Cloning and enzymatic analysis of 22 novel human ubiquitin-specific proteases

We have identified and cloned 22 human cDNAs encoding novel members of the ubiquitin-specific protease (USP) family. Eighteen of the identified proteins contain all structural features characteristic of these cysteine proteinases, whereas four of them have been classified as non-peptidase homologues. Northern blot analysis demonstrated that the identified USPs are broadly and differentially distributed in human tissues, some of them being especially abundant in skeletal muscle or testis. Enzymatic studies performed with the identified USPs revealed that at least twelve of them are deubiquitylating enzymes based on their ability to cleave ubiquitin from a ubiquitin-beta-galactosidase fusion protein. These results provide additional evidence of the extreme complexity and diversity of the USP proteolytic system in human tissues and open the possibility to explore the relevance of their multiple components in the regulation of ubiquitin-mediated pathways in normal and pathological functions.     

Identification, Functional Characterization, and Chromosomal Localization of USP15, a Novel Human Ubiquitin-Specific Protease Related to the UNP Oncoprotein, and a Systematic Nomenclature for Human Ubiquitin-Specific Proteases

We have identified a novel gene, USP15, encoding a human ubiquitin-specific protease (USP). The USP15 protein consists of 952 amino acids with a predicted molecular mass of 109.2 kDa and contains the highly conserved Cys and His boxes present in all members of the UBP family of deubiquitinating enzymes. USP15 shares 60.5% sequence identity and 76% sequence similarity with the human homolog (UNP/Unph/USP4) of the mouse Unp proto-oncogene. Recombinant USP15 demonstrated ubiquitin-specific protease activity against engineered linear fusions of ubiquitin to beta-galactosidase and glutathione S-transferase. USP15 can also cleave the ubiquitin-proline bond, a property previously unique to Unp/UNP. Chromosomal mapping by fluorescence in situ hybridization and radiation hybrid analyses localized the USP15 gene to chromosome band 12q14, a different location than that of UNP (3p21.3). Analysis of expressed sequence tag databases reveals evidence of alternate polyadenylation sites in the USP15 gene and also indicates that the gene may possess an exon/intron structure similar to that of the Unp gene, suggesting they have descended from a common ancestor. A systematic nomenclature for the human USPs is proposed.     

Identification of a novel isopeptidase with dual specificity for ubiquitin- and NEDD8-conjugated proteins

Covalent conjugation of proteins by ubiquitin or ubiquitin-like molecules is an important form of post-translational modification and plays a critical role in many cellular processes. Similar to the concept of phosphorylation and dephosphorylation, these conjugates are regulated by a large number of deconjugating enzymes. Here, we report the cloning of a 2,141-base pair DNA fragment from human placenta cDNA library by a strategy that involves expressed sequence tag data base searching, polymerase chain reaction, and rapid amplification of cDNA ends. Nucleotide sequence analysis revealed that the cloned cDNA contains an open reading frame of 1,143 base pairs encoding a novel protease, USP21, which is composed of 381 residues with a calculated molecular mass of 43 kDa. The human USP21 gene is located on chromosome 1q21 and encodes a member of the ubiquitin-specific protease family with highly conserved Cys and His domains. The activity and specificity of USP21 were determined by using a COS cell expression system in vivo. We showed that USP21 is capable of removing ubiquitin from ubiquitinated proteins as expected. Furthermore, USP21 is capable of removing NEDD8 from NEDD8 conjugates but has no effect on Sentrin-1 conjugates. As expected from its biochemical activity, overexpression of USP21 has a profound growth inhibitory effect on U2OS cells. Thus, USP21 is the first ubiquitin-specific protease shown to have dual specificity for both ubiquitin and NEDD8 and may play an important role in the regulation of cell growth.     

Isolation and characterization of the mouse ubiquitin-specific protease Usp15

We have characterized the mouse ortholog of the human ubiquitin-specific protease USP15. Mouse Usp15 consists of 981 amino acids with a predicted molecular mass of 112 kDa, contains the highly conserved Cys and His boxes present in all members of the UBP family of deubiquitinating enzymes, and is 98% identical/99% similar to human USP15. Usp15 shares 59.5% identity/75.5% sequence similarity with the mouse Unp(Usp4) oncoprotein. Recombinant Usp15 demonstrated ubiquitin-specific protease activity against engineered linear fusions of ubiquitin to glutathione S-transferase. Usp15 can also cleave the ubiquitin-proline bond, as can USP15 and Usp4. Alignment of mouse and human Usp15 and Usp4 protein sequences suggested that Usp15/USP15 may be alternately spliced in a manner analogous to Usp4. Sequence analysis of RT-PCR products from several human and mouse cell lines and tissues revealed alternate splicing in all cells studied. Northern blot analysis of both mouse and human Usp15 revealed two differently sized mRNAs in all tissues examined, owing to alternate polyadenylation sites spaced by 1.5 kb. Chromosomal mapping by interspecific backcross analysis localized the Usp15 gene to the distal region of mouse Chromosome (Chr) 10. This region is syntenic with human Chr 12q24, the location of human USP15, and a different location to Unp(Usp4) (Chr 9). Identification of the mouse Usp15 gene (>69.5 kb) and human USP15 gene (145 kb) sequences in genome databases reveals that both are composed of 22 exons with identical splice sites, and both have an exon/intron structure identical to the mouse Usp4 gene, including the alternately spliced exon. Phylogenetic studies suggest that a sequence currently identified as a chicken Usp4 ortholog is in fact a USP15 ortholog, while bona-fide chicken, cow, and rat Usp4 orthologs can be identified in EST databases.     

USP15 plays an essential role for caspase-3 activation during Paclitaxel-induced apoptosis

Paclitaxel (also known as Taxol) is a well-known anticancer agent that blocks cell mitosis and kills tumor cells, and is often used in clinic to treat cancers. Despite the success of Paclitaxel, the development of drug resistance prevents its clinical applicability. Here, we screened an siRNA library against the entire human genomes using HeLa cells, and have find that lack of USP15 (ubiquitin-specific protease 15) causes Paclitaxel resistance. We also observed the decreased expression of USP15 in Paclitaxel-resistant human ovarian cancer samples. In addition, we have demonstrated that USP15 plays an essential role for stability and activity of caspase-3 during Paclitaxel-induced apoptosis. Thus, USP15 may be a candidate diagnostic marker and therapeutic target for Paclitaxel-resistant cancers.     

GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7

The packaging of eukaryotic genomic DNA into chromatin is modulated through a range of posttranslational histone modifications. Among these, the role of histone ubiquitylation remains poorly understood. Here, we show that the essential Drosophila ubiquitin-specific protease 7 (USP7) contributes to epigenetic silencing of homeotic genes by Polycomb (Pc). We purified USP7 from embryo nuclear extracts as a stable heteromeric complex with guanosine 5'-monophosphate synthetase (GMPS). The USP7-GMPS complex catalyzed the selective deubiquitylation of histone H2B, but not H2A. Biochemical assays confirmed the tight association between USP7 and GMPS in Drosophila embryo extracts. Similar to USP7, mutations in GMPS acted as enhancers of Pc in vivo. USP7 binding to GMPS was required for histone H2B deubiquitylation and strongly augmented deubiquitylation of the human tumor suppressor p53. Thus, GMPS can regulate the activity of a ubiquitin protease. Collectively, these results implicate a biosynthetic enzyme in chromatin control via ubiquitin regulation.     

Regulation of mitochondrial morphology by USP30, a deubiquitinating enzyme present in the mitochondrial outer membrane

Recent studies have suggested that ubiquitination of mitochondrial proteins participates in regulating mitochondrial dynamics in mammalian cells, but it is unclear whether deubiquitination is involved in this process. Here, we identify human ubiquitin-specific protease 30 (USP30) as a deubiquitinating enzyme that is embedded in the mitochondrial outer membrane. Depletion of USP30 expression by RNA interference induced elongated and interconnected mitochondria, depending on the activities of the mitochondrial fusion factors mitofusins, without changing the expression levels of the key regulators for mitochondrial dynamics. Mitochondria were rescued from this abnormal phenotype by ectopic expression of USP30 in a manner dependent on its enzymatic activity. Our findings reveal that USP30 participates in the maintenance of mitochondrial morphology, a finding that provides new insight into the cellular function of deubiquitination.     

TSPYL5 suppresses p53 levels and function by physical interaction with USP7

We have previously reported a gene expression signature that is a powerful predictor of poor clinical outcome in breast cancer. Among the seventy genes in this expression profile is a gene of unknown function: TSPYL5 (TSPY-like 5, also known as KIAA1750). TSPYL5 is located within a small region at chromosome 8q22 that is frequently amplified in breast cancer, which suggests that TSPYL5 has a causal role in breast oncogenesis. Here, we report that high TSPYL5 expression is an independent marker of poor outcome in breast cancer. Mass spectrometric analysis revealed that TSPYL5 interacts with ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease; HAUSP). USP7 is the deubiquitylase for the p53 tumour suppressor and TSPYL5 reduces the activity of USP7 towards p53, resulting in increased p53 ubiquitylation. We demonstrate that TSPYL5 reduces p53 protein levels and inhibits activation of p53-target genes. Furthermore, expression of TSPYL5 overrides p53-dependent proliferation arrest and oncogene-induced senescence, and contributes to oncogenic transformation in multiple cell-based assays. Our data identify TSPYL5 as a suppressor of p53 function through its interaction with USP7.     

Ubiquitin-specific protease 4 is inhibited by its ubiquitin-like domain

USP4 is a member of the ubiquitin-specific protease (USP) family of deubiquitinating enzymes that has a role in spliceosome regulation. Here, we show that the crystal structure of the minimal catalytic domain of USP4 has the conserved USP-like fold with its typical ubiquitin-binding site. A ubiquitin-like (Ubl) domain inserted into the catalytic domain has autoregulatory function. This Ubl domain can bind to the catalytic domain and compete with the ubiquitin substrate, partially inhibiting USP4 activity against different substrates. Interestingly, other USPs, such as USP39, could relieve this inhibition.     

Functional interaction between the ubiquitin-specific protease 25 and the SYK tyrosine kinase

The SYK non-receptor tyrosine kinase is a key effector of immune receptors signaling in hematopoietic cells. Here, we identified and characterized a novel interaction between SYK and the ubiquitin-specific protease 25 (USP25). We report that the second SH2 domain of SYK physically interacts with a tyrosine-rich, C-terminal region of USP25 independently of tyrosine phosphorylation. Moreover, we showed that SYK specifically phosphorylates USP25 and alters its cellular levels. This study thus uncovers a new SYK substrate and reveals a novel SYK function, namely the regulation of USP25 cellular levels.     

The deubiquitinating protein USP24 interacts with DDB2 and regulates DDB2 stability

Damage-specific DNA-binding protein 2 (DDB2) was first isolated as a subunit of the UV-DDB heterodimeric complex that is involved in DNA damage recognition in the nucleotide excision repair pathway (NER). DDB2 is required for efficient repair of CPDs in chromatin and is a component of the CRL4^DDB2 E3 ligase that targets XPC, histones and DDB2 itself for ubiquitination. In this study, a yeast two-hybrid screening of a human cDNA library was performed to identify potential DDB2 cellular partners. We identified a deubiquitinating enzyme, USP24, as a likely DDB2-interacting partner. Interaction between DDB2 and USP24 was confirmed by co-precipitation. Importantly, knockdown of USP24 in two human cell lines decreased the steady-state levels of DDB2, indicating that USP24-mediated DDB2 deubiquitination prevents DDB2 degradation. In addition, we demonstrated that USP24 can cleave an ubiquitinated form of DDB2 in vitro. Taken together, our results suggest that the ubiquitin-specific protease USP24 is a novel regulator of DDB2 stability.     

<Emphasis Type="Italic">Usp9y</Emphasis> (ubiquitin-specific protease 9 gene on the Y) is associated with a functional promoter and encodes an intact open reading frame homologous to <Emphasis Type="Italic">Usp9x</Emphasis> that is under selective constraint

Sequences complementary to the X-linked ubiquitin-specific protease gene Usp9x (Dffrx) have been shown to map to the Sxr^b interval of the mouse Y Chromosome (chr) and to be expressed in a testis-specific manner. In humans, ubiquitously expressed functional homologues (USP9Y and USP9X DFFRY/DFFRX) are present on both sex chromosomes, whereas in mouse it remains to be demonstrated that the Y-linked sequences encode a functional protein. In this paper, it is shown that the Usp9y gene encodes a potentially functional ubiquitin-specific protease possessing a core promoter region that shares several features characteristic of other testis-specific genes. Analysis of synonymous and nonsynonymous nucleotide changes suggests that there is constraint on the amino acid sequence of both the mouse Usp9x and Usp9y genes, a finding that mirrors similar analysis of the human orthologs. Thus, in both mouse and human, selection is acting to maintain the amino acid sequence of the X and Y-linked genes. This indicates that in both species the genes on each sex chromosome continue to encode an important function.     

Increased Expression of Ubiquitin-Specific Protease 4 Participates in Neuronal Apoptosis After Intracerebral Hemorrhage in Adult Rats

Ubiquitinating enzymes catalyze protein ubiquitination, a reversible process countered by deubiquitinating enzyme (DUB) action. Ubiquitin-specific protease 4 (USP4) is a member of the ubiquitin-specific protease (USP) family of DUBs that has a role in spliceosome regulation. In the present study, we demonstrated that USP4 may be involved in neuronal apoptosis in the processes of intracerebral hemorrhage (ICH). We obtained a significant up-regulation of USP4 in neurons adjacent to the hematoma following ICH by the results of Western blot, immunohistochemistry, and immunofluorescence. Increasing USP4 level was found to be accompanied by the up-regulation of active caspase-3, γH2AX, Bax, and decreased expression of Bcl-2. In addition, USP4 co-localized well with γH2AX in the nucleus in the ICH model and hemin-induced apoptosis model. Moreover, in vitro study, knocking down USP4 by USP4-specific siRNA in PC12 cells reduced active caspase-3 expression. All these results above suggested that USP4 may be involved in neuronal apoptosis after ICH.     

The deubiquitinating protein USP24 interacts with DDB2 and regulates DDB2 stability

Damage-specific DNA-binding protein 2 (DDB2) was first isolated as a subunit of the UV-DDB heterodimeric complex that is involved in DNA damage recognition in the nucleotide excision repair pathway (NER). DDB2 is required for efficient repair of CPDs in chromatin and is a component of the CRL4^DDB2 E3 ligase that targets XPC, histones and DDB2 itself for ubiquitination. In this study, a yeast two-hybrid screening of a human cDNA library was performed to identify potential DDB2 cellular partners. We identified a deubiquitinating enzyme, USP24, as a likely DDB2-interacting partner. Interaction between DDB2 and USP24 was confirmed by co-precipitation. Importantly, knockdown of USP24 in two human cell lines decreased the steady-state levels of DDB2, indicating that USP24-mediated DDB2 deubiquitination prevents DDB2 degradation. In addition, we demonstrated that USP24 can cleave an ubiquitinated form of DDB2 in vitro. Taken together, our results suggest that the ubiquitin-specific protease USP24 is a novel regulator of DDB2 stability.     

Ubiquitin-specific protease 53 promotes osteogenic differentiation of human bone marrow-derived mesenchymal stem cells

The ubiquitin protease pathway plays important role in human bone marrow-derived mesenchymal stem cell (hBMSC) differentiation, including osteogenesis. However, the function of deubiquitinating enzymes in osteogenic differentiation of hBMSCs remains poorly understood. In this study, we aimed to investigate the role of ubiquitin-specific protease 53 (USP53) in the osteogenic differentiation of hBMSCs. Based on re-analysis of the Gene Expression Omnibus database, USP53 was selected as a positive regulator of osteogenic differentiation in hBMSCs. Overexpression of USP53 by lentivirus enhanced osteogenesis in hBMSCs, whereas knockdown of USP53 by lentivirus inhibited osteogenesis in hBMSCs. In addition, USP53 overexpression increased the level of active β-catenin and enhanced the osteogenic differentiation of hBMSCs. This effect was reversed by the Wnt/β-catenin inhibitor DKK1. Mass spectrometry showed that USP53 interacted with F-box only protein 31 (FBXO31) to promote proteasomal degradation of β-catenin. Inhibition of the osteogenic differentiation of hBMSCs by FBXO31 was partially rescued by USP53 overexpression. Animal studies showed that hBMSCs with USP53 overexpression significantly promoted bone regeneration in mice with calvarial defects. These results suggested that USP53 may be a target for gene therapy for bone regeneration.Subject terms: Cell signalling, Mesenchymal stem cells     

Ubiquitin-specific Protease 11 (USP11) Deubiquitinates Hybrid Small Ubiquitin-like Modifier (SUMO)-Ubiquitin Chains to Counteract RING Finger Protein 4 (RNF4)

Ring finger protein 4 (RNF4) is a SUMO-targeted ubiquitin E3 ligase with a pivotal function in the DNA damage response (DDR). SUMO interaction motifs (SIMs) in the N-terminal part of RNF4 tightly bind to SUMO polymers, and RNF4 can ubiquitinate these polymers in vitro. Using a proteomic approach, we identified the deubiquitinating enzyme ubiquitin-specific protease 11 (USP11), a known DDR-component, as a functional interactor of RNF4. USP11 can deubiquitinate hybrid SUMO-ubiquitin chains to counteract RNF4. SUMO-enriched nuclear bodies are stabilized by USP11, which functions downstream of RNF4 as a counterbalancing factor. In response to DNA damage induced by methyl methanesulfonate, USP11 could counteract RNF4 to inhibit the dissolution of nuclear bodies. Thus, we provide novel insight into cross-talk between ubiquitin and SUMO and uncover USP11 and RNF4 as a balanced SUMO-targeted ubiquitin ligase/protease pair with a role in the DDR.     

Peroxisomal Import Reduces the Proapoptotic Activity of Deubiquitinating Enzyme USP2

The human deubiquitinating enzyme ubiquitin-specific protease 2 (USP2) regulates multiple cellular pathways, including cell proliferation and apoptosis. As a result of alternative splicing four USP2 isoenzymes are expressed in human cells of which all contain a weak peroxisome targeting signal of type 1 (PTS1) at their C-termini. Here, we systematically analyzed apoptotic effects induced by overexpression and intracellular localization for each isoform. All isoforms exhibit proapoptotic activity and are post-translationally imported into the matrix of peroxisomes in a PEX5-dependent manner. However, a significant fraction of the USP2 pool resides in the cytosol due to a weaker PTS1 and thus low affinity to the PTS receptor PEX5. Blocking of peroxisomal import did not interfere with the proapoptotic activity of USP2, suggesting that the enzyme performs its critical function outside of this compartment. Instead, increase of the efficiency of USP2 import into peroxisomes either by optimization of its peroxisomal targeting signal or by overexpression of the PTS1 receptor did result in a reduction of the apoptotic rate of transfected cells. Our studies suggest that peroxisomal import of USP2 provides additional control over the proapoptotic activity of cytosolic USP2 by spatial separation of the deubiquitinating enzymes from their interaction partners in the cytosol and nucleus.