Deubiquitination Assays Research Articles

Alterations of histone modifications by cobalt compounds

In the present study, we examined the effects of CoCl(2) on multiple histone modifications at the global level. We found that in both human lung carcinoma A549 cells and human bronchial epithelial Beas-2B cells, exposure to CoCl(2) (>/=200 muM) for 24 h increased H3K4me3, H3K9me2, H3K9me3, H3K27me3, H3K36me3, uH2A and uH2B but decreased acetylation at histone H4 (AcH4). Further investigation demonstrated that in A549 cells, the increase in H3K4me3 and H3K27me3 by cobalt ions exposure was probably through enhancing histone methylation processes, as methionine-deficient medium blocked the induction of H3K4me3 and H3K27me3 by cobalt ions, whereas cobalt ions increased H3K9me3 and H3K36me3 by directly inhibiting JMJD2A demethylase activity in vitro, which was probably due to the competition of cobalt ions with iron for binding to the active site of JMJD2A. Furthermore, in vitro ubiquitination and deubiquitination assays revealed that the cobalt-induced histone H2A and H2B ubiquitination is the result of inhibition of deubiquitinating enzyme activity. Microarray data showed that exposed to 200 microM of CoCl(2) for 24 h, A549 cells not only increased but also decreased expression of hundreds of genes involved in different cellular functions, including tumorigenesis. This study is the first to demonstrate that cobalt ions altered epigenetic homeostasis in cells. It also sheds light on the possible mechanisms involved in cobalt-induced alteration of histone modifications, which may lead to altered programs of gene expression and carcinogenesis since cobalt at higher concentrations is a known carcinogen.

USP11 Stabilizes HPV-16E7 and Further Modulates the E7 Biological Activity

HPV-16E7 is a major transforming protein, which has been implicated in the development of cervical cancer. The stability of E7 is thus important to ensure its fully functional status. Using the yeast two-hybrid system, we found that USP11 (ubiquitin-specific protease 11), a member of a protein family that cleaves polyubiquitin chains and/or ubiquitin precursors, interacts and forms a specific complex with HPV-16E7. Our results indicate that the USP11 can greatly increase the steady state level of HPV-16E7 by reducing ubiquitination and attenuating E7 degradation. In contrast, a catalytically inactive mutant of USP11 abolished the deubiquitinating ability and returned E7 to a normal rate of degradation. Moreover, USP11 not only protected E7 from ubiquitination but also influenced E7 function as a modulator of cell growth status. These results suggest that USP11 plays an important role in regulating the levels of E7 protein and subsequently affects the biological function of E7 as well as its contribution to cell transformation by HPV-16E7.

Structural Basis for Ubiquitin Recognition by the Otu1 Ovarian Tumor Domain Protein

Ubiquitination of proteins modifies protein function by either altering their activities, promoting their degradation, or altering their subcellular localization. Deubiquitinating enzymes are proteases that reverse this ubiquitination. Previous studies demonstrate that proteins that contain an ovarian tumor (OTU) domain possess deubiquitinating activity. This domain of approximately 130 amino acids is weakly similar to the papain family of proteases and is highly conserved from yeast to mammals. Here we report structural and functional studies on the OTU domain-containing protein from yeast, Otu1. We show that Otu1 binds polyubiquitin chain analogs more tightly than monoubiquitin and preferentially hydrolyzes longer polyubiquitin chains with Lys(48) linkages, having little or no activity on Lys(63)- and Lys(29)-linked chains. We also show that Otu1 interacts with Cdc48, a regulator of the ER-associated degradation pathway. We also report the x-ray crystal structure of the OTU domain of Otu1 covalently complexed with ubiquitin and carry out structure-guided mutagenesis revealing a novel mode of ubiquitin recognition and a variation on the papain protease catalytic site configuration that appears to be conserved within the OTU family of ubiquitin hydrolases. Together, these studies provide new insights into ubiquitin binding and hydrolysis by yeast Otu1 and other OTU domain-containing proteins.

Inhibition of p97-dependent Protein Degradation by Eeyarestatin I

Elimination of misfolded proteins from the endoplasmic reticulum (ER) by ER-associated degradation involves substrate retrotranslocation from the ER lumen into the cytosol for degradation by the proteasome. For many substrates, retrotranslocation requires the action of ubiquitinating enzymes, which polyubiquitinate substrates emerging from the ER lumen, and of the p97-Ufd1-Npl4 ATPase complex, which hydrolyzes ATP to dislocate polyubiquitinated substrates into the cytosol. Polypeptides extracted by p97 are eventually transferred to the proteasome for destruction. In mammalian cells, ERAD can be blocked by a chemical inhibitor termed Eeyarestatin I, but the mechanism of EerI action is unclear. Here we report that EerI can associate with a p97 complex to inhibit ERAD. The interaction of EerI with the p97 complex appears to negatively influence a deubiquitinating process that is mediated by p97-associated deubiquitinating enzymes. We further show that ataxin-3, a p97-associated deubiquitinating enzyme previously implicated in ER-associated degradation, is among those affected. Interestingly, p97-associated deubiquitination is also involved in degradation of a soluble substrate. Our analyses establish a role for a novel deubiquitinating process in proteasome-dependent protein turnover.

Nickel compounds induce histone ubiquitination by inhibiting histone deubiquitinating enzyme activity

Nickel (Ni) compounds are known carcinogens but underlying mechanisms are not clear. Epigenetic changes are likely to play an important role in nickel ion carcinogenesis. Previous studies have shown epigenetic effects of nickel ions, including the loss of histone acetylation and a pronounced increase in dimethylated H3K9 in nickel-exposed cells. In this study, we demonstrated that both water-soluble and insoluble nickel compounds induce histone ubiquitination (uH2A and uH2B) in a variety of cell lines. Investigations of the mechanism by which nickel increases histone ubiquitination in cells reveal that nickel does not affect cellular levels of the substrates of this modification, i.e., ubiquitin, histones, and other non-histone ubiquitinated proteins. In vitro ubiquitination and deubiquitination assays have been developed to further investigate possible effects of nickel on enzymes responsible for histone ubiquitination. Results from the in vitro assays demonstrate that the presence of nickel did not affect the levels of ubiquitinated histones in the ubiquitinating assay. Instead, the addition of nickel significantly prevents loss of uH2A and uH2B in the deubiquitinating assay, suggesting that nickel-induced histone ubiquitination is the result of inhibition of (a) putative deubiquitinating enzyme(s). Additional supporting evidence comes from the comparison of the response to nickel ions with a known deubiquitinating enzyme inhibitor, iodoacetamide (IAA). This study is the first to demonstrate such effects of nickel ions on histone ubiquitination. It also sheds light on the possible mechanisms involved in altering the steady state of this modification. The study provides further evidence that supports the notion that nickel ions alter epigenetic homeostasis in cells, which may lead to altered programs of gene expression and carcinogenesis.

The Deubiquitinating Enzyme Ubp2 Modulates Rsp5-dependent Lys63-linked Polyubiquitin Conjugates in Saccharomyces cerevisiae

The functions of Lys(63)-linked polyubiquitin chains are poorly understood, as are the enzymes that specifically generate Lys(63)-linked conjugates. Rsp5 is a HECT (homologous to E6AP C terminus) ubiquitin ligase involved in numerous processes, and an associated deubiquitinating enzyme, Ubp2, modulates its activity. A dramatic increase in Lys(63)-linked conjugates was observed in ubp2Delta cells. The formation of these was Rsp5-dependent, and ubp2Delta phenotypes could be suppressed by prevention of formation of Lys(63) conjugates. Cell wall integrity was impaired in rsp5-1 cells and in cells defective in Lys(63)-polyubiquitination, as assayed by calcofluor white sensitivity, and ubp2Delta and rup1Delta mutants suppressed the calcofluor white sensitivity of rsp5-1. A large fraction of the Lys(63) conjugates in ubp2Delta cells bound to Rsp5, and a proteomics approach was used to identify Rsp5 substrates subject to Ubp2 regulation. Two closely related proteins, Csr2 and Ecm21, were among the identified proteins. Both were efficiently Lys(63)-polyubiquitinated by Rsp5 and deubiquitinated by Ubp2. Together, these results indicate that Ubp2 modulates Lys(63)-polyubiquitination of Rsp5 substrates in vivo, including ubiquitination of two newly identified Rsp5 substrates.

A Novel in Vitro Assay for Deubiquitination of IκBα

The ubiquitin-proteasome pathway (UPP) controls a wide range of signal transduction cascades by targeting key regulatory proteins for 26S proteasome-mediated degradation. Several observations suggest that protein deubiquitination may modulate this process; however, few experiments have been performed to test this idea. An excellent model system for studying the regulatory role of the UPP is signal transduction via the nuclear factor-κB (NF-κB) family of transcription factors. The principal inhibitor of NF-κB, IκBα, is polyubiquitinated and degraded in response to diverse stimuli. In this study, we sought to determine whether IκBα deubiquitination also occurs. We established an in vitro deubiquitination assay using polyubiquitinated IκBα as the substrate. Our data provide evidence of an IκBα-directed deubiquitinating activity present in lysates of several cell lines. This activity was inhibited by ubiquitin aldehyde, a specific inhibitor of deubiquitinating enzymes, as well as by alkylating reagents or heat, but was unaffected by the inhibition of several other classes of proteases. Cell lysates and the deubiquitinating enzyme, UCH-L3, hydrolyzed ubiquitin 7-amido-4-methylcoumarin, a model substrate for assaying deubiquitinating activities. However, UCH-L3 had no detectable activity toward ubiquitinated IκBα, thus suggesting a degree of enzymatic specificity in the deubiquitination of IκBα. This assay will be useful for the study of IκBα deubiquitination. Moreover, this assay can be adapted to monitor the deubiquitination of other proteins modified by ubiquitin conjugation.

The human UNP locus at 3p21.31 encodes two tissue-selective, cytoplasmic isoforms with deubiquitinating activity that have reduced expression in small cell lung carcinoma cell lines.

The human Unp gene at 3p21.3 has sequence similarity to ubiquitin proteases and has been suggested to play a role in carcinogenesis of the lung (Gray et al., 1995). To investigate this possibility, we isolated cDNAs from several human tissue libraries and found evidence for two major isoforms, encoding proteins predicted to either contain an internal 47 amino acid segment or not. Both are functional in deubiquitination assays, and mutation of a critical conserved cysteine residue to alanine abolished activity. Unp specifies two closely-migrating transcripts whose relative abundance varies among human adult tissues. Antibodies specific to UNP confirm the presence of at least two endogenous protein isoforms of approximately 105-110 kDa in cell lysates, as predicted from the cDNA sequences. Cellular fractionation and immunocytochemistry revealed UNP expression localized primarily in the cytoplasm. When we examined a panel of lung-derived cell lines for both UNP mRNA and protein expression, we found reduced levels of UNP protein in all four small cell lung carcinoma cell lines tested. These findings directly contradict and offer alternative interpretations to a number of previously published observations on Unp.