Mechanisms of Deubiquitinase Specificity and Regulation.

Protein ubiquitination is one of the most powerful posttranslational modifications of proteins, as it regulates a plethora of cellular processes in distinct manners. Simple monoubiquitination events coexist with more complex forms of polyubiquitination, the latter featuring many different chain architectures. Ubiquitin can be subjected to further posttranslational modifications (e.g., phosphorylation and acetylation) and can also be part of mixed polymers with ubiquitin-like modifiers such as SUMO (small ubiquitin-related modifier) or NEDD8 (neural precursor cell expressed, developmentally downregulated 8). Together, cellular ubiquitination events form a sophisticated and versatile ubiquitin code. Deubiquitinases (DUBs) reverse ubiquitin signals with equally high sophistication. In this review, we conceptualize the many layers of specificity that DUBs encompass to control the ubiquitin code and discuss examples in which DUB specificity has been understood at the molecular level. We further discuss the many mechanisms of DUB regulation with a focus on those that modulate catalytic activity. Our review provides a framework to tackle lingering questions in DUB biology.

Mechanism of Lys6 poly-ubiquitin specificity by the L. pneumophila deubiquitinase LotA.

The versatility of ubiquitination to control vast domains of eukaryotic biology is due, in part, to diversification through differently linked poly-ubiquitin chains. Deciphering signaling roles for some chain types, including those linked via K6, has been stymied by a lack of specificity among the implicated regulatory proteins. Forged through strong evolutionary pressures, pathogenic bacteria have evolved intricate mechanisms to regulate host ubiquitin during infection. Herein, we identify and characterize a deubiquitinase domain of the secreted effector LotA from Legionella pneumophila that specifically regulates K6-linked poly-ubiquitin. We demonstrate the utility of LotA for studying K6 poly-ubiquitin signals. We identify the structural basis of LotA activation and poly-ubiquitin specificity and describe an essential "adaptive" ubiquitin-binding domain. Without LotA activity during infection, the Legionella-containing vacuole becomes decorated with K6 poly-ubiquitin as well as the AAA ATPase VCP/p97/Cdc48. We propose that LotA's deubiquitinase activity guards Legionella-containing vacuole components from ubiquitin-dependent extraction.

Mechanism of activation and regulation of deubiquitinase activity in MINDY1 and MINDY2.

Of the eight distinct polyubiquitin (polyUb) linkages that can be assembled, the roles of K48-linked polyUb (K48-polyUb) are the most established, with K48-polyUb modified proteins being targeted for degradation. MINDY1 and MINDY2 are members of the MINDY family of deubiquitinases (DUBs) that have exquisite specificity for cleaving K48-polyUb, yet we have a poor understanding of their catalytic mechanism. Here, we analyze the crystal structures of MINDY1 and MINDY2 alone and in complex with monoUb, di-, and penta-K48-polyUb, identifying 5 distinct Ub binding sites in the catalytic domain that explain how these DUBs sense both Ub chain length and linkage type to cleave K48-polyUb chains. The activity of MINDY1/2 is inhibited by the Cys-loop, and we find that substrate interaction relieves autoinhibition to activate these DUBs. We also find that MINDY1/2 use a non-canonical catalytic triad composed of Cys-His-Thr. Our findings highlight multiple layers of regulation modulating DUB activity in MINDY1 and MINDY2.

Acetylation-Dependent Deubiquitinase OTUD3 Controls MAVS Activation in Innate Antiviral Immunity.

Accurate regulation of innate immunity is necessary for the host to efficiently respond to invading pathogens and avoid excessive harmful immune pathology. Here we identified OTUD3 as an acetylation-dependent deubiquitinase that restricts innate antiviral immune signaling. OTUD3 deficiency in mice results in enhanced innate immunity, a diminished viral load, and morbidity. OTUD3 directly hydrolyzes lysine 63 (Lys63)-linked polyubiquitination of MAVS and thus shuts off innate antiviral immune response. Notably, the catalytic activity of OTUD3 relies on acetylation of its Lys129 residue. In response to virus infection, the acetylated Lys129 is removed by SIRT1, which promptly inactivates OTUD3 and thus allows timely induction of innate antiviral immunity. Importantly, acetyl-OTUD3 levels are inversely correlated with IFN-ß expression in influenza patients. These findings establish OTUD3 as a repressor of MAVS and uncover a previously unknown regulatory mechanism by which the catalytic activity of OTUD3 is tightly controlled to ensure timely activation of antiviral defense.

Proteasome-Bound UCH37/UCHL5 Debranches Ubiquitin Chains to Promote Degradation.

The linkage, length, and architecture of ubiquitin (Ub) chains are all important variables in providing tight control over many biological paradigms. There are clear roles for branched architectures in regulating proteasome-mediated degradation, but the proteins that selectively recognize and process these atypical chains are unknown. Here, using synthetic and enzyme-derived ubiquitin chains along with intact mass spectrometry, we report that UCH37/UCHL5, a proteasome-associated deubiquitinase, cleaves K48 branched chains. The activity and selectivity toward branched chains is markedly enhanced by the proteasomal Ub receptor RPN13/ADRM1. Using reconstituted proteasome complexes, we find that chain debranching promotes degradation of substrates modified with branched chains under multi-turnover conditions. These results are further supported by proteome-wide pulse-chase experiments, which show that the loss of UCH37 activity impairs global protein turnover. Our work therefore defines UCH37 as a debranching deubiquitinase important for promoting proteasomal degradation.

Dynamic Regulation of Mitochondrial Import by the Ubiquitin System.

Mitochondria import nearly their entire proteome from the cytoplasm by translocating precursor proteins through the translocase of the outer membrane (TOM) complex. Here, we show dynamic regulation of mitochondrial import by the ubiquitin system. Acute pharmacological inhibition or genetic ablation of the mitochondrial deubiquitinase (DUB) USP30 triggers accumulation of Ub-substrates that are normally localized inside the mitochondria. Mitochondrial import of USP30 substrates is impaired in USP30 knockout (KO) cells, suggesting that deubiquitination promotes efficient import. Upstream of USP30, the E3 ligase March5 ubiquitinates mitochondrial proteins whose eventual import depends on USP30. In USP30 KOs, exogenous March5 expression induces accumulation of unimported translocation intermediates that are degraded by the proteasomes. In USP30 KO mice, TOM subunits have reduced abundance across multiple tissues. Together these data highlight how protein import into a subcellular compartment can be regulated by ubiquitination and deubiquitination by E3 ligase and DUB machinery positioned at the gate.

Regulation of Phosphoribosyl-Linked Serine Ubiquitination by Deubiquitinases DupA and DupB.

The family of bacterial SidE enzymes catalyzes non-canonical phosphoribosyl-linked (PR) serine ubiquitination and promotes infectivity of Legionella pneumophila. Here, we describe identification of two bacterial effectors that reverse PR ubiquitination and are thus named deubiquitinases for PR ubiquitination (DUPs; DupA and DupB). Structural analyses revealed that DupA and SidE ubiquitin ligases harbor a highly homologous catalytic phosphodiesterase (PDE) domain. However, unlike SidE ubiquitin ligases, DupA displays increased affinity to PR-ubiquitinated substrates, which allows DupA to cleave PR ubiquitin from substrates. Interfering with DupA-ubiquitin binding switches its activity toward SidE-type ligase. Given the high affinity of DupA to PR-ubiquitinated substrates, we exploited a catalytically inactive DupA mutant to trap and identify more than 180 PR-ubiquitinated host proteins in Legionella-infected cells. Proteins involved in endoplasmic reticulum (ER) fragmentation and membrane recruitment to Legionella-containing vacuoles (LCV) emerged as major SidE targets. The global map of PR-ubiquitinated substrates provides critical insights into host-pathogen interactions during Legionella infection.

Deubiquitinase USP20 promotes breast cancer metastasis by stabilizing SNAI2.

SNAI2/SLUG, a metastasis-promoting transcription factor, is a labile protein that is degraded through the ubiquitin proteasome degradation system. Here, we conducted comprehensive gain- and loss-of-function screens using a human DUB cDNA library of 65 genes and an siRNA library of 98 genes, and identified USP20 as a deubiquitinase (DUB) that regulates SNAI2 ubiquitination and stability. Further investigation of USP20 demonstrated its function in promoting migration, invasion, and metastasis of breast cancer. USP20 positively correlates with SNAI2 protein level in breast tumor samples, and higher USP20 expression is associated with poor prognosis in ER- breast cancer patients.

LC Domain-Mediated Coalescence Is Essential for Otu Enzymatic Activity to Extend Drosophila Lifespan.

In eukaryotic cells, RNA-binding proteins (RBPs) interact with RNAs to form ribonucleoprotein complexes (RNA granules) that have long been thought to regulate RNA fate or activity. Emerging evidence suggests that some RBPs not only bind RNA but also possess enzymatic activity related to ubiquitin regulation, raising important questions of whether these RBP-formed RNA granules regulate ubiquitin signaling and related biological functions. Here, we show that Drosophila Otu binds RNAs and coalesces to membrane-less biomolecular condensates via its intrinsically disordered low-complexity domain, and coalescence represents a functional state for Otu exerting deubiquitinase activity. Notably, coalescence-mediated enzymatic activity of Otu is positively regulated by its bound RNAs and co-partner Bam. Further genetic analysis reveals that the Otu/Bam deubiquitinase complex and dTraf6 constitute a feedback loop to maintain intestinal immune homeostasis during aging, thereby controlling longevity. Thus, regulated biomolecular condensates may represent a mechanism that controls dynamic enzymatic activities and related biological processes.

The deubiquitinase Usp7 in Drosophila melanogaster is required for synaptonemal complex maintenance.

Meiosis is a form of cell division that is essential to sexually reproducing organisms and is therefore highly regulated. Each event of meiosis must occur at the correct developmental stage to ensure that chromosomes are segregated properly during both meiotic divisions. One unique meiosis-specific structure that is tightly regulated in terms of timing of assembly and disassembly is the synaptonemal complex (SC). While the mechanism(s) for assembly and disassembly of the SC are poorly understood in Drosophila melanogaster, posttranslational modifications, including ubiquitination and phosphorylation, are known to play a role. Here, we identify a role for the deubiquitinase Usp7 in the maintenance of the SC in early prophase and show that its function in SC maintenance is independent of the meiotic recombination process. Using two usp7 shRNA constructs that result in different knockdown levels, we have shown that the presence of SC through early/mid-pachytene is critical for normal levels and placement of crossovers.

USP21 deubiquitinase promotes pancreas cancer cell stemness via Wnt pathway activation.

The ubiquitin-specific protease (USP) family is the largest group of cysteine proteases. Cancer genomic analysis identified frequent amplification of USP21 (22%) in human pancreatic ductal adenocarcinoma (PDAC). USP21 overexpression correlates with human PDAC progression, and enforced expression of USP21 accelerates murine PDAC tumor growth and drives PanIN to PDAC progression in immortalized human pancreatic ductal cells. Conversely, depletion of USP21 impairs PDAC tumor growth. Mechanistically, USP21 deubiquitinates and stabilizes the TCF/LEF transcription factor TCF7, which promotes cancer cell stemness. Our work identifies and validates USP21 as a PDAC oncogene, providing a potential druggable target for this intractable disease.

Proteotoxic stress and the ubiquitin proteasome system.

The ubiquitin proteasome system maintains protein homeostasis by regulating the breakdown of misfolded proteins, thereby preventing misfolded protein aggregates. The efficient elimination is vital for preventing damage to the cell by misfolded proteins, known as proteotoxic stress. Proteotoxic stress can lead to the collapse of protein homeostasis and can alter the function of the ubiquitin proteasome system. Conversely, impairment of the ubiquitin proteasome system can also cause proteotoxic stress and disrupt protein homeostasis. This review examines two impacts of proteotoxic stress, 1) disruptions to ubiquitin homeostasis (ubiquitin stress) and 2) disruptions to proteasome homeostasis (proteasome stress). Here, we provide a mechanistic description of the relationship between proteotoxic stress and the ubiquitin proteasome system. This relationship is illustrated by findings from several protein misfolding diseases, mainly neurodegenerative diseases, as well as from basic biology discoveries from yeast to mammals. In addition, we explore the importance of the ubiquitin proteasome system in endoplasmic reticulum quality control, and how proteotoxic stress at this organelle is alleviated. Finally, we highlight how cells utilize the ubiquitin proteasome system to adapt to proteotoxic stress and how the ubiquitin proteasome system can be genetically and pharmacologically manipulated to maintain protein homeostasis.

USP8 promotes cancer progression and extracellular vesicle-mediated CD8+ T cell exhaustion by deubiquitinating the TGF-ß receptor TßRII.

TGF-ß signaling is a key player in tumor progression and immune evasion, and is associated with poor response to cancer immunotherapies. Here, we identified ubiquitin-specific peptidase 8 (USP8) as a metastasis enhancer and a highly active deubiquitinase in aggressive breast tumors. USP8 acts both as a cancer stemness-promoting factor and an activator of the TGF-ß/SMAD signaling pathway. USP8 directly deubiquitinates and stabilizes the type II TGF-ß receptor TßRII, leading to its increased expression in the plasma membrane and in tumor-derived extracellular vesicles (TEVs). Increased USP8 activity was observed in patients resistant to neoadjuvant chemotherapies. USP8 promotes TGF-ß/SMAD-induced epithelial-mesenchymal transition (EMT), invasion, and metastasis in tumor cells. USP8 expression also enables TßRII+ circulating extracellular vesicles (crEVs) to induce T cell exhaustion and chemoimmunotherapy resistance. Pharmacological inhibition of USP8 antagonizes TGF-ß/SMAD signaling, and reduces TßRII stability and the number of TßRII+ crEVs to prevent CD8+ T cell exhaustion and to reactivate anti-tumor immunity. Our findings not only reveal a novel mechanism whereby USP8 regulates the cancer microenvironment but also demonstrate the therapeutic advantages of engineering USP8 inhibitors to simultaneously suppress metastasis and improve the efficacy of cancer immunotherapy.

The Deubiquitinase USP46 Is Essential for Proliferation and Tumor Growth of HPV-Transformed Cancers.

High-risk human papilloma viruses (HPVs) cause cervical, anal, and oropharyngeal cancers, unlike the low-risk HPVs, which cause benign lesions. E6 oncoproteins from the high-risk strains are essential for cell proliferation and transformation in HPV-induced cancers. We report that a cellular deubiquitinase, USP46, is selectively recruited by the E6 of high-risk, but not low-risk, HPV to deubiqutinate and stabilize Cdt2/DTL. Stabilization of Cdt2, a component of the CRL4Cdt2 E3 ubiquitin ligase, limits the level of Set8, an epigenetic writer, and promotes cell proliferation. USP46 is essential for the proliferation of HPV-transformed cells, but not of cells without HPV. Cdt2 is elevated in human cervical cancers and knockdown of USP46 inhibits HPV-transformed tumor growth in xenografts. Recruitment of a cellular deubiquitinase to stabilize key cellular proteins is an important activity of oncogenic E6, and the importance of E6-USP46-Cdt2-Set8 pathway in HPV-induced cancers makes USP46 a target for the therapy of such cancers.

ATXN3 functions as a tumor suppressor through potentiating galectin-9-mediated apoptosis in human colon adenocarcinoma.

While deubiquitinase ATXN3 has been implicated as a potential oncogene in various types of human cancers, its role in colon adenocarcinoma remains understudied. Surprisingly, our findings demonstrate that ATXN3 exerts an antitumor effect in human colon cancers through potentiating Galectin-9-induced apoptosis. CRISPR-mediated ATXN3 deletion unexpectedly intensified colon cancer growth both in vitro and in xenograft colon cancers. At the molecular level, we identified ATXN3 as a bona fide deubiquitinase specifically targeting Galectin-9, as ATXN3 interacted with and inhibited Galectin-9 ubiquitination. Consequently, targeted ATXN3 ablation resulted in reduced Galectin-9 protein expression, thereby diminishing Galectin-9-induced colon cancer apoptosis and cell growth arrest. The ectopic expression of Galectin-9 fully reversed the growth of ATXN3-null colon cancer in mice. Furthermore, immunohistochemistry staining revealed a significant reduction in both ATXN3 and Galectin-9 protein expression, along with a positive correlation between them in human colon cancer. Our study identifies the first Galectin-9-specific deubiquitinase and unveils a tumor-suppressive role of ATXN3 in human colon cancer.

Hotspot site microenvironment in the deubiquitinase OTUB1 drives its stability and aggregation.

Lewy bodies (LB) are aberrant protein accumulations observed in the brain cells of individuals affected by Parkinson's disease (PD). A comprehensive analysis of LB proteome identified over a hundred proteins, many co-enriched with α-synuclein, a major constituent of LB. Within this context, OTUB1, a deubiquitinase detected in LB, exhibits amyloidogenic properties, yet the mechanisms underlying its aggregation remain elusive. In this study, we identify two critical sites in OTUB1-namely, positions 133 and 173-that significantly impact its amyloid aggregation. Substituting alanine at position 133 and lysine at position 173 enhances both thermodynamic and kinetic stability, effectively preventing amyloid aggregation. Remarkably, lysine at position 173 demonstrates the highest stability without compromising enzymatic activity. The increased stability and inhibition of amyloid aggregation are attributed mainly to the changes in the specific microenvironment at the hotspot. In our exploration of the in-vivo co-occurrence of α-synuclein and OTUB1 in LB, we observed a synergistic modulation of each other's aggregation. Collectively, our study unveils the molecular determinants influencing OTUB1 aggregation, shedding light on the role of specific residues in modulating aggregation kinetics and structural transition. These findings contribute valuable insights into the complex interplay of amino acid properties and protein aggregation, with potential implications for understanding broader aspects of protein folding and aggregation phenomena.

Polycomb-dependent histone H2A ubiquitination links developmental disorders with cancer.

Cell identity is tightly controlled by specific transcriptional programs which require post-translational modifications of histones. These histone modifications allow the establishment and maintenance of active and repressed chromatin domains. Histone H2A lysine 119 ubiquitination (H2AK119ub1) has an essential role in building repressive chromatin domains during development. It is regulated by the counteracting activities of the Polycomb repressive complex 1 (PRC1) and the Polycomb repressive-deubiquitinase (PR-DUB) complexes, two multi-subunit ensembles that write and erase this modification, respectively. We have catalogued the recurrent genetic alterations in subunits of the PRC1 and PR-DUB complexes in both neurodevelopmental disorders and cancer. These genetic lesions are often shared across disorders, and we highlight common mechanisms of H2AK119ub1 dysregulation and how they affect development in multiple disease contexts.

USP7 limits CDK1 activity throughout the cell cycle.

Chemical inhibitors of the deubiquitinase USP7 are currently being developed as anticancer agents based on their capacity to stabilize P53. Regardless of this activity, USP7 inhibitors also generate DNA damage in a p53-independent manner. However, the mechanism of this genotoxicity and its contribution to the anticancer effects of USP7 inhibitors are still under debate. Here we show that, surprisingly, even if USP7 inhibitors stop DNA replication, they also induce a widespread activation of CDK1 throughout the cell cycle, which leads to DNA damage and is toxic for mammalian cells. In addition, USP7 interacts with the phosphatase PP2A and supports its active localization in the cytoplasm. Accordingly, inhibition of USP7 or PP2A triggers very similar changes of the phosphoproteome, including a widespread increase in the phosphorylation of CDK1 targets. Importantly, the toxicity of USP7 inhibitors is alleviated by lowering CDK1 activity or by chemical activation of PP2A. Our work reveals that USP7 limits CDK1 activity at all cell cycle stages, providing a novel mechanism that explains the toxicity of USP7 inhibitors through untimely activation of CDK1.

Regulation of ubiquitination and antiviral activity of Cactin by deubiquitinase Usp14 in Drosophila.

Cactin, a highly conserved protein, plays a crucial role in various physiological processes in eukaryotes, including innate immunity. Recently, the function of Cactin in the innate immunity of Drosophila has been explored, revealing that Cactin regulates a non-canonical signaling pathway associated with the Toll and Imd pathways via the Cactin-Deaf1 axis. In addition, Cactin exhibits specific antiviral activity against the Drosophila C virus (DCV) in Drosophila, with an unknown mechanism. During DCV infection, it has been confirmed that the protein level and antiviral activity of Cactin are regulated by ubiquitination. However, the precise ubiquitination and deubiquitination mechanisms of Cactin in Drosophila remain unexplored. In this study, we identified ubiquitin-specific protease 14 (Usp14) as a major deubiquitinase for Cactin through comprehensive deubiquitinase screening. Our results demonstrate that Usp14 interacts with the C_Cactus domain of Cactin via its USP domain. Usp14 efficiently removes K48- and K63-linked polyubiquitin chains from Cactin, thereby preventing its degradation through the ubiquitin-proteasome pathway. Usp14 significantly inhibits DCV replication in Drosophila cells by stabilizing Cactin. Moreover, Usp14-deficient fruit flies exhibit increased susceptibility to DCV infection compared to wild-type flies. Collectively, our findings reveal the regulation of ubiquitination and antiviral activity of Cactin by the deubiquitinase Usp14, providing valuable insights into the modulation of Cactin-mediated antiviral activity in Drosophila.IMPORTANCEViral infections pose a severe threat to human health, marked by high pathogenicity and mortality rates. Innate antiviral pathways, such as Toll, Imd, and JAK-STAT, are generally conserved across insects and mammals. Recently, the multi-functionality of Cactin in innate immunity has been identified in Drosophila. In addition to regulating a non-canonical signaling pathway through the Cactin-Deaf1 axis, Cactin exhibits specialized antiviral activity against the Drosophila C virus (DCV) with an unknown mechanism. A previous study emphasized the significance of the Cactin level, regulated by the ubiquitin-proteasome pathway, in modulating antiviral signaling. However, the regulatory mechanisms governing Cactin remain unexplored. In this study, we demonstrate that Usp14 stabilizes Cactin by preventing its ubiquitination and subsequent degradation. Furthermore, Usp14 plays a crucial role in regulating the antiviral function mediated by Cactin. Therefore, our findings elucidate the regulatory mechanism of Cactin in Drosophila, offering a potential target for the prevention and treatment of viral infections.

Mouse endothelial OTUD1 promotes angiotensin II-induced vascular remodeling by deubiquitinating SMAD3.

Understanding the molecular mechanisms of pathological vascular remodeling is important for treating cardiovascular diseases and complications. Recent studies have highlighted a role of deubiquitinases in vascular pathophysiology. Here, we investigate the role of a deubiquitinase, OTUD1, in angiotensin II (Ang II)-induced vascular remodeling. We detect upregulated OTUD1 in the vascular endothelium of Ang II-challenged mice and show that OTUD1 deletion attenuates vascular remodeling, collagen deposition, and EndMT. Conversely, OTUD1 overexpression aggravates these pathological changes both in vivo and in vitro. Mechanistically, SMAD3 is identified as a substrate of OTUD1 using co-immunoprecipitation followed by LC-MS/MS. We find that OTUD1 stabilizes SMAD3 and facilitates SMAD3/SMAD4 complex formation and subsequent nuclear translocation through both K48- and K63-linked deubiquitination. OTUD1-mediated SMAD3 activation regulates transcription of genes involved in vascular EndMT and remodeling in HUVECs. Finally, SMAD3 inhibition reverses OTUD1-promoted vascular remodeling. Our findings demonstrate that endothelial OTUD1 promotes Ang II-induced vascular remodeling by deubiquitinating SMAD3. We identify SMAD3 as a target of OTUD1 and propose OTUD1 as a potential therapeutic target for diseases related to vascular remodeling.