Embryonic Demise Caused by Targeted Disruption of a Cysteine Protease Dub-2

Background

A plethora of biological metabolisms are regulated by the mechanisms of ubiquitination, wherein this process is balanced with the action of deubiquitination system. Dub-2 is an IL-2-inducible, immediate-early gene that encodes a deubiquitinating enzyme with growth regulatory activity. DUB-2 presumably removes ubiquitin from ubiquitin-conjugated target proteins regulating ubiquitin-mediated proteolysis, but its specific target proteins are unknown yet.

Methodology/Principal Findings

To elucidate the functional role of Dub-2, we generated genetically modified mice by introducing neo cassette into the second exon of Dub-2 and then homologous recombination was done to completely abrogate the activity of DUB-2 proteins. We generated Dub-2+/− heterozygous mice showing a normal phenotype and are fertile, whereas new born mouse of Dub-2−/− homozygous alleles could not survive. In addition, Dub-2−/− embryo could not be seen between E6.5 and E12.5 stages. Furthermore, the number of embryos showing normal embryonic development for further stages is decreased in heterozygotes. Even embryonic stem cells from inner cell mass of Dub-2−/− embryos could not be established.

Conclusions

Our study suggests that the targeted disruption of Dub-2 may cause embryonic lethality during early gestation, possibly due to the failure of cell proliferation during hatching process.     

Deubiquitinating enzyme USP36 contains the PEST motif and is polyubiquitinated

The ubiquitin-mediated protein degradation pathway has been emphasized for the regulation of numerous cellular mechanisms and the significance of deubiquitination, mediated by deubiquitinating (DUB) enzymes, has been emerging as an essential regulatory step to control these cellular mechanisms. Previously, we demonstrated a human DUB enzyme, HeLa DUB-1, expressed in human ovarian cancer cells. Here, we report human USP36, which has the extension of the C-terminal region of HeLa DUB-1 and has conserved amino acid domains as previously shown in other DUBs. Human USP36, encoding a DUB enzyme, was isolated from ovarian cancer cells using RT-PCR and characterized. We identified DUB enzyme activity of USP36 by analyzing its capability to cleave the ubiquitin. Interestingly, structural and immunoprecipitation analyses revealed for the first time that USP36 contains the PEST motif and is polyubiquitinated.     

The murine DUB-1 gene is specifically induced by the betac subunit of interleukin-3 receptor.

Cytokines regulate cell growth and differentiation by inducing the expression of specific target genes. We have recently isolated a cytokine-inducible, immediate-early cDNA, DUB-1, that encodes a deubiquitinating enzyme. The DUB-1 mRNA was specifically induced by the receptors for interleukin-3, granulocyte-macrophage colony-stimulating factor, and interleukin-5, suggesting a role for the beta common (betac subunit known to be shared by these receptors. In order to identify the mechanism of cytokine induction, we isolated a murine genomic clone for DUB-1 containing a functional promoter region. The DUB-1 gene contains two exons, and the nucleotide sequence of its coding region is identical to the sequence of DUB-1 cDNA. Various regions of the 5' flanking region of the DUB-1 gene were assayed for cytokine-inducible activity. An enhancer region that retains the beta c-specific inducible activity of the DUB-1 gene was identified. Enhancer activity was localized to a 112-bp fragment located 1.4 kb upstream from the ATG start codon. Gel mobility shift assays revealed two specific protein complexes that bound to this minimal enhancer region. One complex was induced by betac signaling, while the other was noninducible. Finally, the membrane-proximal region of human betac was required for DUB-1 induction. In conclusion, DUB-1 is the first example of an immediate-early gene that is induced by a specific subunit of a cytokine receptor. Further analysis of the DUB-1 enhancer element may reveal specific determinants of a betac-specific signaling pathway.     

The deubiquitinating enzyme DUB2A enhances CSF3 signalling by attenuating lysosomal routing of the CSF3 receptor

Ubiquitination of the CSF3R [CSF3 (colony-stimulating factor 3) receptor] occurs after activated CSF3Rs are internalized and reside in early endosomes. CSF3R ubiquitination is crucial for lysosomal routing and degradation. The E3 ligase SOCS3 (suppressor of cytokine signalling 3) has been shown to play a major role in this process. Deubiquitinating enzymes remove ubiquitin moieties from target proteins by proteolytic cleavage. Two of these enzymes, AMSH [associated molecule with the SH3 domain of STAM (signal transducing adaptor molecule)] and UBPY (ubiquitin isopeptidase Y), interact with the general endosomal sorting machinery. Whether deubiquitinating enzymes control CSF3R trafficking from early towards late endosomes is unknown. In the present study, we asked whether AMSH, UBPY or a murine family of deubiquitinating enzymes could fulfil such a role. This DUB family (deubiquitin enzyme family) comprises four members (DUB1, DUB1A, DUB2 and DUB2A), which were originally described as being haematopoietic-specific and cytokine-inducible, but their function in cytokine receptor routing and signalling has remained largely unknown. We show that DUB2A expression is induced by CSF3 in myeloid 32D cells and that DUB2 decreases ubiquitination and lysosomal degradation of the CSF3R, leading to prolonged signalling. These results support a model in which CSF3R ubiquitination is dynamically controlled at the early endosome by feedback mechanisms involving CSF3-induced E3 ligase (SOCS3) and deubiquitinase (DUB2A) activities.     

DUB-1, a fate determinant of dynein heavy chain in B-lymphocytes, is regulated by the ubiquitin-proteasome pathway

Ubiquitination and deubiquitination of post-translational modification play counter roles in determining the fate of protein function in eukaryotic system for maintaining the cellular homeostasis. Even though novel family members of growth-regulating deubiquitinating enzymes (DUB-1 and DUB-2) have been identified, their target proteins and functions are poorly understood. Dub genes encoding DUB-1 and DUB-2 are immediate-early genes and are induced in response to cytokine stimuli rapidly and transiently. In order to explore the possible proteins regulated by DUB-1, we performed the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis followed by immunoprecipitation. We confirmed that DUB-1 interacts with dynein heavy chain, which is known to regulate the movement of organelles and microtubule binding ability. In addition, structural and immunoprecipitation analyses revealed that DUB-1 contains a putative PEST motif and is polyubiquitinated, indicating that DUB-1 is also regulated by the ubiquitin-proteasome pathway.     

Deubiquitinating enzyme regulation of the p53 pathway: A lesson from Otub1

Deubiquitination has emerged as an important mechanism of p53 regulation. A number of deubiquitinating enzymes(DUBs) from the ubiquitin-specific protease family have been shown to regulate the p53-MDM2-MDMX networks. We recently reported that Otub1, a DUB from the OTU-domain containing protease family, is a novel p53 regulator. Interestingly, Otub1 abrogates p53 ubiquitination and stabilizes and activates p53 in cells independently of its deubiquitinating enzyme activity. Instead, it does so by inhibiting the MDM2 cognate ubiquitin-conjugating enzyme(E2) UbcH5. Otub1 also regulates other biological signaling through this non-canonical mechanism, suppression of E2, including the inhibition of DNA-damage-induced chromatin ubiquitination. Thus, Otub1 evolves as a unique DUB that mainly suppresses E2 to regulate substrates. Here we review the current progress made towards the understanding of the complex regulation of the p53 tumor suppressor pathway by DUBs, the biological function of Otub1 including its positive regulation of p53, and the mechanistic insights into how Otub1 suppresses E2.     

JosD1, a Membrane-targeted Deubiquitinating Enzyme,Is Activated by Ubiquitination and Regulates Membrane Dynamics,Cell Motility,and Endocytosis

The functional diversity of deubiquitinating enzymes (DUBs) is not well understood. The MJD family of DUBs consists of four cysteine proteases that share a catalytic “Josephin” domain. The family is named after the DUB ATXN3, which causes the neurodegenerative disease Machado-Joseph disease. The two closely related Josephin domain-containing (JosD) proteins 1 and 2 consist of little more than the Josephin domain. To gain insight into the properties of Josephin domains, we investigated JosD1 and JosD2. JosD1 and JosD2 were found to differ fundamentally in many respects. In vitro, only JosD2 can cleave ubiquitin chains. In contrast, JosD1 cleaves ubiquitin chains only after it is monoubiquitinated, a form of posttranslational-dependent regulation shared with ATXN3. A significant fraction of JosD1 is monoubiquitinated in diverse mouse tissues. In cell-based studies, JosD2 localizes to the cytoplasm whereas JosD1 preferentially localizes to the plasma membrane, particularly when ubiquitinated. The membrane occupancy by JosD1 suggests that it could participate in membrane-dependent events such as cell motility and endocytosis. Indeed, time-lapse imaging revealed that JosD1 enhances membrane dynamics and cell motility. JosD1 also influences endocytosis in cultured cells by increasing the uptake of endocytic markers of macropinocytosis while decreasing those for clathrin- and caveolae-mediated endocytosis. Our results establish that two closely related DUBs differ markedly in activity and function and that JosD1, a membrane-associated DUB whose activity is regulated by ubiquitination, helps regulate membrane dynamics, cell motility, and endocytosis.     

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.     

The DUB/USP17 deubiquitinating enzymes, a multigene family within a tandemly repeated sequence

Here we report the identification of 10 human, 1 murine, and 2 rat ORFs, all of which represent additional members of the DUB/USP17 family of deubiquitinating enzymes. In addition, we demonstrate that this family constitutes part of a tandemly repeated sequence conserved throughout humans, mice, and rats. Furthermore, upon examination of the known family members we have found that the multiple genes observed, in contrast to other gene families, have arisen due to the independent expansion of an ancestral sequence within each species. This premise is further strengthened by the observation that the murine and rat genes span two exons while their human counterparts have one. These observations, in conjunction with previous work demonstrating that the DUB/USP17's are cytokine inducible and that they regulate both cell growth and survival, suggest that the DUB/USP17's are a large highly conserved family of genes that may play an important role in controlling cell fate.     

Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20

Nuclear factor κB (NF-κB) activation in tumor necrosis factor, interleukin-1, and Toll-like receptor pathways requires Lys63-linked nondegradative polyubiquitination. A20 is a specific feedback inhibitor of NF-κB activation in these pathways that possesses dual ubiquitin-editing functions. While the N-terminal domain of A20 is a deubiquitinating enzyme (DUB) for Lys63-linked polyubiquitinated signaling mediators such as TRAF6 and RIP, its C-terminal domain is a ubiquitin ligase (E3) for Lys48-linked degradative polyubiquitination of the same substrates. To elucidate the molecular basis for the DUB activity of A20, we determined its crystal structure and performed a series of biochemical and cell biological studies. The structure reveals the potential catalytic mechanism of A20, which may be significantly different from papain-like cysteine proteases. Ubiquitin can be docked onto a conserved A20 surface; this interaction exhibits charge complementarity and no steric clash. Surprisingly, A20 does not have specificity for Lys63-linked polyubiquitin chains. Instead, it effectively removes Lys63-linked polyubiquitin chains from TRAF6 without dissembling the chains themselves. Our studies suggest that A20 does not act as a general DUB but has the specificity for particular polyubiquitinated substrates to assure its fidelity in regulating NF-κB activation in the tumor necrosis factor, interleukin-1, and Toll-like receptor pathways.     

Two novel protein-tyrosine kinases, each with a second phosphotransferase-related catalytic domain, define a new class of protein kinase.

The protein-tyrosine kinases (PTKs) are a burgeoning family of proteins, each of which bears a conserved domain of 250 to 300 amino acids capable of phosphorylating substrate proteins on tyrosine residues. We recently exploited the existence of two highly conserved sequence elements within the catalytic domain to generate PTK-specific degenerate oligonucleotide primers (A. F. Wilks, Proc. Natl. Acad. Sci. USA 86:1603-1607, 1989). By application of the polymerase chain reaction, portions of the catalytic domains of several novel PTKs were amplified. We describe here the primary sequence of one of these new PTKs, JAK1 (from Janus kinase), a member of a new class of PTK characterized by the presence of a second phosphotransferase-related domain immediately N terminal to the PTK domain. The second phosphotransferase domain bears all the hallmarks of a protein kinase, although its structure differs significantly from that of the PTK and threonine/serine kinase family members. A second member of this family (JAK2) has been partially characterized and exhibits a similar array of kinase-related domains. JAK1 is a large, widely expressed membrane-associated phosphoprotein of approximately 130,000 Da. The PTK activity of JAK1 has been located in the C-terminal PTK-like domain. The role of the second kinaselike domain is unknown.     

Molecular cloning of rabbit CAP-50, a calcyclin-associated annexin protein.

CAP-50 is a member of annexin family proteins which binds specifically to calcyclin in a Ca2+ dependent manner (Tokumitsu. H., Mizutani. A., Minami. H., Kobayashi. R., and Hidaka. H. (1992) J. Biol. Chem. 267,8919-8924). The cDNA representing the rabbit form of this protein has been cloned from rabbit lung cDNA library. Sequence analysis of two overlapping clones revealed a 81-nucleotides 5'-nontranslated region, 1512-nucleotides of open reading frame, a 672-nucleotides 3'-nontranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of portions of the deduced amino acid sequence with eight sequences of proteolytic peptides obtained from rabbit lung protein. CAP-50 cDNA encodes a 503 residue protein with a calculated M(r) of 54,043 and shows that the protein is composed of four imperfect repeats and hydrophobic N-terminal region. C-terminal region including four imperfect repeats shows 58.1% identity with human synexin (annexin VII), 48.0% identity with annexin I, 47.4% identity with annexin II, 60.1% identity with annexin IV, 54.5% identity with annexin V. Hydrophobic N-terminal region composed of 202 amino acid residues is not homologous with other annexin proteins suggesting that CAP-50 is a novel member of annexin family proteins.     

Reversible monoubiquitination of PCNA: A novel slant on regulating translesion DNA synthesis

Deubiquitination of monoubiquitinated proteins is effected by a large number of deubiquitinating enzymes (DUBs) that function as cysteine proteases in eukaryotic cells. Huang et al. (2006) report the regulation of monoubiquitination of PCNA by autocleavage of a DUB called USP1 in cells exposed to UV radiation.     

Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild‐type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight‐binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30‐fold higher affinity of Ubp8^C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.     

Chalcone-based small-molecule inhibitors attenuate malignant phenotype via targeting deubiquitinating enzymes

The ubiquitin-proteasome system (UPS) is usurped by many if not all cancers to regulate their survival, proliferation, invasion, angiogenesis and metastasis. Bioflavonoids curcumin and chalcones exhibit anti-neoplastic selectivity through inhibition of the 26S proteasome-activity within the UPS. Here, we provide evidence for a novel mechanism of action of chalcone-based derivatives AM146, RA-9 and RA-14, which exert anticancer activity by targeting deubiquitinating enzymes (DUB) without affecting 20S proteasome catalytic-core activity. The presence of the α,β-unsaturated carbonyl group susceptible to nucleophilic attack from the sulfhydryl of cysteines in the active sites of DUB determines the capacity of novel small-molecules to act as cell-permeable, partly selective DUB inhibitors and induce rapid accumulation of polyubiquitinated proteins and deplete the pool of free ubiquitin. These chalcone-derivatives directly suppress activity of DUB UCH-L1, UCH-L3, USP2, USP5 and USP8, which are known to regulate the turnover and stability of key regulators of cell survival and proliferation. Inhibition of DUB-activity mediated by these compounds downregulates cell-cycle promoters, e.g., cyclin D1 and upregulates tumor suppressors p53, p27Kip1 and p16Ink4A. These changes are associated with arrest in S-G₂/M, abrogated anchorage-dependent growth and onset of apoptosis in breast, ovarian and cervical cancer cells without noticeable alterations in primary human cells. Altogether, this work provides evidence of antitumor activity of novel chalcone-based derivatives mediated by their DUB-targeting capacity; supports the development of pharmaceuticals to directly target DUB as a most efficient strategy compared with proteasome inhibition and also provides a clear rationale for the clinical evaluation of these novel small-molecule DUB inhibitors.     

Chalcone-based small-molecule inhibitors attenuate malignant phenotype via targeting deubiquitinating enzymes

The ubiquitin-proteasome system (UPS) is usurped by many if not all cancers to regulate their survival, proliferation, invasion, angiogenesis and metastasis. Bioflavonoids curcumin and chalcones exhibit anti-neoplastic selectivity through inhibition of the 26S proteasome-activity within the UPS. Here, we provide evidence for a novel mechanism of action of chalcone-based derivatives AM146, RA-9 and RA-14, which exert anticancer activity by targeting deubiquitinating enzymes (DUB) without affecting 20S proteasome catalytic-core activity. The presence of the α,β-unsaturated carbonyl group susceptible to nucleophilic attack from the sulfhydryl of cysteines in the active sites of DUB determines the capacity of novel small-molecules to act as cell-permeable, partly selective DUB inhibitors and induce rapid accumulation of polyubiquitinated proteins and deplete the pool of free ubiquitin. These chalcone derivatives directly suppress activity of DUB UCH-L1, UCH-L3, USP2, USP5 and USP8, which are known to regulate the turnover and stability of key regulators of cell survival and proliferation. Inhibition of DUB-activity mediated by these compounds downregulates cell cycle promoters, e.g., cyclin D1 and upregulates tumor suppressors p53, p27Kip1 and p16Ink4A. These changes are associated with arrest in S-G2/M, abrogated anchorage-dependent growth and onset of apoptosis in breast, ovarian and cervical cancer cells without noticeable alterations in primary human cells. Altogether, this work provides evidence of antitumor activity of novel chalcone-based derivatives mediated by their DUB-targeting capacity; supports the development of pharmaceuticals to directly target DUB as a most efficient strategy compared with proteasome inhibition and also provides a clear rationale for the clinical evaluation of these novel small-molecule DUB inhibitors.

Note: Please see the Erratum, Expression of Concern, and subsequent Comment published regarding this Report.     

Arterivirus and nairovirus ovarian tumor domain-containing Deubiquitinases target activated RIG-I to control innate immune signaling

The innate immune response constitutes the first line of defense against viral infection and is extensively regulated through ubiquitination. The removal of ubiquitin from innate immunity signaling factors by deubiquitinating enzymes (DUBs) therefore provides a potential opportunity for viruses to evade this host defense system. It was previously found that specific proteases encoded by the unrelated arteri- and nairoviruses resemble the ovarian tumor domain-containing (OTU) family of DUBs. In arteriviruses, this domain has been characterized before as a papain-like protease (PLP2) that is also involved in replicase polyprotein processing. In nairoviruses, the DUB resides in the polymerase protein but is not essential for RNA replication. Using both in vitro and cell-based assays, we now show that PLP2 DUB activity is conserved in all members of the arterivirus family and that both arteri- and nairovirus DUBs inhibit RIG-I-mediated innate immune signaling when overexpressed. The potential relevance of RIG-I-like receptor (RLR) signaling for the innate immune response against arterivirus infection is supported by our finding that in mouse embryonic fibroblasts, the production of beta interferon primarily depends on the recognition of arterivirus RNA by the pattern-recognition receptor MDA5. Interestingly, we also found that both arteri- and nairovirus DUBs inhibit RIG-I ubiquitination upon overexpression, suggesting that both MDA5 and RIG-I have a role in countering infection by arteriviruses. Taken together, our results support the hypothesis that arteri- and nairoviruses employ their deubiquitinating potential to inactivate cellular proteins involved in RLR-mediated innate immune signaling, as exemplified by the deubiquitination of RIG-I.     

Differential Regulation of JAMM Domain Deubiquitinating Enzyme Activity within the RAP80 Complex

BRCC36 is a JAMM (JAB1/MPN/Mov34 metalloenzyme) domain, lysine 63-ubiquitin (K63-Ub)-specific deubiquitinating enzyme (DUB) and a member of two protein complexes: the DNA damage-responsive BRCA1-RAP80 complex, and the cytoplasmic BRCC36 isopeptidase complex (BRISC). The presence of several identical constituents in both complexes suggests common regulatory mechanisms and potential competition between K63-Ub-related signaling in cytoplasmic and nuclear compartments. Surprisingly, we discover that BRCC36 DUB activity requires different interactions within the context of each complex. Abraxas and BRCC45 were essential for BRCC36 DUB activity within the RAP80 complex, whereas KIAA0157/Abro was the only interaction required for DUB activity within the BRISC. Poh1 also required protein interactions for activity, suggesting a common regulatory mechanism for JAMM domain DUBs. Finally, BRISC deficiency enhanced formation of the BRCA1-RAP80 complex in vivo, increasing BRCA1 levels at DNA double strand breaks. These findings reveal that JAMM domain DUB activity and K63-Ub levels are regulated by multiple mechanisms within the cell.