The potential role of ubiquitin c-terminal hydrolases in oncogenesis

Deubiquitinating enzymes (DUBs), capable of removing ubiquitin (Ub) from protein substrates, are involved in numerous biological processes. The ubiquitin C-terminal hydrolases (UCHs) subfamily of DUBs consists of four members: UCH-L1, UCH-L3, UCH37 and BRCA1-associated protein-1 (BAP1). UCH-L1 possesses deubiquitinating activity and dimerization-dependent ubiquitin ligase activity, and functions as a mono-ubiquitin stabilizer; UCH-L3 does both deubiquitinating and deneddylating activity, except dimerization or ligase activity, and unlike UCH-L1, can interact with Lys48-linked Ub dimers to protect it from degradation and in the meanwhile to inhibit its hydrolase activity; UCH37 is responsible for the deubiquitinating activity in the 19S proteasome regulatory complex, and as indicated by the recent study, UCH37 is also associated with the human Ino80 chromatin-remodeling complex (hINO80) in the nucleus and can be activated via transient association of 19S regulatory particle- or proteasome-bound hRpn13 with hINO80; BAP1, binding to the wild-type BRCA1 RING finger domain, is regarded as a tumor suppressor, but for such suppressing activity, as demonstrated otherwise, both deubiquitinating activity and nucleus localization are required. There is growing evidence that UCH enzymes and human malignancies are closely correlated. Previous studies have shown that UCH enzymes play a crucial role in some signalings and cell-cycle regulation. In this review, we provided an insight into the relation between UCH enzymes and oncogenesis.     

The deubiquitinating enzyme UCH-L3 regulates the apical membrane recycling of the epithelial sodium channel

The epithelial sodium channel (ENaC) is ubiquitinated by the E3 ligase Nedd4-2 at the apical membranes of polarized cortical collecting duct (CCD) epithelial cells. This leads to ENaC endocytosis and possible degradation. Because ENaC is known to recycle at the apical membranes of CCD cells, deubiquitinating enzymes (DUBs) are likely involved in regulating ENaC surface density by facilitating ENaC recycling as opposed to degradation. Using a chemical probe approach to tag active DUBs, we identified ubiquitin C-terminal hydrolase (UCH) isoform L3 as the predominant DUB in endosomal compartments of CCD cells. Blocking UCH-L3 activity or reducing its expression by selective knockdown increased ENaC ubiquitination and resulted in its removal from the apical membranes of CCD cells. Functionally this caused a rapid reduction in transepithelial Na(+) currents across the CCD epithelia. Surface biotinylation demonstrated the loss of ENaC from the apical surface when UCH-L3 was inhibited. Whole cell or apical surface immunoprecipitation demonstrated increased ENaC ubiquitination with UCH-L3 inhibition. This constitutes a novel function for UCH in epithelia and in the regulation of ion channels and demonstrates the dynamic regulation of apically located ENaC by recycling, which is facilitated by this DUB.     

An atlas of altered expression of deubiquitinating enzymes in human cancer

Background

Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin (Ub) or ubiquitin-like gene products, remodel polyubiquitin(-like) chains on target proteins, and counteract protein ubiquitination exerted by E3 ubiquitin-ligases. A wealth of studies has established the relevance of DUBs to the control of physiological processes whose subversion is known to cause cellular transformation, including cell cycle progression, DNA repair, endocytosis and signal transduction. Altered expression of DUBs might, therefore, subvert both the proteolytic and signaling functions of the Ub system.

Methodology/Principal Findings

In this study, we report the first comprehensive screening of DUB dysregulation in human cancers by in situ hybridization on tissue microarrays (ISH-TMA). ISH-TMA has proven to be a reliable methodology to conduct this kind of study, particularly because it allows the precise identification of the cellular origin of the signals. Thus, signals associated with the tumor component can be distinguished from those associated with the tumor microenvironment. Specimens derived from various normal and malignant tumor tissues were analyzed, and the “normal” samples were derived, whenever possible, from the same patients from whom tumors were obtained. Of the ∼90 DUBs encoded by the human genome, 33 were found to be expressed in at least one of the analyzed tissues, of which 22 were altered in cancers. Selected DUBs were subjected to further validation, by analyzing their expression in large cohorts of tumor samples. This analysis unveiled significant correlations between DUB expression and relevant clinical and pathological parameters, which were in some cases indicative of aggressive disease.

Conclusions/Significance

The results presented here demonstrate that DUB dysregulation is a frequent event in cancer, and have implications for therapeutic approaches based on DUB inhibition.     

Engagement of ubiquitination and de-ubiquitination at rostral ventrolateral medulla in experimental brain death

Background

Whereas brain death is a vitally important clinical phenomenon, our contemporary understanding on its underlying cellular mechanisms remains elusive. This study evaluated whether the ubiquitin-proteasome system (UPS) in the rostral ventrolateral medulla (RVLM), a neural substrate that our laboratory identified previously to be intimately related to brain death, is engaged in this fatal process.

Methods

We performed proteomics, Western Blot, real-time PCR, ELISA and pharmacological experiments in conjunction with a clinically relevant experimental endotoxemia model of brain death based on intravenous administration of Escherichia coli lipopolysaccharide in adult male Sprague–Dawley rats.

Results

Proteomics, Western blot and enzyme activity analyses demonstrated that polyubiquitination was preserved and de-ubiquitination by ubiquitin C-terminal hydrolase isozyme-L1 (UCH-L1) was sustained, alongside increased monoubiquitin availability or proteasome activity in RVLM over the course of experimental endotoxemia. However, real-time PCR revealed no significant alteration in proteasome subunit alpha type-1, ubiquitin or UCH-L1 at mRNA level. Functionally, whereas microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) potentiated survival, an inhibitor of ubiquitin-recycling (ubiquitin aldehyde) or an UCH-L1 inhibitor exacerbated mortality.

Conclusions

We proposed previously that the progression towards brain death entails a tug-of-war between pro-death and pro-life programs in RVLM. It is conceivable that ubiquitination or de-ubiquitination in RVLM participate in brain death by regulating the degradation of the proteins involved in those programs.     

Characterisation of a Plancitoxin-1-Like DNase II Gene in Trichinella spiralis

Background

Deoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the Trichinella spiralis (T. spiralis) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"XM_003370715.1","term_id":"339256039","term_text":"XM_003370715.1"}}XM_003370715.1) contains the HKD motif in its C-terminus domain.

Methodology/Principal Findings

In this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from T. spiralis were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (Ts-Pt, 885 bp) and has been deposited in GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"KF984291","term_id":"594551321","term_text":"KF984291"}}KF984291. The recombinant protein (rTs-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that rTs-Pt was recognised by serum from T. spiralis-infected mice; the anti-rTs-Pt serum recognised crude antigens but not ES antigens. The Ts-Pt gene was examined at all T. spiralis developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. rTs-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C.

Conclusions

This study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity.     

Crystal structure of the de-ubiquitinating enzyme UCH37 (human UCH-L5) catalytic domain

Ubiquitin C-terminal hydrolases (UCHs) are one of five sub-families of de-ubiquitinating enzymes (DUBs) that hydrolyze the C-terminal peptide bond of ubiquitin. UCH37 (also called UCH-L5) is the only UCH family protease that interacts with the 19S proteasome regulatory complex and disassembles Lys48-linked poly-ubiquitin from the distal end of the chain. The structures of three UCHs, UCH-L1, UCH-L3, and YUH1, have been determined by X-ray crystallography. However, little is known about their physiological substrates. These enzymes do not hydrolyze large adducts of ubiquitin such as proteins. To identify and characterize the hydrolytic specificities of their substrates, the crystal structure of the UCH37 catalytic domain (UCH-domain) was determined and compared with that of the other UCHs. The overall folding patterns are similar in these UCHs. However, helix-3 is collapsed in UCH37 and the pattern of electrostatic potential on the surface of the putative substrate-binding site (P′-site) is different. Helix-3 comprises an edge of the P′-site. As a result, the P′-site is wider than that in other UCHs. These differences indicate that UCH37 can interact with larger adducts such as ubiquitin.     

A fluorescence assay for elucidating the substrate specificities of deubiquitinating enzymes

Ubiquitin C-terminal hydrolases (UCHs) are a representative family of deubiquitinating enzymes (DUBs), which specifically cleave ubiquitin (Ub) chains or extensions. Here we present a convenient method for characterizing the substrate specificities of various UCHs by fluorescently mutated Ub-fusion proteins (Ub(F45W)-Xaa) and di-ubiquitin chains (Ub(F45W)-diUb). After removal of the intact substrate by Ni(2+)-NTA affinity, the enzymatic activities of UCHs were quantitatively determined by recording fluorescence of the Ub(F45W) product. The results show that three UCHs, i.e. UCH-L1, UCH-L3 and UCH37/UCH-L5, are distinct in their substrate specificities for the Ub-fusions and diUb chains. This assay method may also be applied to study the enzymatic activities and substrate specificities of other DUBs.     

The interaction between ubiquitin C-terminal hydrolase 37 and glucose-regulated protein 78 in hepatocellular carcinoma

The ubiquitin C-terminal hydrolase (UCH) is a subfamily of deubiquitinating enzymes, which consists of four members: UCH-L1, UCH-L3, UCH37, and BRCA1-associated protein-1. Although there is growing evidence that UCH enzymes and human malignancies are closely correlated, there have been few studies on UCH37, especially on its interactions with other proteins. In the current study, a functional proteomic analysis was performed to screen UCH37-interacting proteins in hepatocellular carcinoma (HCC), and glucose-regulated protein 78 was identified as one interacting with UCH37, which was confirmed by co-immunoprecipitation and confocal laser scanning microscopy analysis, suggesting that their interaction could provide a new insight into the mechanism of HCC.     

OTUB1 overexpression in mesangial cells is a novel regulator in the pathogenesis of glomerulonephritis through the decrease of DCN level

Background

OTUB1 is a member of OTUs (Ovarian-tumor-domain-containing proteases), a deubiquitinating enzymes family (DUBs), which was shown as a proteasome-associated DUB to be involved in the proteins Ub-dependent degradation. It has been reported that OTUB1 was expressed in kidney tissue. But its concrete cellular location and function in the kidney remain unclear. Decorin (DCN) in mesangial cells (MC) is considered to be a potentially important factor for antagonizing glomerulonephritides, and its degradation is mediated by ubiquitination. The aim of this study is to investigate the role of OTUB1 expression in MC and its relationship with DCN during glomerulonephritis.

Methodology/Principal Findings

Using quantitative RT-PCR and Western blot, we demonstrated that OTUB1 mRNA and protein were constitutively expressed in cultured rat MC and found to be upregulated by the stimulation of IL-1β or ATS. OTUB1 overexpression was detected in the mesangial area of glomeruli in some immunocomplex mediated nephritides such as IgA nephropathy, acute diffuse proliferative glomerulonephritis and lupus nephritis by immunohistochemistry. The immunoprecipitation assay demonstrated that OTUB1 interacted with DCN. The overexpression of OTUB1 enhanced the ubiquitination and degradation of DCN in MC.

Conclusion/Significance

These data showed the inflammatory injury could up-regulate OTUB1 expression in MC, which might attribute the promoting effect of OTUB1 on glomerulonephritides to the decrease of DCN level.     

Ubiquitin dimers control the hydrolase activity of UCH-L3

Ubiquitin (Ub) carboxy terminal hydrolase (UCH)-L1 and UCH-L3 are two of the deubiquitinating enzymes expressed in the brain. Both gad mice, which lack UCH-L1 expression and Uchl3 knockout mice exhibit neurodegeneration, although at distinct areas. These phenotypes indicate the importance of UCH-L1 and UCH-L3 in the regulation of the central nervous system. However, molecular substrates and the molecular regulators of UCH-L1 and UCH-L3 remain poorly identified. Here we show that Ub dimers interact non-covalently with UCH-L3 in vitro and in cells. These interactions were not observed with UCH-L1 in cells. In vitro, K48-linked Ub dimers pronouncedly inhibited the hydrolase activity of UCH-L3, while mono-Ub, a previously identified interacting protein, inhibited the hydrolase activity of UCH-L1. These results indicate that mono-Ub and Ub dimers may regulate the enzymatic functions of UCH-L1 and UCH-L3, respectively, in vivo.     

Partially Protective Immunity Induced by a 20 kDa Protein Secreted by Trichinella spiralis Stichocytes

Background

Trichinella spiralis infection induces protective immunity against re-infection in animal models. Identification of the antigens eliciting acquired immunity during infection is important for vaccine development against Trichinella infection and immunodiagnosis.

Methods and Findings

The T. spiralis adult cDNA library was immunoscreened with sera from pigs experimentally infected with 20,000 infective T. spiralis larvae. Total 43 positive clones encoding for 28 proteins were identified; one of the immunodominant proteins was 20 kDa Ts-ES-1 secreted by Trichinella stichocytes and existing in the excretory/secretory (ES) products of T. spiralis adult and muscle larval worms. Ts-ES-1 contains 172 amino acids with a typical signal peptide in the first 20 amino acids. The expression of Ts-ES-1 was detected in both the adult and muscle larval stages at the mRNA and protein expression levels. Mice immunized with recombinant Ts-ES-1 (rTs-ES-1) formulated with ISA50v2 adjuvant exhibited a significant worm reduction in both the adult worm (27%) and muscle larvae burden (42.1%) after a challenge with T. spiralis compared to the adjuvant control group (p<0.01). The rTs-ES-1-induced protection was associated with a high level of specific anti-Ts-ES-1 IgG antibodies and a Th1/Th2 mixed immune response.

Conclusion

The newly identified rTs-ES-1 is an immunodominant protein secreted by Trichinella stichocytes during natural infection and enables to the induction of partial protective immunity in vaccinated mice against Trichinella infection. Therefore, rTs-ES-1 is a potential candidate for vaccine development against trichinellosis.     

A Global Census of Fission Yeast Deubiquitinating Enzyme Localization and Interaction Networks Reveals Distinct Compartmentalization Profiles and Overlapping Functions in Endocytosis and Polarity

Ubiquitination and deubiquitination are reciprocal processes that tune protein stability, function, and/or localization. The removal of ubiquitin and remodeling of ubiquitin chains is catalyzed by deubiquitinating enzymes (DUBs), which are cysteine proteases or metalloproteases. Although ubiquitination has been extensively studied for decades, the complexity of cellular roles for deubiquitinating enzymes has only recently been explored, and there are still several gaps in our understanding of when, where, and how these enzymes function to modulate the fate of polypeptides. To address these questions we performed a systematic analysis of the 20 Schizosaccharomyces pombe DUBs using confocal microscopy, proteomics, and enzymatic activity assays. Our results reveal that S. pombe DUBs are present in almost all cell compartments, and the majority are part of stable protein complexes essential for their function. Interestingly, DUB partners identified by our study include the homolog of a putative tumor suppressor gene not previously linked to the ubiquitin pathway, and two conserved tryptophan-aspartate (WD) repeat proteins that regulate Ubp9, a DUB that we show participates in endocytosis, actin dynamics, and cell polarity. In order to understand how DUB activity affects these processes we constructed multiple DUB mutants and find that a quintuple deletion of ubp4 ubp5 ubp9 ubp15 sst2/amsh displays severe growth, polarity, and endocytosis defects. This mutant allowed the identification of two common substrates for five cytoplasmic DUBs. Through these studies, a common regulatory theme emerged in which DUB localization and/or activity is modulated by interacting partners. Despite apparently distinct cytoplasmic localization patterns, several DUBs cooperate in regulating endocytosis and cell polarity. These studies provide a framework for dissecting DUB signaling pathways in S. pombe and may shed light on DUB functions in metazoans.     

A double-edged sword role for ubiquitin-proteasome system in brain stem cardiovascular regulation during experimental brain death

Background

Brain stem cardiovascular regulatory dysfunction during brain death is underpinned by an upregulation of nitric oxide synthase II (NOS II) in rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from blood pressure of comatose patients that disappears before brain death ensues. Furthermore, the ubiquitin-proteasome system (UPS) may be involved in the synthesis and degradation of NOS II. We assessed the hypothesis that the UPS participates in brain stem cardiovascular regulation during brain death by engaging in both synthesis and degradation of NOS II in RVLM.

Methodology/Principal Findings

In a clinically relevant experimental model of brain death using Sprague-Dawley rats, pretreatment by microinjection into the bilateral RVLM of proteasome inhibitors (lactacystin or proteasome inhibitor II) antagonized the hypotension and reduction in the life-and-death signal elicited by intravenous administration of Escherichia coli lipopolysaccharide (LPS). On the other hand, pretreatment with an inhibitor of ubiquitin-recycling (ubiquitin aldehyde) or ubiquitin C-terminal hydrolase isozyme L1 (UCH-L1) potentiated the elicited hypotension and blunted the prevalence of the life-and-death signal. Real-time polymerase chain reaction, Western blot, electrophoresis mobility shift assay, chromatin immunoprecipitation and co-immunoprecipitation experiments further showed that the proteasome inhibitors antagonized the augmented nuclear presence of NF-κB or binding between NF-κB and nos II promoter and blunted the reduced cytosolic presence of phosphorylated IκB. The already impeded NOS II protein expression by proteasome inhibitor II was further reduced after gene-knockdown of NF-κB in RVLM. In animals pretreated with UCH-L1 inhibitor and died before significant increase in nos II mRNA occurred, NOS II protein expression in RVLM was considerably elevated.

Conclusions/Significance

We conclude that UPS participates in the defunct and maintained brain stem cardiovascular regulation during experimental brain death by engaging in both synthesis and degradation of NOS II at RVLM. Our results provide information on new therapeutic initiatives against this fatal eventuality.