Posttranslational modification by ubiquitin controls multiple cellular functions and is counteracted by the activities of deubiquitinating enzymes. UBPy (USP8) is a growth-regulated ubiquitin isopeptidase that interacts with the HRS-STAM complex. Using Cre-loxP-mediated gene targeting in mice, we show that lack of UBPy results in embryonic lethality, whereas its conditional inactivation in adults causes fatal liver failure. The defect is accompanied by a strong reduction or absence of several growth factor receptor tyrosine kinases (RTKs), like epidermal growth factor receptor, hepatocyte growth factor receptor (c-met), and ERBB3. UBPydeficient cells exhibit aberrantly enlarged early endosomes colocalizing with enhanced ubiquitination and have reduced levels of HRS and STAM2. Congruently immortalized cells gradually stop proliferation upon induced deletion of UBPy. These results unveil a central and nonredundant role of UBPy in growth regulation, endosomal sorting, and the control of RTKs in vivo.Posttranslational modification of proteins by mono-or polyubiquitination represents a central mechanism for modulating a wide range of cellular functions, like protein stability, intracellular transport, protein interactions, and transcriptional activity. Ubiquitin is covalently bound to substrates by the activity of E1, E2, and E3 conjugating enzymes (17,44,53). Analogous to other posttranslational modifications, ubiquitination is a reversible process counteracted by deubiquitinating enzymes (DUBs), which cleave the isopeptide linkage between the protein substrate and the ubiquitin residue (14). While the processes and biological consequences of ubiquitin conjugation have been intensively studied, the role of DUBs is just beginning to emerge. Based on structural predictions, the human genome contains more than 90 putative DUBs. These fall into the subclasses of ubiquitin C-terminal hydrolases, ubiquitin-specific proteases (USPs), Machado Joseph disease protein domain proteases, ovarian tumor proteases, and JAMM motif proteases (1, 37, 42). The USPs, with more than 50 members, comprise the largest class of DUBs. Members of the USP family have been associated with the regulation of different cellular pathways. USP7 (HAUSP) regulates p53 stability by deubiquitination of p53 and Mdm2 (32, 33), USP2a was reported to regulate the stability of fatty acid synthase (12), and USP1 has been shown to deubiquitinate the monoubiquitinated proteins Fanconia anemia protein FANCD2 (41) and DNA replication processivity factor PCNA (19).USP8 (UBPy/HUMORF8) was first described as a growthregulated ubiquitin isopeptidase that accumulates upon growth stimulation. Protein levels of UBPy decrease, when cells undergo growth arrest by contact inhibition, suggesting a possible role in the control of mammalian-cell proliferation (40). An oncogenic fusion product of the 5Ј end of phosphatidylinositol 3-kinase p85␤ fused to the 3Ј end of UBPy, which contains the catalytic domain, was isolated from a patient with chronic myelogenous leukemia (21). B...

UBP43/USP18 was described as a specific protease that removes conjugated ubiquitin-like modifier ISG15 from target proteins. The severe phenotype of UBP43 ؊/؊ mice characterized by premature death, brain cell injury, and deregulated STAT1 signaling was ascribed to an enhanced conjugation of ISG15. In contrast, no phenotypic changes were detected in ISG15 ؊/؊ mice. To verify the role of ISG15 in the phenotype of UBP43؊/؊ mice, we employed mice deficient for both ISG15 and UBP43. Here, we show that the phenotype of UBP43 ؊/؊ mice was not rescued by the absence of ISG15, as evident from unchanged mortality, neurological symptoms, and occurrence of hydrocephalus. Also, the reported hypersensitivity of UBP43 ؊/؊ mice to an interferon inducer, poly(I · C), was ISG15 independent. Furthermore, no evidence for a role of ISG15 in the modulation of STAT1 signaling or in the resistance against lymphocytic choriomeningitis virus and vesicular stomatitis virus was found. Presented results clearly demonstrate that the phenotypic alterations of UBP43 ؊/؊ mice are not caused by the lack of ISG15 deconjugation and must be due to another, non-ISG15-mediated molecular mechanism.Postranslational modification of proteins by covalently linked ubiquitin and ubiquitin-like modifiers (UBLs) is a rapid and versatile regulatory mechanism involved in many cellular processes (5,14). ISG15 is a 15-kDa interferon (IFN)-induced UBL which bears two ubiquitin-like domains, each about 30% homologous to ubiquitin (1,3,6,12). Analogous to ubiquitin and other UBLs, ISG15 is conjugated by an isopeptide bond to target proteins (7). Correspondingly, an ISG15-specific enzymatic cascade that consists of an activating UBE1L (E1) and a conjugating UbcH8 (E2) enzyme was recently identified (18,19). Several ISG15-modified substrates such as serpin 2a (4), JAK1, STAT1, phospholipase C␥1, and ERK1 (8) were reported, but rigorous evidence for an ISG15 covalently linked to these proteins, with the exception of serpin 2a, has not yet been found. Most recently, a large number (158) of ISG15 target proteins that function in diverse cellular pathways were identified by mass spectroscopy (20).Previous work from several laboratories implicated ISG15 and ISGylation in diverse biological activities, ranging from a role in pregnancy to antiviral and antitumor defense (11,17). Speculations on the biological significance of ISG15 were further amplified by analyses of mice lacking UBP43/USP18 (16), a protease that removes conjugated ISG15 from substrate proteins (9). Mice with a targeted deletion of UBP43 (UBP43 Ϫ/Ϫ ) have elevated levels of ISG15 conjugates and develop brain injury due to necrosis of ependymal cells, accompanied by hydrocephalus and early death (16). Investigators reported a prolonged STAT1 phosphorylation in the absence of UBP43 and claimed that ISG15 modification is an important factor in the regulation of the JAK-STAT pathway and IFN signaling (10, 17). Most recently, some evidence was presented for a role of UBP43 and possibly ISGylation in innate immun...

The modification of proteins by ubiquitin has a major role in cells of the immune system and is counteracted by various deubiquitinating enzymes (DUBs) with poorly defined functions. Here we identified the ubiquitin-specific protease USP8 as a regulatory component of the T cell antigen receptor (TCR) signalosome that interacted with the adaptor Gads and the regulatory molecule 14-3-3 . Caspasedependent processing of USP8 occurred after stimulation of the TCR. T cell-specific deletion of USP8 in mice revealed that USP8 was essential for thymocyte maturation and upregulation of the gene encoding the cytokine receptor IL-7R mediated by the transcription factor Foxo1. Mice with T cell-specific USP8 deficiency developed colitis that was promoted by disturbed T cell homeostasis, a predominance of CD8(+) T cells in the intestine and impaired regulatory T cell function. Collectively, our data reveal an unexpected role for USP8 as an immunomodulatory DUB in T cells. Ubiquitin-modification is counteracted by deubiquitinating enzymes (DUBs). Here we identify 32 USP8 as a novel component of the TCR signalosome that interacts with the adapter 33 molecule GADS and 14-3-3β. Upon TCR-stimulation USP8 is processed in a caspase-34 dependent manner. T-cell-specific deletion in mice (USP8 f/f CD4-Cre) revealed that USP8 is 35 essential for thymocyte transition to the CD4 + and CD8 + single positive stages and critical for 36 TCR and Foxo1-mediated IL7Rα upregulation. In vivo reconstitution showed that enzymatic 37 activity and SH3-binding are essential for USP8 function in T-cells. USP8 f/f CD4-Cre mice 38 DOI

Aims: To investigate treatment of moderate-to-severe ulcerative colitis (UC) using real-world German health insurance claims data. Materials and methods: A retrospective, longitudinal cohort study was conducted from a German statutory health insurance database for adult patients with UC indexed on biologic therapy initiation (2013-2015). Anonymized data were evaluated for 12 months prior to (baseline) through 24 months after (follow-up) indexing. Biologic dose escalations, steroid and immunosuppressant use, healthcare resource utilization (HCRU) and direct healthcare costs were evaluated, with significant differences assessed across and between index biologics. Descriptive statistics, chi-square or Fisher's exact tests, and analysis of variance were performed. Results: The analysis included 304 patients (adalimumab, n ¼ 125; golimumab, n ¼ 47; infliximab, n ¼ 114; vedolizumab, n ¼ 18). Demographic and clinical characteristics were similar across biologics. Dose escalations occurred in 58% of patients (73% of patients receiving adalimumab), with 41% receiving subsequent de-escalation. Steroids were used during follow-up by 74% of patients; 25% received steroids >14 weeks after indexing. Overall, 41% of patients received an immunosuppressant during follow-up. Steroid and immunosuppressant use were similar across biologics. Total direct healthcare costs were higher during follow-up than baseline and differed significantly across treatments (p < .05), with highest costs for golimumab. Biologic costs contributed to a major portion of follow-up costs. HCRU and costs for most resources were higher in the first 12-month follow-up period than baseline. All resource use except gastroenterology visits returned to, or below, baseline levels 13-24 months post-index date. Limitations: There was potential for inappropriate inclusion/exclusion due to miscoding. Patients may have received biologics >12 months prior to the index date. Biologic originators and biosimilars could not be differentiated. Conclusions: These data suggest that control with current biologics is suboptimal. Further treatment options that provide sustained steroid-free remission for this patient population without the need for dose escalations or concomitant therapies may be warranted.

Background Screening for cervical cancer precursors by Papanicolaou cytology is a public health success story; however, its low sensitivity entails unnecessary referrals to colposcopy of healthy women with equivocal (ASCUS) or mild dysplasia (LSIL) cytology.Objective We assessed the accuracy of p16/Ki-67 immuno-testing for triage of low grade cervical cytology.Search strategy We systematically searched Medline, Embase, CRD and Cochrane databases, and handsearched key references.Selection criteria Eligible studies included women with ASCUS or LSIL cervical cytology who had undergone p16/Ki-67 testing and subsequent verification by colposcopy-directed biopsies and histologic analysis.Data collection and analysis We extracted data on patient characteristics and test conduct, diagnostic accuracy measures and assessed the methodological quality of the studies. R software was used to perform a bivariate analysis of test performance data.Main results Five eligible studies were identified. Four of the studies had high risk of bias. In the LSIL subgroup, the sensitivity of p16/Ki-67 testing ranged from 0.86 to 0.98, compared with 0.92-0.96 of high-risk HPV testing (hrHPV); specificity ranged from 0.43 to 0.68 versus 0.19 to 0.37, respectively. In the ASCUS subgroup, sensitivity ranged from 0.64 to 0.92 (p16/Ki67 test) versus 0.91 to 0.97 (hrHPV); specificity ranged from 0.53 to 0.81 versus 0.26 to 0.44, respectively.Authors' conclusions p16/Ki-67 testing cannot be recommended for triage women with ASCUS or LSIL cytology due to insufficient high-quality evidence. Further studies on test performance and the impact of p16/Ki-67-based triage on health outcomes are needed for a definitive evaluation of its clinical utility.Keywords Cervical cancer screening, human papillomavirus, molecular diagnosis and prognosis, p16INK4A, Ki-67.Please cite this paper as: Kisser A, Zechmeister-Koss I. A systematic review of p16/Ki-67 immuno-testing for triage of low grade cervical cytology. BJOG 2015;122:64-70.

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