The role of UBL domains in ubiquitin-specific proteases

Abstract Ubiquitin conjugation and deconjugation provides a powerful signalling system to change the fate of its target enzymes. Ubiquitination levels are organized through a balance between ubiquitinating E1, E2 and E3 enzymes and deubiquitination by DUBs (deubiquitinating enzymes). These enzymes are tightly regulated to control their activity. In the present article, we discuss the different ways in which DUBs of the USP (ubiquitin-specific protease) family are regulated by internal domains with a UBL (ubiquitin-like) fold. The UBL domain in USP14 is important for its localization at the proteasome, which enhances catalysis. In contrast, a UBL domain in USP4 binds to the catalytic domain and competes with ubiquitin binding. In this process, the UBL domain mimics ubiquitin and partially inhibits catalysis. In USP7, there are five consecutive UBL domains, of which the last two affect catalytic activity. Surprisingly, they do not act like ubiquitin and activate catalysis rather than inhibiting it. These C-terminal UBL domains promote a conformational change that allows ubiquitin binding and organizes the catalytic centre. Thus it seems that UBL domains have different functions in different USPs. Other proteins can modulate the roles of UBL domains in USP4 and USP7. On one hand, the inhibition of USP4 can be relieved when the UBL is sequestered by another USP. On the other, the activation of USP7 is increased, when the UBL-activated state is stabilized by allosteric binding of GMP synthetase. Altogether, UBL domains appear to be able to regulate catalytic activity in USPs, but they can use widely different mechanisms of action, in which they may, as in USP4, or may not, as in USP7, use the direct resemblance to ubiquitin

Xpert human papillomavirus test is a promising cervical cancer screening test for HIV-seropositive women

This study investigated the performance of Cepheid Xpert human papillomavirus (HPV) assay in South African human immunodeficiency virus (HIV)-infected women and compared its performance with that of hybrid capture-2 (hc2). Methods: Stored cervical specimens from HIV-infected women that had previously been tested using hc2 were tested using Xpert. Results: The overall HR-HPV prevalence was found to be 62.0% (720/1161) by Xpert and 61.2% (711/1161) by hc2. 13.6% (158/1161) were HPV16 positive, 18.8% (218/1161) were HPV18/45, 37.3% (434/1161) were HPV31/33/35/52/58, 12.7% (147/1161) were HPV51/59 and 23.3% (270/1161) were HPV39/68/56/66. Overall agreement with hc2 was 90%; Cohen's kappa was 0.78 (95% CI 0.74-0.82) indicating substantial agreement. Detection of HPV16, HPV18/45, and HPV31/33/35/52/58 were independently associated with cervical intraepithelial neoplasia (CIN)-2+ (P<0.0001 for each); while HPV51/59 and HPV39/68/56/66 were not. Women infected with HPV16, HPV18/45 or HPV31/33/35/52/58 were found to have significantly higher amounts of HPV DNA detected for those with CIN2+ compared to those without CIN2+, P<0.0001 for each. Xpert and hc2 were similarly sensitive (88.3% and 91.5%, respectively) and specific (48.4% and 51.0%) for CIN2+ and CIN3 (sensitivity: 95.8% and 97.9%; specificity: 41.4% and 42.8%). Conclusions: Xpert is a promising screening test in HIV-infected women that performs similarly to hc2

Structure and catalytic regulatory function of ubiquitin specific protease 11 N-terminal and ubiquitin-like domains

The ubiquitin specific protease 11 (USP11) is implicated in DNA repair, viral RNA replication, and TGFβ signaling. We report the first characterization of the USP11 domain architecture and its role in regulating the enzymatic activity. USP11 consists of an N-terminal "domain present in USPs" (DUSP) and "ubiquitin-like" (UBL) domain, together referred to as DU domains, and the catalytic domain harboring a second UBL domain. Crystal structures of the DU domains show a tandem arrangement with a shortened β-hairpin at the two-domain interface and altered surface characteristics compared to the homologues USP4 and USP15. A conserved VEVY motif is a signature feature at the two-domain interface that shapes a potential protein interaction site. Small angle X-ray scattering and gel filtration experiments are consistent with the USP11DU domains and full-length USP11 being monomeric. Unexpectedly, we reveal, through kinetic assays of a series of deletion mutants, that the catalytic activity of USP11 is not regulated through intramolecular autoinhibition or activation by the N-terminal DU or UBL domains. Moreover, ubiquitin chain cleavage assays with all eight linkages reveal a preference for Lys(63)-, Lys(6)-, Lys(33)-, and Lys(11)-linked chains over Lys(27)-, Lys(29)-, and Lys(48)-linked and linear chains consistent with USP11's function in DNA repair pathways that is mediated by the protease domain. Our data support a model whereby USP11 domains outside the catalytic core domain serve as protein interaction or trafficking modules rather than a direct regulatory function of the proteolytic activity. This highlights the diversity of USPs in substrate recognition and regulation of ubiquitin deconjugation

Estimating the burden of cervical disease among HIV-infected women accessing screening services in South Africa: A model-based analysis

Background. Cervical cancer remains the second most common cancer among women worldwide, with much of the global burden occurring in low- and middle-income countries. HIV-infected women are at increased risk of human papillomavirus infection, preinvasive cervical disease and invasive cervical cancer (ICC). Funded through the United States President’s Emergency Plan for AIDS Relief (PEPFAR) and working in collaboration with the South African (SA) Department of Health, our team supports cervical screening integrated within public sector HIV clinics in SA.Objectives. To estimate the burden of cervical disease among HIV-infected women accessing screening services supported through our programme.Methods. We constructed conditional probability models to estimate the burden of grade 1 and grades 2/3 cervical intraepithelial lesions (CIN1 and CIN2/3) and ICC among two cohorts: one consisting of 3 190 HIV-infected women for whom only cytology results were available for analysis, and another consisting of 75 358 HIV-infected women for whom neither cytology nor histology results were available. Parameter estimates for the models were derived from routinely collected programmatic data and published clinical trials.Results. Between January 2009 and November 2015, 75 358 HIV-infected women underwent Pap smear screening in public sector clinics supported by our cervical cancer prevention programme. Based on modelling analysis, we estimate that 46 123 cases of CIN1 (range 45 500 - 49 608), 13 598 cases of CIN2/3 (range 12 749 - 14 828), and 104 cases of ICC (range 61 - 186) occurred in this population.Conclusions. Our findings highlight the magnitude of cervical disease among HIV-infected women in SA.

Screening of DUB activity and specificity by MALDI-TOF mass spectrometry

Deubiquitylases (DUBs) are key regulators of the ubiquitin system which cleave ubiquitin moieties from proteins and polyubiquitin chains. Several DUBs have been implicated in various diseases and are attractive drug targets. We have developed a sensitive and fast assay to quantify in vitro DUB enzyme activity using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Unlike other current assays, this method uses unmodified substrates, such as diubiquitin topoisomers. By analyzing 42 human DUBs against all diubiquitin topoisomers we provide an extensive characterization of DUB activity and specificity. Our results confirm the high specificity of many members of the OTU and JAMM DUB families and highlight that all USPs tested display low linkage selectivity. We also demonstrate that this assay can be deployed to assess the potency and specificity of DUB inhibitors by profiling 11 compounds against a panel of 32 DUBs

Lopinavir/ritonavir monotherapy after virologic failure of first-line antiretroviral therapy in resource-limited settings

To evaluate virologic response rates of lopinavir/ritonavir (LPV/r) monotherapy as second-line antiretroviral treatment (ART) among adults in resource-limited settings (RLS)

Improved Neuropsychological and Neurological Functioning Across Three Antiretroviral Regimens in Diverse Resource-Limited Settings: AIDS Clinical Trials Group Study A5199, the International Neurological Study

Background. AIDS Clinical Trials Group (ACTG) A5199 compared the neurological and neuropsychological (NP) effects of 3 antiretroviral regimens in participants infected with human immunodeficiency virus type 1 (HIV-1) in resource-limited settings

Recent advances in understanding Cushing disease: resistance to glucocorticoid negative feedback and somatic USP8 mutations

Cushing’s disease is a rare disease with a characteristic phenotype due to significant hypercortisolism driven by over-secretion of adrenocorticotropic hormone and to high morbidity and mortality if untreated. It is caused by a corticotroph adenoma of the pituitary, but the exact mechanisms leading to tumorigenesis are not clear. Recent advances in molecular biology such as the discovery of somatic mutations of the ubiquitin-specific peptidase 8 (USP8) gene allow new insights into the pathogenesis, which could be translated into exciting and much-needed therapeutic applications

TRIP13 and APC15 drive mitotic exit by turnover of interphase- and unattached kinetochore-produced MCC

The mitotic checkpoint ensures accurate chromosome segregation through assembly of the mitotic checkpoint complex (MCC), a soluble inhibitor of the anaphase-promoting complex/cyclosome (APC/C) produced by unattached kinetochores. MCC is also assembled during interphase by Mad1/Mad2 bound at nuclear pores, thereby preventing premature mitotic exit prior to kinetochore maturation and checkpoint activation. Using degron tagging to rapidly deplete the AAA+ ATPase TRIP13, we show that its catalytic activity is required to maintain a pool of open-state Mad2 for MCC assembly, thereby supporting mitotic checkpoint activation, but is also required for timely mitotic exit through catalytic disassembly of MCC. Strikingly, combining TRIP13 depletion with elimination of APC15-dependent Cdc20 ubiquitination/degradation results in a complete inability to exit mitosis, even when MCC assembly at unattached kinetochores is prevented. Thus, mitotic exit requires MCC produced either in interphase or mitosis to be disassembled by TRIP13-catalyzed removal of Mad2 or APC15-driven ubiquitination/degradation of its Cdc20 subunit