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Deubiquitinating enzymes are key regulators in the ubiquitin system and an emerging class of drug targets. These proteases disassemble polyubiquitin chains and many deubiquitinases show selectivity for specific polyubiquitin linkages. However, most biochemical insights originate from studies of single diubiquitin linkages in isolation, whereas in cells all linkages coexist. To better mimick this diubiquitin substrate competition, we develop a multiplexed mass spectrometry-based deubiquitinase assay that can probe all ubiquitin linkage types simultaneously to quantify deubiquitinase activity in the presence of all potential diubiquitin substrates. For this, all eight native diubiquitins are generated and each linkage type is designed with a distinct molecular weight by incorporating neutron-encoded amino acids. Overall, 22 deubiquitinases are profiled, providing a three-dimensional overview of deubiquitinase linkage selectivity over time and enzyme concentration., (© 2023. The Author(s).)

The proteasome recognizes ubiquitinated proteins and can also edit ubiquitin marks, allowing substrates to be rejected based on ubiquitin chain topology. In yeast, editing is mediated by deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome through deubiquitination and a noncatalytic effect. Here, we report cryo-EM structures of the proteasome bound to Ubp6, based on which we identify mutants in Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations define a conserved region that we term the ILR element. The ILR is found within the BL1 loop, which obstructs the catalytic groove in free Ubp6. Rpt1-ILR interaction opens the groove by rearranging not only BL1 but also a previously undescribed network of three interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition are linked in that they are eliminated by the same mutations. Ubp6 and ubiquitin together drive proteasomes into a unique conformation associated with proteasome inhibition. Thus, a multicomponent allosteric switch exerts simultaneous control over both Ubp6 and the proteasome., (© 2022. The Author(s).)

The COVID-19 pandemic caused by SARS-CoV-2 is a continuous challenge worldwide, and there is an urgent need to map the landscape of immunogenic and immunodominant epitopes recognized by CD8 + T cells. Here, we analyze samples from 31 patients with COVID-19 for CD8 + T cell recognition of 500 peptide-HLA class I complexes, restricted by 10 common HLA alleles. We identify 18 CD8 + T cell recognized SARS-CoV-2 epitopes, including an epitope with immunodominant features derived from ORF1ab and restricted by HLA-A*01:01. In-depth characterization of SARS-CoV-2-specific CD8 + T cell responses of patients with acute critical and severe disease reveals high expression of NKG2A, lack of cytokine production and a gene expression profile inhibiting T cell re-activation and migration while sustaining survival. SARS-CoV-2-specific CD8 + T cell responses are detectable up to 5 months after recovery from critical and severe disease, and these responses convert from dysfunctional effector to functional memory CD8 + T cells during convalescence.

The endosomal system is a highly dynamic multifunctional organelle, whose complexity is regulated in part by reversible ubiquitylation. Despite the wide-ranging influence of ubiquitin in endosomal processes, relatively few enzymes utilizing ubiquitin have been described to control endosome integrity and function. Here we reveal the deubiquitylating enzyme (DUB) ubiquitin-specific protease 32 (USP32) as a powerful player in this context. Loss of USP32 inhibits late endosome (LE) transport and recycling of LE cargos, resulting in dispersion and swelling of the late compartment. Using SILAC-based ubiquitome profiling we identify the small GTPase Rab7-the logistical centerpiece of LE biology-as a substrate of USP32. Mechanistic studies reveal that LE transport effector RILP prefers ubiquitylation-deficient Rab7, while retromer-mediated LE recycling benefits from an intact cycle of Rab7 ubiquitylation. Collectively, our observations suggest that reversible ubiquitylation helps switch Rab7 between its various functions, thereby maintaining global spatiotemporal order in the endosomal system.

USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal tail near the catalytic site. Here we delineate how the target protein promotes the activation of USP7. Using NMR analysis and biochemistry we describe the order of activation steps, showing that ubiquitin binding is an instrumental step in USP7 activation. Using chemically synthesised p53-peptides we also demonstrate how the correct ubiquitinated substrate increases catalytic activity. We then used transient reaction kinetic modelling to define how the USP7 multistep mechanism is driven by target recognition. Our data show how this pleiotropic DUB can gain specificity for its cellular targets.

Signal transduction by small ubiquitin-like modifier (SUMO) regulates a myriad of nuclear processes. Here we report on the role of SUMO in mitosis in human cell lines. Knocking down the SUMO conjugation machinery results in a delay in mitosis and defects in mitotic chromosome separation. Searching for relevant SUMOylated proteins in mitosis, we identify the anaphase-promoting complex/cyclosome (APC/C), a master regulator of metaphase to anaphase transition. The APC4 subunit is the major SUMO target in the complex, containing SUMO acceptor lysines at positions 772 and 798. SUMOylation is crucial for accurate progression of cells through mitosis and increases APC/C ubiquitylation activity toward a subset of its targets, including the newly identified target KIF18B. Combined, our findings demonstrate the importance of SUMO signal transduction for genome integrity during mitotic progression and reveal how SUMO and ubiquitin cooperate to drive mitosis.

Deubiquitinating enzymes (DUBs) regulate ubiquitin signaling by trimming ubiquitin chains or removing ubiquitin from modified substrates. Similar activities exist for ubiquitin-related modifiers, although the enzymes involved are usually not related. Here, we report human ZUFSP (also known as ZUP1 and C6orf113) and fission yeast Mug105 as founding members of a DUB family different from the six known DUB classes. The crystal structure of human ZUFSP in covalent complex with propargylated ubiquitin shows that the DUB family shares a fold with UFM1- and Atg8-specific proteases, but uses a different active site more similar to canonical DUB enzymes. ZUFSP family members differ widely in linkage specificity through differential use of modular ubiquitin-binding domains (UBDs). While the minimalistic Mug105 prefers K48 chains, ZUFSP uses multiple UBDs for its K63-specific endo-DUB activity. K63 specificity, localization, and protein interaction network suggest a role for ZUFSP in DNA damage response.

The deubiquitinating enzyme BAP1 is an important tumor suppressor that has drawn attention in the clinic since its loss leads to a variety of cancers. BAP1 is activated by ASXL1 to deubiquitinate mono-ubiquitinated H2A at K119 in Polycomb gene repression, but the mechanism of this reaction remains poorly defined. Here we show that the BAP1 C-terminal extension is important for H2A deubiquitination by auto-recruiting BAP1 to nucleosomes in a process that does not require the nucleosome acidic patch. This initial encounter-like complex is unproductive and needs to be activated by the DEUBAD domains of ASXL1, ASXL2 or ASXL3 to increase BAP1's affinity for ubiquitin on H2A, to drive the deubiquitination reaction. The reaction is specific for Polycomb modifications of H2A as the complex cannot deubiquitinate the DNA damage-dependent ubiquitination at H2A K13/15. Our results contribute to the molecular understanding of this important tumor suppressor.

DNA topoisomerase II inhibitors are a major class of cancer chemotherapeutics, which are thought to eliminate cancer cells by inducing DNA double-strand breaks. Here we identify a novel activity for the anthracycline class of DNA topoisomerase II inhibitors: histone eviction from open chromosomal areas. We show that anthracyclines promote histone eviction irrespective of their ability to induce DNA double-strand breaks. The histone variant H2AX, which is a key component of the DNA damage response, is also evicted by anthracyclines, and H2AX eviction is associated with attenuated DNA repair. Histone eviction deregulates the transcriptome in cancer cells and organs such as the heart, and can drive apoptosis of topoisomerase-negative acute myeloid leukaemia blasts in patients. We define a novel mechanism of action of anthracycline anticancer drugs doxorubicin and daunorubicin on chromatin biology, with important consequences for DNA damage responses, epigenetics, transcription, side effects and cancer therapy.

Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis.