Ubiquitin-specific Processing Protease Research Articles

Pheromone-dependent Ubiquitination of the Mitogen-activated Protein Kinase Kinase Ste7

Many cell signaling pathways are regulated by phosphorylation, ubiquitination, and degradation of constituent proteins. As with phosphorylation, protein ubiquitination can be reversed, through the action of ubiquitin-specific processing proteases (UBPs). Here we have analyzed 15 UBP disruption mutants in the yeast Saccharomyces cerevisiae and identified one (ubp3 Delta) that acts specifically in the pheromone response pathway. Upon pheromone stimulation, ubp3 Delta mutants accumulate unconjugated polyubiquitin chains as well as polyubiquitinated forms of the mitogen-activated protein kinase kinase Ste7. The ubp3 Delta mutants exhibit a potentiated response to pheromone, as measured by in vivo MAP kinase activity, transcriptional induction, and cell cycle arrest. Signaling is likewise enhanced upon direct activation of Ste4 (G protein beta subunit) and Ste11 (Ste7 kinase) but not the downstream transcription factor Ste12. These findings reveal a mechanism by which pheromone-triggered ubiquitination of Ste7 can modulate the pheromone response in vivo.

HAUSP Stabilizes p53

The tumor suppressor p53 is regulated by phosphorylation and controlled proteolysis by the ubiquitin-proteasome system. Li et al. show that herpesvirus-associated ubiquitin-specific protease (HAUSP) interacted with p53 and promoted its accumulation, even in conditions where the p53-ubiquitin ligase Mdm2 is overexpressed. Overexpression of HAUSP inhibited colony formation by lung carcinoma cells and inhibited growth of mouse embryo fibroblasts. This growth inhibition was dependent on the cells expressing p53. In addition to growth inhibition, HAUSP promoted p53-dependent apoptosis. HAUSP is a member of the ubiquitin-specific processing protease (UBP) family of deubiquitination enzymes (DUBs). The ability of HAUSP to influence p53 stability required the deubiquitinating activity and HAUSP decreased the detectable ubiquitinated p53 in Western blots. In an in vitro assay, HAUSP was able to directly deubiquitinate p53. In normal cells, the expression of a dominant-negative form of HAUSP that can bind p53, but not deubiquitinate it, resulted in accumulation of ubiquitinated p53 species (when the proteasome was inhibited). In addition, p53 was destabilized compared with that in cells that did not express the mutated HAUSP. The concentration of p53 appears to be regulated both by processes that promote ubiquitination and subsequent degradation and by processes that promote deubiquitination and subsequent stabilization. M. Li, D. Chen, A. Shiloh, J. Luo, A. Y. Nikolaev, J. Qin, W. Gu, Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature 416 , 648-653 (2002). [Online Journal]

HAUSP Stabilizes p53

The tumor suppressor p53 is regulated by phosphorylation and controlled proteolysis by the ubiquitin-proteasome system. Li et al. show that herpesvirus-associated ubiquitin-specific protease (HAUSP) interacted with p53 and promoted its accumulation, even in conditions where the p53-ubiquitin ligase Mdm2 is overexpressed. Overexpression of HAUSP inhibited colony formation by lung carcinoma cells and inhibited growth of mouse embryo fibroblasts. This growth inhibition was dependent on the cells expressing p53. In addition to growth inhibition, HAUSP promoted p53-dependent apoptosis. HAUSP is a member of the ubiquitin-specific processing protease (UBP) family of deubiquitination enzymes (DUBs). The ability of HAUSP to influence p53 stability required the deubiquitinating activity and HAUSP decreased the detectable ubiquitinated p53 in Western blots. In an in vitro assay, HAUSP was able to directly deubiquitinate p53. In normal cells, the expression of a dominant-negative form of HAUSP that can bind p53, but not deubiquitinate it, resulted in accumulation of ubiquitinated p53 species (when the proteasome was inhibited). In addition, p53 was destabilized compared with that in cells that did not express the mutated HAUSP. The concentration of p53 appears to be regulated both by processes that promote ubiquitination and subsequent degradation and by processes that promote deubiquitination and subsequent stabilization.M. Li, D. Chen, A. Shiloh, J. Luo, A. Y. Nikolaev, J. Qin, W. Gu, Deubiquitination of p53 by HAUSP is an important pathway for p53 stabilization. Nature 416, 648-653 (2002). [Online Journal]

Linking RNA Turnover to the Ubiquitin Cycle

Regulation of messenger RNA (mRNA) stability is one mechanism by which cells can alter their protein constituents rapidly. The AU-rich element (ARE) in the 3′ untranslated region (UTR) of mRNAs promotes degradation. Laroia et al. investigated the role that ubiquitination and the proteasome play in controlling ARE-mediated RNA instability. Cells were transfected with a reporter gene containing a functional ARE or a mutated ARE, the so-called GC reporter, and either the ubiquitin-specific processing protease (UBP) UBPY or UNP. UBPY promotes proteasome-mediated degradation by recycling ubiquitin from degraded peptide fragments; UNP inhibits degradation by removing ubiquitin from native or insufficiently denatured proteins. In addition to monitoring the expression of the reporter genes, the amount of p53 (a ubiquitinated, unstable protein) and eIF4 (a stable, nonubiquitinated protein) were also monitored. Coexpression of the reporter with UBPY promoted degradation of the ARE reporter transcript and inhibited expression of the reporter protein, and coexpression of UNP inhibited ARE-mediated mRNA degradation and increased expression of the reporter protein. Neither UNP nor UBPY affected expression from or stability of the GC reporter transcript. Overwhelming the cell with ARE-containing transcripts can inhibit ARE-mediated degradation. Overexpression of UBPY can overcome this inhibition, suggesting that ubiquitination may be limiting in this situation. Stimulation of ARE-mediated degradation by UBPY was blocked if the cells were treated with a drug to inhibit the proteasome; thus, it is ubiquitin-mediated protein degradation that is important for ARE-mediated mRNA instability. These results were all obtained in transfected cells with reporter constructs; it will be interesting to see if the deubiquitinating proteins (many of which are encoded by ARE-containing transcripts) regulate mRNA stability in vivo. G. Laroia, B. Sarkar, R. J. Schneider, Ubiquitin-dependent mechanism regulates rapid turnover of AU-rich cytokine mRNAs. Proc. Natl. Acad. Sci. U.S.A. 99 , 1842-1846 (2002). [Abstract] [Full Text]

Linking RNA Turnover to the Ubiquitin Cycle

Regulation of messenger RNA (mRNA) stability is one mechanism by which cells can alter their protein constituents rapidly. The AU-rich element (ARE) in the 3′ untranslated region (UTR) of mRNAs promotes degradation. Laroia et al. investigated the role that ubiquitination and the proteasome play in controlling ARE-mediated RNA instability. Cells were transfected with a reporter gene containing a functional ARE or a mutated ARE, the so-called GC reporter, and either the ubiquitin-specific processing protease (UBP) UBPY or UNP. UBPY promotes proteasome-mediated degradation by recycling ubiquitin from degraded peptide fragments; UNP inhibits degradation by removing ubiquitin from native or insufficiently denatured proteins. In addition to monitoring the expression of the reporter genes, the amount of p53 (a ubiquitinated, unstable protein) and eIF4 (a stable, nonubiquitinated protein) were also monitored. Coexpression of the reporter with UBPY promoted degradation of the ARE reporter transcript and inhibited expression of the reporter protein, and coexpression of UNP inhibited ARE-mediated mRNA degradation and increased expression of the reporter protein. Neither UNP nor UBPY affected expression from or stability of the GC reporter transcript. Overwhelming the cell with ARE-containing transcripts can inhibit ARE-mediated degradation. Overexpression of UBPY can overcome this inhibition, suggesting that ubiquitination may be limiting in this situation. Stimulation of ARE-mediated degradation by UBPY was blocked if the cells were treated with a drug to inhibit the proteasome; thus, it is ubiquitin-mediated protein degradation that is important for ARE-mediated mRNA instability. These results were all obtained in transfected cells with reporter constructs; it will be interesting to see if the deubiquitinating proteins (many of which are encoded by ARE-containing transcripts) regulate mRNA stability in vivo. G. Laroia, B. Sarkar, R. J. Schneider, Ubiquitin-dependent mechanism regulates rapid turnover of AU-rich cytokine mRNAs. Proc. Natl. Acad. Sci. U.S.A. 99, 1842-1846 (2002).[Abstract][Full Text]

The deubiquitinating enzyme Ubp21p of fission yeast stabilizes a mutant form of protein kinase Prp4p.

The protein kinase Prp4p of Schizosaccharomyces pombe is involved in control of the formation of active spliceosomes, phosphorylating the spliceosomal component Prp1p. The kinase domain of Prp4p is closely related to cyclin-dependent kinases (CDKs) and mitogen-activated kinases (MAPKs). A mutational analysis of the highly conserved amino acid sequence ALKHP in subdomain XI of this kinase showed that structural features of this sequence are important for the function of the kinase. We identified ubp21 as a high-copy-number suppressor of a mutation in the ALKHP motif. Characterization of this gene revealed that it encodes a deubiquitinating enzyme belonging to the family of ubiquitin-specific processing proteases (Ubps). The results presented in this report are consistent with the notion that the deubiquitinating activity of Ubp21p may be involved in regulating the steady-state levels of proteins including Prp4p.

Ubiquitination of a Novel Deubiquitinating Enzyme Requires Direct Binding to von Hippel-Lindau Tumor Suppressor Protein

von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome caused by germline mutations of the VHL gene. Recent studies suggest that VHL protein (pVHL) is a component of an E3 ubiquitin ligase, but the detailed biological function of pVHL remains to be determined. To further elucidate the biological functions of pVHL, we searched pVHL-interacting proteins using yeast two-hybrid screening. A novel protein named VHL-interacting deubiquitinating enzyme 1 (VDU1) was identified as being able to directly interact with pVHL in vitro and in vivo. We have determined the full-length cDNA of this enzyme, which includes two putative subtypes. Type I consists of 942 amino acids, and type II consists of 911 amino acids with predicted molecular masses of 107 and 103 kDa, respectively. We have also cloned a mouse homologue of this enzyme. Sequence analysis reveals that this protein is conserved between human and mouse and contains the signature motifs of the ubiquitin-specific processing protease family. Enzymatic function studies demonstrate its deubiquitinating activity. We have determined that the VDU1-interacting region in pVHL is located in its beta-domain, and several naturally occurring mutations located in this domain disrupt the interaction between pVHL and VDU1 protein. Co-immunoprecipitation demonstrates that VDU1 can be recruited into the pVHL-elongin C-elongin B complex. Finally, we demonstrate that VDU1 is able to be ubiquitinated via a pVHL-dependent pathway for proteasomal degradation, and VHL mutations that disrupt the interaction between VDU1 and pVHL abrogate the ubiquitination of VDU1. Our findings indicate that VDU1, a novel ubiquitin-specific processing protease, is a downstream target for ubiquitination and degradation by pVHL E3 ligase. Targeted degradation of VDU1 by pVHL could be crucial for regulating the ubiquitin-proteasome degradation pathway.

Molecular cloning and complete cDNA sequence of UBH1 in mouse testis.

We have identified a full-length mouse UBH1 cDNA, encoding a putative deubiquitinating enzyme, from the testis by RT-PCR using primers prepared from sequences conserved amongst deubiquitinating enzymes. Sequence analysis predicts that the UBH1 cDNA encodes a 355 amino acid polypeptide with the molecular weight of approximately 39 kDa containing the highly conserved Cys, Asp, and His domains characteristic of the ubiquitin-specific processing proteases. Biochemical assay revealed that the mouse UBH1 has deubiquitinating enzyme activity and sequence analysis showed 98.3% amino acid identity with human UBH1.

Imprinting and Evolution of Two Kruppel-type Zinc-Finger Genes, ZIM3 and ZNF264, Located in the PEG3/USP29 Imprinted Domain

We have isolated Kruppel-type (C2H2) zinc-finger genes, ZIM3 (zinc-finger gene 3 from imprinted domain) and ZNF264, located downstream of human and mouse USP29 genes (encoding ubiquitin-specific processing protease 29). In human, both ZIM3 and ZNF264 encode zinc-finger proteins with Kruppel-associated box (KRAB) A and B domains at the amino-terminal regions of the predicted proteins. In contrast, mouse Zim3 and Zfp264 seem to have lost protein-coding capability based on the lack of open reading frames (ORFs) in their cDNA sequences. In particular, the 3′ end of the Zim3 transcript overlaps with the coding region of the adjacent gene Usp29 in an antisense orientation, indicating the conversion of mouse Zim3 into an antisense transcript gene for Usp29. The expression patterns of ZIM3 and ZNF264 have been largely conserved between human and mouse, with testis-specific expression of ZIM3 and ubiquitous expression of ZNF264, but high expression levels in adult testes in both species. Our studies also demonstrate that both mouse genes are imprinted with maternal expression of Zim3 in adult testes and paternal expression of Zfp264 in neonatal and adult brain. The reciprocal imprinting of two neighboring mouse genes, Zim3 and Zfp264, is consistent with a pattern observed frequently in other imprinted domains, and suggests that the imprinting of these two genes might be coregulated.

Divergent N-terminal Sequences of a Deubiquitinating Enzyme Modulate Substrate Specificity

Ubiquitin-specific processing proteases (UBPs) are characterized by a conserved core domain with surrounding divergent sequences, particularly at the N-terminal end. We previously cloned two isoforms of a testis UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini that target the two isoforms to different subcellular locations (Lin, H., Keriel, A., Morales, C. R., Bedard, N., Zhao, Q., Hingamp, P., Lefrancois, S., Combaret, L., and Wing, S. S. (2000) Mol. Cell. Biol. 20, 6568-6578). To determine whether the N termini also influence the biochemical functions of the UBP, we expressed UBP-t1, UBP-t2, and the common core domain, UBP core, in Escherichia coli. The three isoforms cleaved branched triubiquitin at >20-fold faster rates than linear diubiquitin, suggesting that UBP-testis functions as an isopeptidase. Both N-terminal extensions inhibited the ability of UBP-core to generate free ubiquitin when linked in a peptide bond with itself, another peptide, or to small adducts. The N-terminal extension of UBP-t2 increased the ability of UBP-core to cleave branched triubiquitin. UBP-core removed ubiquitin from testis ubiquitinated proteins more rapidly than UBP-t2 and UBP-t1. Thus, UBP enzymes appear to contain a catalytic core domain, the activities and specificities of which can be modulated by N-terminal extensions. These divergent N termini can alter localization and confer multiple functions to the various members of the large UBP family.

Divergent N-terminal sequences target an inducible testis deubiquitinating enzyme to distinct subcellular structures.

Ubiquitin-specific processing proteases (UBPs) presently form the largest enzyme family in the ubiquitin system, characterized by a core region containing conserved motifs surrounded by divergent sequences, most commonly at the N-terminal end. The functions of these divergent sequences remain unclear. We identified two isoforms of a novel testis-specific UBP, UBP-t1 and UBP-t2, which contain identical core regions but distinct N termini, thereby permitting dissection of the functions of these two regions. Both isoforms were germ cell specific and developmentally regulated. Immunocytochemistry revealed that UBP-t1 was induced in step 16 to 19 spermatids while UBP-t2 was expressed in step 18 to 19 spermatids. Immunoelectron microscopy showed that UBP-t1 was found in the nucleus while UBP-t2 was extranuclear and was found in residual bodies. For the first time, we show that the differential subcellular localization was due to the distinct N-terminal sequences. When transfected into COS-7 cells, the core region was expressed throughout the cell but the UBP-t1 and UBP-t2 isoforms were concentrated in the nucleus and the perinuclear region, respectively. Fusions of each N-terminal end with green fluorescent protein yielded the same subcellular localization as the native proteins, indicating that the N-terminal ends were sufficient for determining differential localization. Interestingly, UBP-t2 colocalized with anti-gamma-tubulin immunoreactivity, indicating that like several other components of the ubiquitin system, a deubiquitinating enzyme is associated with the centrosome. Regulated expression and alternative N termini can confer specificity of UBP function by restricting its temporal and spatial loci of action.

Tissue-specificity, functional characterization and subcellular localization of a rat ubiquitin-specific processing protease, UBP109, whose mRNA expression is developmentally regulated.

A cDNA encoding an ubiquitin-specific processing protease, UBP109, in rat skeletal muscle was cloned and its product was characterized. Northern analysis revealed that UBP109 mRNA is highly expressed in testis and spleen, compared with other tissues. Furthermore, in situ hybridization showed that the level of UBP109 mRNA in liver, spinal cord and brain dramatically changed during embryonic development, indicating that the expression of UBP109 mRNA is developmentally regulated. UBP109 was expressed in Escherichia coli and purified to apparent homogeneity using a (125)I-labelled ubiquitin-peptide fusion as a substrate. The purified enzyme cleaved at the C-terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes. UBP109 also released free ubiquitin from poly-His-tagged penta-ubiquitin. Moreover, it released free ubiquitin from poly-ubiquitinated protein conjugates of rabbit reticulocytes. In addition, UBP109 localized to both the cytoplasm and the nucleus and, among three putative nuclear localization sequences, only the one located near the C-terminus is responsible for nuclear localization. These results suggest that UBP109 may play an important role in generation of free ubiquitin from its precursors and its recycling from poly-ubiquitinated protein conjugates, and hence in regulation of ubiquitin-mediated cellular processes, particularly related to embryonic development.

Tissue-specificity, functional characterization and subcellular localization of a rat ubiquitin-specific processing protease, UBP109, whose mRNA expression is developmentally regulated

A cDNA encoding an ubiquitin-specific processing protease, UBP109, in rat skeletal muscle was cloned and its product was characterized. Northern analysis revealed that UBP109 mRNA is highly expressed in testis and spleen, compared with other tissues. Furthermore, in situ hybridization showed that the level of UBP109 mRNA in liver, spinal cord and brain dramatically changed during embryonic development, indicating that the expression of UBP109 mRNA is developmentally regulated. UBP109 was expressed in Escherichia coli and purified to apparent homogeneity using a 125I-labelled ubiquitin-peptide fusion as a substrate. The purified enzyme cleaved at the C-terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their sizes. UBP109 also released free ubiquitin from poly-His-tagged penta-ubiquitin. Moreover, it released free ubiquitin from poly-ubiquitinated protein conjugates of rabbit reticulocytes. In addition, UBP109 localized to both the cytoplasm and the nucleus and, among three putative nuclear localization sequences, only the one located near the C-terminus is responsible for nuclear localization. These results suggest that UBP109 may play an important role in generation of free ubiquitin from its precursors and its recycling from poly-ubiquitinated protein conjugates, and hence in regulation of ubiquitin-mediated cellular processes, particularly related to embryonic development.

Sequencing, tissue distribution and chromosomal assignment of a novel ubiquitin-specific protease USP23

We have identified human and mouse cDNAs encoding a novel ubiquitin-specific protease designated USP23. Both cDNAs encode a 62-kDa protein containing the highly conserved His and Cys domains characteristic of the C19 cysteine protease family of ubiquitin-specific processing proteases (UCH-2). Human tissue Northern blots revealed USP23 to be ubiquitously expressed, whereas USP12, its closest human paralogue, displayed a more restricted expression pattern. The human USP23 gene mapped to chromosome 1q22.

A ubiquitin-protein ligase (E3) mutation of Saccharomyces cerevisiae suppressed by co-overexpression of two ubiquitin-specific processing proteases.

To isolate mutations related to the ubiquitin system, I constructed a plasmid carrying the YUH1 and UBP1 genes (genes of ubiquitin-specific processing proteases) whose expressions were under the control of the galactose-inducible GAL1-GAL10 promoter. Cells of a strain carrying the plasmid were mutagenized with ethyl methanesulfonate. One mutant, which showed galactose-dependent growth at a high temperature (37 degrees C), was isolated from about 380,000 mutagenized colonies. The mutation responsible for galactose-dependent growth at 37 degrees C was a single nuclear recessive mutation designated as uby1-1. UBP1 and YUH1 as well as the GAL1-GAL10 promoter are required to suppress uby1-1. At the restrictive temperature, a uby1-1 mutant did not arrest at a specific phase of the cell cycle, but still lost viability. Even at the permissive temperature (30 degrees C), the uby1-1 mutant grew somewhat slowly and showed pleiotropic phenotypes including hypersensitivity to stresses such as cadmium and canavanine, and sporulation defects. The genomic DNA fragments in a single-copy plasmid which complemented uby1-1 were isolated. Chromosomal mapping, sequencing and subcloning analyses indicated that the gene complementing uby1-1 is RSP5, which encodes a ubiquitin-protein ligase (E3) homologous to E6-AP (E6 associated protein). Deletion, complementation and linkage analyses revealed that UBY1 and RSP5 are the same gene. Therefore, the E3 protein encoded by RSP5 (UBY1) is required for vegetative growth, sporulation and stress response. The present procedure using suppression by co-overexpression of two cloned genes will be useful to isolate mutations of related genes and to analyze biochemical pathways and gene-interactions.

Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3, and functional analysis of the UBP gene family

In eukaryotes, both natural and engineered ubiquitin (Ub) fusions to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin. YUH1 and UBP1, the genes for two ubiquitin-specific proteases of the yeast Saccharomyces cerevisiae, have been cloned previously and shown to encode nonhomologous proteins. Using an Escherichia coli-based genetic screen, we have isolated two other yeast genes for ubiquitin-specific proteases, named UBP2 and UBP3. Ubp2 (1,264 residues), Ubp3 (912 residues), and the previously cloned Ubp1 (809 residues) are largely dissimilar except for two short regions containing Cys and His which encompass their putative active sites. Neither of these proteases has sequence similarities to Yuh1. Both Ubp2 and the previously identified Ubp1 cleave in vitro at the C terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size, poly-Ub being the exception. However, both Ubp1 and Ubp2 are also capable of cleaving poly-Ub when coexpressed with it in E. coli, suggesting that such cleavage is largely cotranslational. Although inactive in E. coli extracts, Ubp3 was active with all of the tested ubiquitin fusions except poly-Ub when coexpressed with them in E. coli. Null yuh1 ubp1 ubp2 ubp3 quadruple mutants are viable and retain the ability to deubiquitinate ubiquitin fusions, indicating the presence of at least one more ubiquitin-specific processing protease in S. cerevisiae.

Cloning and functional analysis of the ubiquitin-specific protease gene UBP1 of Saccharomyces cerevisiae

In eukaryotes, both natural and engineered fusions of ubiquitin to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin. Using the method of sib selection, and taking advantage of the fact that bacteria such as Escherichia coli lack ubiquitin-specific enzymes, we have cloned a gene, named UBP1, of the yeast Saccharomyces cerevisiae that encodes a ubiquitin-specific processing protease. With the exception of polyubiquitin, the UBP1 protease cleaves at the carboxyl terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size or the presence of an amino-terminal ubiquitin extension. These properties of UBP1 distinguish it from the previously cloned yeast protease YUH1, which deubiquitinates relatively short ubiquitin fusions but is virtually inactive with longer fusions such as ubiquitin-beta-galactosidase. The amino acid sequence of the 809-residue UBP1 lacks significant similarities to other known proteins, including the 236-residue YUH1 protease. Null ubp1 mutants are viable, and retain the ability to deubiquitinate ubiquitin-beta-galactosidase, indicating that the family of ubiquitin-specific proteases in yeast is not limited to UBP1 and YUH1.