X-linked ubiquitin-specific peptidase 11 increases tauopathy vulnerability in women.

Although women experience significantly higher tau burden and increased risk for Alzheimer's disease (AD) than men, the underlying mechanism for this vulnerability has not been explained. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that X-linked ubiquitin specific peptidase 11 (USP11) augments pathological tau aggregation via tau deubiquitination initiated at lysine-281. Removal of ubiquitin provides access for enzymatic tau acetylation at lysines 281 and 274. USP11 escapes complete X-inactivation, and female mice and people both exhibit higher USP11 levels than males. Genetic elimination of usp11 in a tauopathy mouse model preferentially protects females from acetylated tau accumulation, tau pathology, and cognitive impairment. USP11 levels also strongly associate positively with tau pathology in females but not males. Thus, inhibiting USP11-mediated tau deubiquitination may provide an effective therapeutic opportunity to protect women from increased vulnerability to AD and other tauopathies.

K29-linked free polyubiquitin chains affect ribosome biogenesis and direct ribosomal proteins to the intranuclear quality control compartment.

Ribosome assembly requires precise coordination between the production and assembly of ribosomal components. Mutations in ribosomal proteins that inhibit the assembly process or ribosome function are often associated with ribosomopathies, some of which are linked to defects in proteostasis. In this study, we examine the interplay between several yeast proteostasis enzymes, including deubiquitylases (DUBs) Ubp2 and Ubp14, and E3 ligases Ufd4 and Hul5, and we explore their roles in the regulation of the cellular levels of K29-linked unanchored polyubiquitin (polyUb) chains. Accumulating K29-linked unanchored polyUb chains associate with maturing ribosomes to disrupt their assembly, activate the ribosome assembly stress response (RASTR), and lead to the sequestration of ribosomal proteins at the intranuclear quality control compartment (INQ). These findings reveal the physiological relevance of INQ and provide insights into mechanisms of cellular toxicity associated with ribosomopathies.

Deubiquitinases: From mechanisms to their inhibition by small molecules.

Deubiquitinases (DUBs) are specialized proteases that remove ubiquitin from substrates or cleave within ubiquitin chains to regulate ubiquitylation and therefore play important roles in eukaryotic biology. Dysregulation of DUBs is implicated in several human diseases, highlighting the importance of DUB function. In addition, many pathogenic bacteria and viruses encode and deploy DUBs to manipulate host immune responses and establish infectious diseases in humans and animals. Hence, therapeutic targeting of DUBs is an increasingly explored area that requires an in-depth mechanistic understanding of human and pathogenic DUBs. In this review, we summarize the multiple layers of regulation that control autoinhibition, activation, and substrate specificity of DUBs. We discuss different strategies to inhibit DUBs and the progress in developing selective small-molecule DUB inhibitors. Finally, we propose a classification system of DUB inhibitors based on their mode of action.

Goldilocks meets Polycomb.

The Polycomb system modulates chromatin structure to maintain gene repression during cell differentiation. Polycomb repression involves methylation of histone H3K27 (H3K27me3) by Polycomb repressive complex 2 (PRC2), monoubiquitylation of H2A (H2Aub1) by noncanonical PRC1 (ncPRC1), and chromatin compaction by canonical PRC1 (cPRC1), which is independent of its enzymatic activity. Puzzlingly, Polycomb repression also requires deubiquitylation of H2Aub1 by Polycomb repressive deubiquitinase (PR-DUB). In this issue of Genes & Development, Bonnet and colleagues (pp. 1046-1061) resolve this paradox by showing that high levels of H2Aub1 in Drosophila lacking PR-DUB activity promotes open chromatin and gene expression in spite of normal H3K27me3 levels and PRC binding. Pertinently, gene repression is restored by concomitant loss of PRC1 E3 ubiquitin ligase activity but depends on its chromatin compaction activity. These findings suggest that PR-DUB ensures just-right levels of H2Aub1 to allow chromatin compaction by cPRC1.

PR-DUB preserves Polycomb repression by preventing excessive accumulation of H2Aub1, an antagonist of chromatin compaction.

The Polycomb repressive complexes PRC1, PRC2, and PR-DUB repress target genes by modifying their chromatin. In Drosophila, PRC1 compacts chromatin and monoubiquitinates histone H2A at lysine 118 (H2Aub1), whereas PR-DUB is a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains unclear. We show that in early embryos, H2Aub1 is enriched at Polycomb target genes, where it facilitates H3K27me3 deposition by PRC2 to mark genes for repression. During subsequent stages of development, H2Aub1 becomes depleted from these genes and is no longer enriched when Polycomb maintains them repressed. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high levels of H2Aub1 accumulate at Polycomb target genes, and Polycomb repression breaks down. These high H2Aub1 levels do not diminish Polycomb protein complex binding or H3K27 trimethylation but increase DNA accessibility. We show that H2Aub1 interferes with nucleosome stacking and chromatin fiber folding in vitro. Consistent with this, Polycomb repression defects in PR-DUB mutants are exacerbated by reducing PRC1 chromatin compaction activity, but Polycomb repression is restored if PRC1 E3 ligase activity is removed. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, although deposited by PRC1, antagonizes PRC1-mediated chromatin compaction.

A comprehensive phenotypic CRISPR-Cas9 screen of the ubiquitin pathway uncovers roles of ubiquitin ligases in mitosis.

The human ubiquitin proteasome system, composed of over 700 ubiquitin ligases (E3s) and deubiquitinases (DUBs), has been difficult to characterize systematically and phenotypically. We performed chemical-genetic CRISPR-Cas9 screens to identify E3s/DUBs whose loss renders cells sensitive or resistant to 41 compounds targeting a broad range of biological processes, including cell cycle progression, genome stability, metabolism, and vesicular transport. Genes and compounds clustered functionally, with inhibitors of related pathways interacting similarly with E3s/DUBs. Some genes, such as FBXW7, showed interactions with many of the compounds. Others, such as RNF25 and FBXO42, showed interactions primarily with a single compound (methyl methanesulfonate for RNF25) or a set of related compounds (the mitotic cluster for FBXO42). Mutation of several E3s with sensitivity to mitotic inhibitors led to increased aberrant mitoses, suggesting a role for these genes in cell cycle regulation. Our comprehensive CRISPR-Cas9 screen uncovered 466 gene-compound interactions covering 25% of the interrogated E3s/DUBs.

BAP1 enhances Polycomb repression by counteracting widespread H2AK119ub1 deposition and chromatin condensation.

BAP1 is mutated or deleted in many cancer types, including mesothelioma, uveal melanoma, and cholangiocarcinoma. It is the catalytic subunit of the PR-DUB complex, which removes PRC1-mediated H2AK119ub1, essential for maintaining transcriptional repression. However, the precise relationship between BAP1 and Polycombs remains elusive. Using embryonic stem cells, we show that BAP1 restricts H2AK119ub1 deposition to Polycomb target sites. This increases the stability of Polycomb with their targets and prevents diffuse accumulation of H2AK119ub1 and H3K27me3. Loss of BAP1 results in a broad increase in H2AK119ub1 levels that is primarily dependent on PCGF3/5-PRC1 complexes. This titrates PRC2 away from its targets and stimulates H3K27me3 accumulation across the genome, leading to a general chromatin compaction. This provides evidence for a unifying model that resolves the apparent contradiction between BAP1 catalytic activity and its role in vivo, uncovering molecular vulnerabilities that could be useful for BAP1-related pathologies.

Ubiquitin system mutations in neurological diseases.

Neuronal ubiquitin balance impacts the fate of countless cellular proteins, and its disruption is associated with various neurological disorders. The ubiquitin system is critical for proper neuronal cell state transitions and the clearance of misfolded or aggregated proteins that threaten cellular integrity. This article reviews the state of and recent advancements in our understanding of the disruptions to components of the ubiquitin system, in particular E3 ligases and deubiquitylases, in neurodevelopmental and neurodegenerative diseases. Specific focus is on enzymes with recent progress in their characterization, including identifying enzyme-substrate pairs, the use of stem cell and animal models, and the development of therapeutics for ubiquitin-related diseases.

Deubiquitinating enzyme mutagenesis screens identify a USP43-dependent HIF-1 transcriptional response.

The ubiquitination and proteasome-mediated degradation of Hypoxia Inducible Factors (HIFs) is central to metazoan oxygen-sensing, but the involvement of deubiquitinating enzymes (DUBs) in HIF signalling is less clear. Here, using a bespoke DUBs sgRNA library we conduct CRISPR/Cas9 mutagenesis screens to determine how DUBs are involved in HIF signalling. Alongside defining DUBs involved in HIF activation or suppression, we identify USP43 as a DUB required for efficient activation of a HIF response. USP43 is hypoxia regulated and selectively associates with the HIF-1α isoform, and while USP43 does not alter HIF-1α stability, it facilitates HIF-1 nuclear accumulation and binding to its target genes. Mechanistically, USP43 associates with 14-3-3 proteins in a hypoxia and phosphorylation dependent manner to increase the nuclear pool of HIF-1. Together, our results highlight the multifunctionality of DUBs, illustrating that they can provide important signalling functions alongside their catalytic roles.

The SAGA chromatin-modifying complex: the sum of its parts is greater than the whole.

There are many large protein complexes involved in transcription in a chromatin context. However, recent studies on the SAGA coactivator complex are generating new paradigms for how the components of these complexes function, both independently and in concert. This review highlights the initial discovery of the canonical SAGA complex 23 years ago, our evolving understanding of its modular structure and the relevance of its modular nature for its coactivator function in gene regulation.

Differential Oligomerization of the Deubiquitinases USP25 and USP28 Regulates Their Activities.

Deubiquitinases have emerged as promising drug targets for cancer therapy. The two DUBs USP25 and USP28 share high similarity but vary in their cellular functions. USP28 is known for its tumor-promoting role, whereas USP25 is a regulator of the innate immune system and, recently, a role in tumorigenesis was proposed. We solved the structures of the catalytic domains of both proteins and established substantial differences in their activities. While USP28 is a constitutively active dimer, USP25 presents an auto-inhibited tetramer. Our data indicate that the activation of USP25 is not achieved through substrate or ubiquitin binding. USP25 cancer-associated mutations lead to activation in vitro and in vivo, thereby providing a functional link between auto-inhibition and the cancer-promoting role of the enzyme. Our work led to the identification of significant differences between USP25 and USP28 and provided the molecular basis for the development of new and highly specific anti-cancer drugs.

Targeting the Plasmodium falciparum UCHL3 ubiquitin hydrolase using chemically constrained peptides.

The ubiquitin-proteasome system is essential to all eukaryotes and has been shown to be critical to parasite survival as well, including Plasmodium falciparum, the causative agent of the deadliest form of malarial disease. Despite the central role of the ubiquitin-proteasome pathway to parasite viability across its entire life-cycle, specific inhibitors targeting the individual enzymes mediating ubiquitin attachment and removal do not currently exist. The ability to disrupt P. falciparum growth at multiple developmental stages is particularly attractive as this could potentially prevent both disease pathology, caused by asexually dividing parasites, as well as transmission which is mediated by sexually differentiated parasites. The deubiquitinating enzyme PfUCHL3 is an essential protein, transcribed across both human and mosquito developmental stages. PfUCHL3 is considered hard to drug by conventional methods given the high level of homology of its active site to human UCHL3 as well as to other UCH domain enzymes. Here, we apply the RaPID mRNA display technology and identify constrained peptides capable of binding to PfUCHL3 with nanomolar affinities. The two lead peptides were found to selectively inhibit the deubiquitinase activity of PfUCHL3 versus HsUCHL3. NMR spectroscopy revealed that the peptides do not act by binding to the active site but instead block binding of the ubiquitin substrate. We demonstrate that this approach can be used to target essential protein-protein interactions within the Plasmodium ubiquitin pathway, enabling the application of chemically constrained peptides as a novel class of antimalarial therapeutics.

Crosstalk between Lys63- and Lys11-polyubiquitin signaling at DNA damage sites is driven by Cezanne.

The establishment of polyubiquitin conjugates with distinct linkages play important roles in the DNA damage response. Much remains unknown about the regulation of linkage-specific ubiquitin signaling at sites of DNA damage. Here we reveal that Cezanne (also known as Otud7B) deubiquitinating enzyme promotes the recruitment of Rap80/BRCA1-A complex by binding to Lys63-polyubiquitin and targeting Lys11-polyubiquitin. Using a ubiquitin binding domain protein array screen, we identify that the UBA domains of Cezanne and Cezanne2 (also known as Otud7A) selectively bind to Lys63-linked polyubiquitin. Increased Lys11-linkage ubiquitination due to lack of Cezanne DUB activity compromises the recruitment of Rap80/BRCA1-A. Cezanne2 interacts with Cezanne, facilitating Cezanne in the recruitment of Rap80/BRCA1-A, Rad18, and 53BP1, in cellular resistance to ionizing radiation and DNA repair. Our work presents a model that Cezanne serves as a "reader" of the Lys63-linkage polyubiquitin at DNA damage sites and an "eraser" of the Lys11-linkage ubiquitination, indicating a crosstalk between linkage-specific ubiquitination at DNA damage sites.

Assembly and function of branched ubiquitin chains.

Post-translational modification with ubiquitin is required for cell division, differentiation, and survival in all eukaryotes. As part of an intricate signaling code, ubiquitin is attached to its targets as single molecules or polymeric chains, with the distinct modifications encoding a wide range of outcomes. After early work focused on homotypic ubiquitin chains, such as the K48-linked polymers that drive proteasomal degradation, recent studies noted abundant conjugates that contained ubiquitin molecules modified on two or more sites. Such branched ubiquitin chains are produced in response to specific signals and they exert functions that are critical for cellular and organismal homeostasis. In this review, we will discuss our rapidly evolving understanding of the assembly and function of branched ubiquitin chains.

Structural basis for the bi-specificity of USP25 and USP28 inhibitors.

The development of cancer therapeutics is often hindered by the fact that specific oncogenes cannot be directly pharmaceutically addressed. Targeting deubiquitylases that stabilize these oncogenes provides a promising alternative. USP28 and USP25 have been identified as such target deubiquitylases, and several small-molecule inhibitors indiscriminately inhibiting both enzymes have been developed. To obtain insights into their mode of inhibition, we structurally and functionally characterized USP28 in the presence of the three different inhibitors AZ1, Vismodegib and FT206. The compounds bind into a common pocket acting as a molecular sink. Our analysis provides an explanation why the two enzymes are inhibited with similar potency while other deubiquitylases are not affected. Furthermore, a key glutamate residue at position 366/373 in USP28/USP25 plays a central structural role for pocket stability and thereby for inhibition and activity. Obstructing the inhibitor-binding pocket by mutation of this glutamate may provide a tool to accelerate future drug development efforts for selective inhibitors of either USP28 or USP25 targeting distinct binding pockets.

ZUFSP Deubiquitylates K63-Linked Polyubiquitin Chains to Promote Genome Stability.

Deubiquitylating enzymes (DUBs) enhance the dynamics of the versatile ubiquitin (Ub) code by reversing and regulating cellular ubiquitylation processes at multiple levels. Here we discovered that the uncharacterized human protein ZUFSP (zinc finger with UFM1-specific peptidase domain protein/C6orf113/ZUP1), which has been annotated as a potentially inactive UFM1 protease, and its fission yeast homolog Mug105 define a previously unrecognized class of evolutionarily conserved cysteine protease DUBs. Human ZUFSP selectively interacts with and cleaves long K63-linked poly-Ub chains by means of tandem Ub-binding domains, whereas it displays poor activity toward mono- or di-Ub substrates. In cells, ZUFSP is recruited to and regulates K63-Ub conjugates at genotoxic stress sites, promoting chromosome stability upon replication stress in a manner dependent on its catalytic activity. Our findings establish ZUFSP as a new type of linkage-selective cysteine peptidase DUB with a role in genome maintenance pathways.

DUB3 Promotes BET Inhibitor Resistance and Cancer Progression by Deubiquitinating BRD4.

The bromodomain and extra-terminal domain (BET) protein BRD4 is emerging as a promising anticancer therapeutic target. However, resistance to BET inhibitors often occurs, and it has been linked to aberrant degradation of BRD4 protein in cancer. Here, we demonstrate that the deubiquitinase DUB3 binds to BRD4 and promotes its deubiquitination and stabilization. Expression of DUB3 is transcriptionally repressed by the NCOR2-HDAC10 complex. The NCOR2 gene is frequently deleted in castration-resistant prostate cancer patient specimens, and loss of NCOR2 induces elevation of DUB3 and BRD4 proteins in cancer cells. DUB3-proficient prostate cancer cells are resistant to the BET inhibitor JQ1 in vitro and in mice, but this effect is diminished by DUB3 inhibitory agents such as CDK4/6 inhibitor in a RB-independent manner. Our findings identify a previously unrecognized mechanism causing BRD4 upregulation and drug resistance, suggesting that DUB3 is a viable therapeutic target to overcome BET inhibitor resistance in cancer.

Discovery and Characterization of ZUFSP/ZUP1, a Distinct Deubiquitinase Class Important for Genome Stability.

Deubiquitinating enzymes (DUBs) are important regulators of ubiquitin signaling. Here, we report the discovery of deubiquitinating activity in ZUFSP/C6orf113. High-resolution crystal structures of ZUFSP in complex with ubiquitin reveal several distinctive features of ubiquitin recognition and catalysis. Our analyses reveal that ZUFSP is a novel DUB with no homology to any known DUBs, leading us to classify ZUFSP as the seventh DUB family. Intriguingly, the minimal catalytic domain does not cleave polyubiquitin. We identify two ubiquitin binding domains in ZUFSP: a ZHA (ZUFSP helical arm) that binds to the distal ubiquitin and an atypical UBZ domain in ZUFSP that binds to polyubiquitin. Importantly, both domains are essential for ZUFSP to selectively cleave K63-linked polyubiquitin. We show that ZUFSP localizes to DNA lesions, where it plays an important role in genome stability pathways, functioning to prevent spontaneous DNA damage and also promote cellular survival in response to exogenous DNA damage.

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.

Oligomerization-driven MLKL ubiquitylation antagonizes necroptosis.

Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor-interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis-specific multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin-insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane-located deubiquitylating enzyme. Oligomerization-induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.