providing a potential rationale for this pathway. These results may have implications for the epidemiologic association between arsenic exposure and diabetes.

Despite much evidence of the involvement of the proteasomeubiquitin signaling system in temperature stress response, the dynamics of the ubiquitylome during cold response has not yet been studied. Here, we have compared quantitative ubiquitylomes from a strain deficient in proteasome substrate recruitment and a reference strain during cold response. We have observed that a large group of proteins showing increased ubiquitylation in the proteasome mutant at low temperature is comprised by reverses suppressor of Ty-phenotype 5 (Rsp5)-regulated plasma membrane proteins. Analysis of internalization and degradation of plasma membrane proteins at low temperature showed that the proteasome becomes determinant for this process, whereas, at 30 °C, the proteasome is dispensable. Moreover, our observations indicate that proteasomes have increased capacity to interact with lysine 63-polyubiquitylated proteins during low temperature in vivo. These unanticipated observations indicate that, during cold response, there is a proteolytic cellular reprogramming in which the proteasome acquires a role in the endocytic-vacuolar pathway., This work was supported by Spanish Government (Ministry of Economy and Competitiveness) Grants BFU2009-06985 and BFU2012-35716, Agència de Gestió d'Ajuts Universitaris i de Recerca Grants 2009SGR-1482 and FI-DGR 2013, and European Molecular Biology Organization Fellowship ASTF 363.00-2010

Stress responses are adaptive cellular programs that identify and mitigate potentially dangerous threats. Misfolded proteins are a ubiquitous and clinically relevant stress. Trivalent metalloids, such as arsenic, have been proposed to cause protein misfolding. Using tandem mass tag-based mass spectrometry, we show that trivalent arsenic results in widespread reorganization of the cell from an anabolic to a catabolic state. Both major pathways of protein degradation, the proteasome and autophagy, show increased abundance of pathway components and increased functional output, and are required for survival. Remarkably, cells also showed a down-regulation of ribosomes at the protein level. That this represented an adaptive response and not an adverse toxic effect was indicated by enhanced survival of ribosome mutants after arsenic exposure. These results suggest that a major source of toxicity of trivalent arsenic derives from misfolding of newly synthesized proteins and identifies ribosome reduction as a rapid, effective, and reversible proteotoxic stress response.

Characterizing a protein's function often requires a description of the cellular state in its absence. Multiplexing in mass spectrometry-based proteomics has now achieved the ability to globally measure protein expression levels in yeast from 10 cell states simultaneously. We applied this approach to quantify expression differences in wild type and nine deubiquitylating enzyme (DUB) knockout strains with the goal of creating "information networks" that might provide deeper, mechanistic insights into a protein's biological role. In total, more than 3700 proteins were quantified with high reproducibility across three biological replicates (30 samples in all). DUB mutants demonstrated different proteomics profiles, consistent with distinct roles for each family member. These included differences in total ubiquitin levels and specific chain linkages. Moreover, specific expression changes suggested novel functions for several DUB family members. For instance, the ubp3Δ mutant showed large expression changes for members of the cytochrome C oxidase complex, consistent with a role for Ubp3 in mitochondrial regulation. Several DUBs also showed broad expression changes for phosphate transporters as well as other components of the inorganic phosphate signaling pathway, suggesting a role for these DUBs in regulating phosphate metabolism. These data highlight the potential of multiplexed proteome-wide analyses for biological investigation and provide a framework for further study of the DUB family. Our methods are readily applicable to the entire collection of yeast deletion mutants and may help facilitate systematic analysis of yeast and other organisms.

In human and porcine cysticercosis caused by the tapeworm Taenia solium, the larval stage (cysts) can infest several tissues including the central nervous system (CNS) and the skeletal muscles (SM). The cyst’s proteomics changes associated with the tissue localization in the host tissues have been poorly studied. Quantitative multiplexed proteomics has the power to evaluate global proteome changes in response to different conditions. Here, using a TMT-multiplexed strategy we identified and quantified over 4,200 proteins in cysts obtained from the SM and CNS of pigs, of which 891 were host proteins. To our knowledge, this is the most extensive intermixing of host and parasite proteins reported for tapeworm infections.Several antigens in cysticercosis, i.e., GP50, paramyosin and a calcium-binding protein were enriched in skeletal muscle cysts. Our results suggested the occurrence of tissue-enriched antigen that could be useful in the improvement of the immunodiagnosis for cysticercosis. Using several algorithms for epitope detection, we selected 42 highly antigenic proteins enriched for each tissue localization of the cysts. Taking into account the fold changes and the antigen/epitope contents, we selected 10 proteins and produced synthetic peptides from the best epitopes. Nine peptides were recognized by serum antibodies of cysticercotic pigs, suggesting that those peptides are antigens. Mixtures of peptides derived from SM and CNS cysts yielded better results than mixtures of peptides derived from a single tissue location, however the identification of the ‘optimal’ tissue-enriched antigens remains to be discovered. Through machine learning technologies, we determined that a reliable immunodiagnostic test for porcine cysticercosis required at least five different antigenic determinants., Author summary Human and porcine cysticercosis caused by Taenia solium is a parasite disease still endemic in developing countries. The cysts can be located in different host tissues, including different organs of the central nervous system and the skeletal muscles. The molecular mechanisms associated with the tissue localization of the cysts are not well understood. Here, we described the proteome changes of the cysts obtained from different host tissues from infected pigs using quantitative multiplex proteomics. We explored the diversity of host proteins identified in the cyst’s protein extracts and we also explored the immune-localization of several host-related proteins within the cysts, and propose their possible function. We identified several proteins and antigens enriched for a given tissue localization. Several synthetic peptides designed from these tissue-enriched antigens were tested trough ELISA. Using a combination of peptide mixtures and machine learning technologies we were able to distinguish non cysticercotic and cysticercotic pig’s sera. The tissue-enriched proteins/antigens could be useful for the development of improved immuno-diagnostic tests capable of discriminate the tissue-localization of the cysts.

Protein misfolding is a universal threat to cells. The ubiquitin-proteasome system mediates a cellular stress response capable of eliminating misfolded proteins. Here we identify Cuz1/Ynl155w as a component of the ubiquitin system, capable of interacting with both the proteasome and Cdc48. Cuz1/Ynl155w is regulated by the transcription factor Rpn4, and is required for cells to survive exposure to the trivalent metalloids arsenic and antimony. A related protein, Yor052c, shows similar phenotypes, suggesting a multicomponent stress response pathway. Cuz1/Ynl155w functions as a zinc-dependent ubiquitin-binding protein. Thus, Cuz1/Ynl155w is proposed to protect cells from metalloid-induced proteotoxicity by delivering ubiquitinated substrates to Cdc48 and the proteasome for destruction.

Progression through the G1 phase of the cell cycle is controlled by diverse cyclin-dependent kinases (CDKs) that might be associated to numerous cyclin isoforms. Given such complexity, regulation of cyclin degradation should be crucial for coordinating progression through the cell cycle. In Saccharomyces cerevisiae, SCF is the only E3 ligase known to date to be involved in G1 cyclin degradation. Here, we report the design of a genetic screening that uncovered Dma1 as another E3 ligase that targets G1 cyclins in yeast. We show that the cyclin Pcl1 is ubiquitinated in vitro and in vivo by Dma1, and accordingly, is stabilized in dma1 mutants. We demonstrate that Pcl1 must be phosphorylated by its own CDK to efficiently interact with Dma1 and undergo degradation. A nonphosphorylatable version of Pcl1 accumulates throughout the cell cycle, demonstrating the physiological relevance of the proposed mechanism. Finally, we present evidence that the levels of Pcl1 and Cln2 are independently controlled in response to nutrient availability. This new previously unknown mechanism for G1 cyclin degradation that we report here could help elucidate the specific roles of the redundant CDK-cyclin complexes in G1., This work was supported by Spanish Ministry of Science and Innovation Grant BFU 2009-09728, now known as the Ministry of Economy and Competitiveness.

Tumours exist in a hypoxic microenvironment and must limit excessive oxygen consumption. Hypoxia-inducible factor (HIF) controls mitochondrial oxygen consumption, but how/if tumours regulate non-mitochondrial oxygen consumption (NMOC) is unknown. Protein-tyrosine phosphatase-1B (PTP1B) is required for Her2/Neu-driven breast cancer (BC) in mice, although the underlying mechanism and human relevance remain unclear. We found that PTP1B-deficient HER2(+) xenografts have increased hypoxia, necrosis and impaired growth. In vitro, PTP1B deficiency sensitizes HER2(+) BC lines to hypoxia by increasing NMOC by α-KG-dependent dioxygenases (α-KGDDs). The moyamoya disease gene product RNF213, an E3 ligase, is negatively regulated by PTP1B in HER2(+) BC cells. RNF213 knockdown reverses the effects of PTP1B deficiency on α-KGDDs, NMOC and hypoxia-induced death of HER2(+) BC cells, and partially restores tumorigenicity. We conclude that PTP1B acts via RNF213 to suppress α-KGDD activity and NMOC. This PTP1B/RNF213/α-KGDD pathway is critical for survival of HER2(+) BC, and possibly other malignancies, in the hypoxic tumour microenvironment.

System-wide quantitative analysis of ubiquitylomes has proven to be a valuable tool for elucidating targets and mechanisms of the ubiquitin-driven signaling systems, as well as gaining insights into neurodegenerative diseases and cancer. Current mass spectrometry methods for ubiquitylome detection require large amounts of starting material and rely on stochastic data collection to increase replicate analyses. We describe a method compatible with cell line and tissue samples for large-scale quantification of 5,000-9,000 ubiquitylation forms across ten samples simultaneously. Using this method, we reveal site-specific ubiquitylation in mammalian brain and liver tissues, as well as in cancer cells undergoing proteasome inhibition. To demonstrate the power of the approach for signal-dependent ubiquitylation, we examined protein and ubiquitylation dynamics for mitochondria undergoing PARKIN- and PINK1-dependent mitophagy. This analysis revealed the largest collection of PARKIN- and PINK1-dependent ubiquitylation targets to date in a single experiment, and it also revealed a subset of proteins recruited to the mitochondria during mitophagy.

El pdf del artículo es al manuscrito de autor (PubMed).-- et al., The proteasome recognizes its substrates via a diverse set of ubiquitin receptors, including subunits Rpn10/S5a and Rpn13. In addition, shuttling factors, such as Rad23, recruit substrates to the proteasome by delivering ubiquitinated proteins. Despite the increasing understanding of the factors involved in this process, the regulation of substrate delivery remains largely unexplored. Here we report that Rpn10 is monoubiquitinated in vivo and that this modification has profound effects on proteasome function. Monoubiquitination regulates the capacity of Rpn10 to interact with substrates by inhibiting Rpn10's ubiquitin-interacting motif (UIM). We show that Rsp5, a member of NEDD4 ubiquitin-protein ligase family, and Ubp2, a deubiquitinating enzyme, control the levels of Rpn10 monoubiquitination in vivo. Notably, monoubiquitination of Rpn10 is decreased under stress conditions, suggesting a mechanism of control of receptor availability mediated by the Rsp5-Ubp2 system. Our results reveal an unanticipated link between monoubiquitination signal and regulation of proteasome function., This work was performed in the Institute of Molecular Biology of Barcelona (IBMB), supported by Spanish Government (MICINN) grant BFU2006-02928. MS analysis was performed at Harvard Medical School (HMS). We acknowledge National Institutes of Health (NIH) grants GM065592 (D.F.) and GM67945 (S.G).

Deubiquitination is a crucial mechanism in ubiquitin-mediated signalling networks. The importance of Dubs (deubiquitinating enzymes) as regulators of diverse cellular processes is becoming ever clearer as new roles are elucidated and new pathways are shown to be affected by this mechanism. Recent work, reviewed in the present paper, provides new perspective on the widening influence of Dubs and a new tool to focus studies of not only Dub interactions, but also potentially many more cellular systems. © The Authors Journal compilation., This work was supported by the Spanish Government Ministerio de Educación y Ciencia (grant number BFU2006-02928).

Despite the progress made in understanding the roles of proteasome polyubiquitin receptors, such as the subunits Rpn10 (regulatory particle non-ATPase 10) and Rpn13, and the transient interactors Rad23 (radiation sensitivity abnormal 23) and Dsk2 (dual-specificity protein kinase 2), the mechanisms involved in their regulation are virtually unknown. Rpn10, which is found in the cell in proteasome-bound and-unbound pools, interacts with Dsk2, and this interaction has been proposed to regulate the amount of Dsk2 that gains access to the proteasome. Rpn10 monoubiquitination has emerged as a conserved mechanism with a strong effect on Rpn10 function. In the present study, we show that functional yeast proteasomes have the capacity to associate and dissociate with Rpn10 and that Rpn10 monoubiquitination decreases the Rpn10-proteasome and Rpn10-Dsk2 associations. Remarkably, this process facilitates the formation of Dsk2-proteasomes in vivo. Therefore, Rpn10 monoubiquitination acts as mechanism that serves to switch the proteasome from an 'Rpn10 high/Dsk2 low' state to an 'Rpn10 low/Dsk2 high' state. Interestingly, Rpn10-ubiquitin, with an inactivated ubiquitin-interacting motif (UIM), and Dsk2, with an inactive ubiquitin-like domain (UBL), show temperaturedependent phenotypes with multiple functional interactions.

The ubiquitin-proteasome system has emerged in the last decades as a new paradigm in cell physiology. Ubiquitin is found in fundamental levels of cell regulation, as a target for degradation to the proteasome or as a signal that controls protein function in a complex manner. Even though many aspects of the ubiquitin system remain unexplored, the contributions on the field uncover that ubiquitin represents one of the most sophisticated codes in cellular biology. The proteasome is an ATP-dependent protease that degrades a large number of protein substrates in the cell. The proteasome recruits substrates by a number of receptors that interact with polyubiquitin. Recently, it has been shown that one of these receptors, Rpn10, is regulated by monoubiquitination. In this chapter, we show an overview of the central aspects of the pathway and describe the methodology to characterize in vitro the monoubiquitination of proteasome subunits.

The ubiquitin-proteasome system has emerged in the last decades as a new paradigm in cell physiology. Ubiquitin is found in fundamental levels of cell regulation, as a target for degradation to the proteasome or as a signal that controls protein function in a complex manner. Even though many aspects of the ubiquitin system remain unexplored, the contributions on the field uncover that ubiquitin represents one of the most sophisticated codes in cellular biology. The proteasome is an ATP-dependent protease that degrades a large number of protein substrates in the cell. The proteasome recruits substrates by a number of receptors that interact with polyubiquitin. Recently, it has been shown that one of these receptors, Rpn10, is regulated by monoubiquitination. In this chapter, we show an overview of the central aspects of the pathway and describe the methodology to characterize in vitro the monoubiquitination of proteasome subunits. © 2012 Springer Science+Business Media New York.