Regulation of USP28 Deubiquitinating Activity by SUMO Conjugation

USP28 (ubiquitin-specific protease 28) is a deubiquitinating enzyme that has been implicated in the DNA damage response, the regulation of Myc signaling, and cancer progression. The half-life stability of major regulators of critical cellular pathways depends on the activities of specific ubiquitin E3 ligases that target them for proteosomal degradation and deubiquitinating enzymes that promote their stabilization. One function of the post-translational small ubiquitin modifier (SUMO) is the regulation of enzymatic activity of protein targets. In this work, we demonstrate that the SUMO modification of the N-terminal domain of USP28 negatively regulates its deubiquitinating activity, revealing a role for the N-terminal region as a regulatory module in the control of USP28 activity. Despite the presence of ubiquitin-binding domains in the N-terminal domain, its truncation does not impair deubiquitinating activity on diubiquitin or polyubiquitin chain substrates. In contrast to other characterized USP deubiquitinases, our results indicate that USP28 has a chain preference activity for Lys¹¹, Lys⁴⁸, and Lys⁶³ diubiquitin linkages.     

Mutant ubiquitin (UBB+1) associated with neurodegenerative disorders is hydrolyzed by ubiquitin C-terminal hydrolase L3 (UCH-L3)

Mutant ubiquitin (UBB⁺¹) accumulates in the hallmarks of tauopathies and polyglutamine diseases. We show that the deubiquitinating enzyme YUH1 of Saccharomyces cerevisiae and its mouse and human ortholog UCH-L3 are able to hydrolyze the C-terminal extension of UBB⁺¹. This yields another dysfunctional ubiquitin molecule (UB^G76Y) with biochemical properties similar to full length UBB⁺¹. UBB⁺¹ may be detected in post-mortem tissue due to impaired C-terminal truncation of UBB⁺¹. Although the level of UCH-L3 protein in several neurodegenerative diseases is unchanged, we show that in vitro oxidation of recombinant UCH-L3 impairs its deubiquitinating activity. We postulate that impaired UCH-L3 function may contribute to the accumulation of full length UBB⁺¹ in various pathologies.     

Crystal structure of a UBP-family deubiquitinating enzyme in isolation and in complex with ubiquitin aldehyde

The ubiquitin-specific processing protease (UBP) family of deubiquitinating enzymes plays an essential role in numerous cellular processes. HAUSP, a representative UBP, specifically deubiquitinates and hence stabilizes the tumor suppressor protein p53. Here, we report the crystal structures of the 40 kDa catalytic core domain of HAUSP in isolation and in complex with ubiquitin aldehyde. These studies reveal that the UBP deubiquitinating enzymes exhibit a conserved three-domain architecture, comprising Fingers, Palm, and Thumb. The leaving ubiquitin moiety is specifically coordinated by the Fingers, with its C terminus placed in the active site between the Palm and the Thumb. Binding by ubiquitin aldehyde induces a drastic conformational change in the active site that realigns the catalytic triad residues for catalysis.     

The Arabidopsis mitochondrial membrane‐bound ubiquitin protease UBP27 contributes to mitochondrial morphogenesis

Mitochondria are essential organelles with dynamic morphology and function. Post‐translational modifications (PTMs), which include protein ubiquitination, are critically involved in animal and yeast mitochondrial dynamics. How PTMs contribute to plant mitochondrial dynamics is just beginning to be elucidated, and mitochondrial enzymes involved in ubiquitination have not been reported from plants. In this study, we identified an Arabidopsis mitochondrial localized ubiquitin protease, UBP27, through a screen that combined bioinformatics and fluorescent fusion protein targeting analysis. We characterized UBP27 with respect to its membrane topology and enzymatic activities, and analysed the mitochondrial morphological changes in UBP27T‐DNA insertion mutants and overexpression lines. We have shown that UBP27 is embedded in the mitochondrial outer membrane with an N_in–C_out orientation and possesses ubiquitin protease activities in vitro. UBP27 demonstrates similar sub‐cellular localization, domain structure, membrane topology and enzymatic activities with two mitochondrial deubiquitinases, yeast ScUBP16 and human HsUSP30, which indicated that these proteins are functional orthologues in eukaryotes. Although loss‐of‐function mutants of UBP27 do not show obvious phenotypes in plant growth and mitochondrial morphology, UBP27 overexpression can change mitochondrial morphology from rod to spherical shape and reduce the mitochondrial association of dynamin‐related protein 3 (DRP3) proteins, large GTPases that serve as the main mitochondrial fission factors. Thus, our study has uncovered a plant ubiquitin protease that plays a role in mitochondrial morphogenesis possibly through modulation of the function of organelle division proteins.     

The Ubiquitin Receptor DA1 Regulates Seed and Organ Size by Modulating the Stability of the Ubiquitin-Specific Protease UBP15/SOD2 in Arabidopsis

Although the control of organ size is a fundamental question in developmental biology, little is known about the genetic and molecular mechanisms that determine the final size of seeds in plants. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligases DA2 and ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to restrict seed growth in Arabidopsis thaliana. Here, we describe UBIQUITIN-SPECIFIC PROTEASE15 (UBP15), encoded by SUPPRESSOR2 OF DA1 (SOD2), which acts maternally to regulate seed size by promoting cell proliferation in the integuments of ovules and developing seeds. The sod2/ubp15 mutants form small seeds, while overexpression of UBP15 increases seed size of wild-type plants. Genetic analyses indicate that UBP15 functions antagonistically in a common pathway with DA1 to influence seed size, but does so independently of DA2 and EOD1. Further results reveal that DA1 physically associates with UBP15 in vitro and in vivo and modulates the stability of UBP15. Therefore, our findings establish a genetic and molecular framework for the regulation of seed size by four ubiquitin-related proteins DA1, DA2, EOD1, and UBP15 and suggest that they are promising targets for increasing seed size in crops.     

The USP1/UAF1 complex promotes double-strand break repair through homologous recombination

Protein ubiquitination plays a key role in the regulation of a variety of DNA repair mechanisms. Protein ubiquitination is controlled by the coordinate activity of ubiquitin ligases and deubiquitinating enzymes (DUBs). The deubiquitinating enzyme USP1 regulates DNA repair and the Fanconi anemia pathway through its association with its WD40 binding partner, UAF1, and through its deubiquitination of two critical DNA repair proteins, FANCD2-Ub and PCNA-Ub. To investigate the function of USP1 and UAF1, we generated USP1^−/−, UAF1^−/−/−, and USP1^−/− UAF1^−/−/− chicken DT40 cell clones. These three clones showed similar sensitivities to chemical cross-linking agents, to a topoisomerase poison, camptothecin, and to an inhibitor of poly(ADP-ribose) polymerase (PARP), indicating that the USP1/UAF1 complex is a regulator of the cellular response to DNA damage. The hypersensitivity to both camptothecin and a PARP inhibitor suggests that the USP1/UAF1 complex promotes homologous recombination (HR)-mediated double-strand break (DSB) repair. To gain insight into the mechanism of the USP1/UAF1 complex in HR, we inactivated the nonhomologous end-joining (NHEJ) pathway in UAF1-deficient cells. Disruption of NHEJ in UAF1-deficient cells restored cellular resistance to camptothecin and the PARP inhibitor. Our results indicate that the USP1/UAF1 complex promotes HR, at least in part by suppressing NHEJ.     

Interaction of the Deubiquitinating Enzyme Ubp2 and the E3 Ligase Rsp5 Is Required for Transporter/Receptor Sorting in the Multivesicular Body Pathway

Protein ubiquitination is essential for many events linked to intracellular protein trafficking. We sought to elucidate the possible involvement of the S. cerevisiae deubiquitinating enzyme Ubp2 in transporter and receptor trafficking after we (this study) and others established that affinity purified Ubp2 interacts stably with the E3 ubiquitin ligase Rsp5 and the (ubiquitin associated) UBA domain containing protein Rup1. UBP2 interacts genetically with RSP5, while Rup1 facilitates the tethering of Ubp2 to Rsp5 via a PPPSY motif. Using the uracil permease Fur4 as a model reporter system, we establish a role for Ubp2 in membrane protein turnover. Similar to hypomorphic rsp5 alleles, cells deleted for UBP2 exhibited a temporal stabilization of Fur4 at the plasma membrane, indicative of perturbed protein trafficking. This defect was ubiquitin dependent, as a Fur4 N-terminal ubiquitin fusion construct bypassed the block and restored sorting in the mutant. Moreover, the defect was absent in conditions where recycling was absent, implicating Ubp2 in sorting at the multivesicular body. Taken together, our data suggest a previously overlooked role for Ubp2 as a positive regulator of Rsp5-mediated membrane protein trafficking subsequent to endocytosis.     

At UBP3 and At UBP4 are two closely related Arabidopsis thaliana ubiquitin-specific proteases present in the nucleus

The ubiquitin-specific proteases (UBPs) are a class of enzymes vital to the ubiquitin pathway. These enzymes cleave ubiquitin at its C-terminus from two types of substrates containing (i) ubiquitin in an α-amino linkage, as found in the primary ubiquitin translation products, polyubiquitin and ubiquitin-ribosomal fusion proteins, or (ii) ubiquitin in an ?-amino linkage, as found in multiubiquitin chains either unattached or conjugated to cellular proteins. We have isolated cDNAs for two Arabidopsis thaliana genes, AtUBP3 and AtUBP4, which encode UBPs that are 93% identical. These two cDNAs represent the only two members of this subgroup and encode the smallest UBPs described to date in any organism. Using in vivo assays in Escherichia coli that allow the coexpression of a UBP with a putative substrate, we have shown that AtUBP3 and AtUBP4 can specifically deubiquitinate the artificial substrate Ub-X-β-gal but cannot act upon the natural α-amino-linked ubiquitin fusions Arabidopsis Ub-CEP52 and Arabidopsis polyubiquitin. Affinity-purified antibody prepared against AtUBP3 expressed in E. coli recognizes both AtUBP3 and AtUBP4. AtUBP3 and/or AtUBP4 are present in all Arabidopsis organs examined and at multiple developmental stages. Subcellular localization studies show that AtUBP3 and/or AtUBP4 are present in nuclear extracts. Possible physiological roles for these UBPs are discussed.     

Structural Basis for Ovarian Tumor Domain-containing Protein 1 (OTU1) Binding to p97/Valosin-containing Protein (VCP)

Valosin-containing protein (VCP), also known as p97, is an AAA⁺ ATPase that plays an essential role in a broad array of cellular processes including the endoplasmic reticulum-associated degradation (ERAD) pathway. Recently, ERAD-specific deubiquitinating enzymes have been reported to be physically associated with VCP, although the exact mechanism is not yet clear. Among these enzymes is ovarian tumor domain-containing protein 1 (OTU1). Here, we report the structural basis for interaction between VCP and OTU1. The crystal structure of the ubiquitin regulatory X-like (UBXL) domain of OTU1 (UBXL_OTU1) complexed to the N-terminal domain of VCP (N_VCP) at 1.8-Å resolution reveals that UBXL_OTU1 adopts a ubiquitin-like fold and binds at the interface of two subdomains of N_VCP using the ³⁹GYPP⁴² loop of UBXL_OTU1 with the two prolines in cis- and trans-configurations, respectively. A mutagenesis study shows that this loop is not only critical for the interaction with VCP but also for its role in the ERAD pathway. Negative staining EM shows that one molecule of OTU1 binds to one VCP hexamer, and isothermal titration calorimetry suggests that the two proteins bind with a K_D of 0.71 μm. Analytical size exclusion chromatography and isothermal titration calorimetry demonstrates that OTU1 can bind VCP in both the presence and absence of a heterodimer formed by ubiquitin fusion degradation protein 1 and nuclear localization protein 4.     

Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14

The ubiquitin-specific processing protease (UBP) family of deubiquitinating enzymes plays an essential role in numerous cellular processes. Mammalian USP14 (Ubp6 in yeast) is unique among known UBP enzymes in that it is activated catalytically upon specific association with the 26S proteasome. Here, we report the crystal structures of the 45-kDa catalytic domain of USP14 in isolation and in a complex with ubiquitin aldehyde, which reveal distinct structural features. In the absence of ubiquitin binding, the catalytic cleft leading to the active site of USP14 is blocked by two surface loops. Binding by ubiquitin induces a significant conformational change that translocates the two surface loops thereby allowing access of the ubiquitin C-terminus to the active site. These structural observations, in conjunction with biochemical characterization, identify important regulatory mechanisms for USP14.     

Decoding the Ubiquitin-Mediated Pathway of Arthropod Disease Vectors

Protein regulation by ubiquitin has been extensively described in model organisms. However, characterization of the ubiquitin machinery in disease vectors remains mostly unknown. This fundamental gap in knowledge presents a concern because new therapeutics are needed to control vector-borne diseases, and targeting the ubiquitin machinery as a means for disease intervention has been already adopted in the clinic. In this study, we employed a bioinformatics approach to uncover the ubiquitin-mediated pathway in the genomes of Anopheles gambiae, Aedes aegypti, Culex quinquefasciatus, Ixodes scapularis, Pediculus humanus and Rhodnius prolixus. We observed that (1) disease vectors encode a lower percentage of ubiquitin-related genes when compared to Drosophila melanogaster, Mus musculus and Homo sapiens but not Saccharomyces cerevisiae; (2) overall, there are more proteins categorized as E3 ubiquitin ligases when compared to E2-conjugating or E1-activating enzymes; (3) the ubiquitin machinery within the three mosquito genomes is highly similar; (4) ubiquitin genes are more than doubled in the Chagas disease vector (R. prolixus) when compared to other arthropod vectors; (5) the deer tick I. scapularis and the body louse (P. humanus) genomes carry low numbers of E1-activating enzymes and HECT-type E3 ubiquitin ligases; (6) R. prolixus have low numbers of RING-type E3 ubiquitin ligases; and (7) C. quinquefasciatus present elevated numbers of predicted F-box E3 ubiquitin ligases, JAB and UCH deubiquitinases. Taken together, these findings provide novel opportunities to study the interaction between a pathogen and an arthropod vector.     

The solution structure of the ZnF UBP domain of USP33/VDU1

USP33/VDU1 is a deubiquitinating enzyme that binds to the von Hippel-Lindau tumor suppressor protein. It also regulates thyroid hormone activation by deubiquitinating type 2 iodothyronine deiodinase. USP33/VDU1 contains a ZF UBP domain, a protein module found in many proteins in the ubiquitin-proteasome system. Several ZF UBP domains have been shown to bind ubiquitin, and a structure of a complex of the ZF UBP domain of isoT/USP5 and ubiquitin is available. In the present work, the solution structure of the ZF UBP domain of USP33/VDU1 has been determined by NMR spectroscopy. The structure differs from that of the USP5 domain, which contains only one of the three Zn ions present in the USP33/VDU1 structure. The USP33/VDU1 ZnF UBP domain does not bind to ubiquitin.     

Hse1, a component of the yeast Hrs-STAM ubiquitin-sorting complex, associates with ubiquitin peptidases and a ligase to control sorting efficiency into multivesicular bodies

Ubiquitinated integral membrane proteins are delivered to the interior of the lysosome/vacuole for degradation. This process relies on specific ubiquitination of potential cargo and recognition of that Ub-cargo by sorting receptors at multiple compartments. We show that the endosomal Hse1-Vps27 sorting receptor binds to ubiquitin peptidases and the ubiquitin ligase Rsp5. Hse1 is linked to Rsp5 directly via a PY element within its C-terminus and through a novel protein Hua1, which recruits a complex of Rsp5, Rup1, and Ubp2. The SH3 domain of Hse1 also binds to the deubiquitinating protein Ubp7. Functional analysis shows that when both modes of Rsp5 association with Hse1 are altered, sorting of cargo that requires efficient ubiquitination for entry into the MVB is blocked, whereas sorting of cargo containing an in-frame addition of ubiquitin is normal. Further deletion of Ubp7 restores sorting of cargo when the Rsp5:Hse1 interaction is compromised suggesting that both ubiquitin ligases and peptidases associate with the Hse1-Vps27 sorting complex to control the ubiquitination status and sorting efficiency of cargo proteins. Additionally, we find that disruption of UBP2 and RUP1 inhibits MVB sorting of some cargos suggesting that Rsp5 requires association with Ubp2 to properly ubiquitinate cargo for efficient MVB sorting.     

Structure of the HHARI Catalytic Domain Shows Glimpses of a HECT E3 Ligase

The ubiquitin-signaling pathway utilizes E1 activating, E2 conjugating, and E3 ligase enzymes to sequentially transfer the small modifier protein ubiquitin to a substrate protein. During the last step of this cascade different types of E3 ligases either act as scaffolds to recruit an E2 enzyme and substrate (RING), or form an ubiquitin-thioester intermediate prior to transferring ubiquitin to a substrate (HECT). The RING-inBetweenRING-RING (RBR) proteins constitute a unique group of E3 ubiquitin ligases that includes the Human Homologue of Drosophila Ariadne (HHARI). These E3 ligases are proposed to use a hybrid RING/HECT mechanism whereby the enzyme uses facets of both the RING and HECT enzymes to transfer ubiquitin to a substrate. We now present the solution structure of the HHARI RING2 domain, the key portion of this E3 ligase required for the RING/HECT hybrid mechanism. The structure shows the domain possesses two Zn²⁺-binding sites and a single exposed cysteine used for ubiquitin catalysis. A structural comparison of the RING2 domain with the HECT E3 ligase NEDD4 reveals a near mirror image of the cysteine and histidine residues in the catalytic site. Further, a tandem pair of aromatic residues exists near the C-terminus of the HHARI RING2 domain that is conserved in other RBR E3 ligases. One of these aromatic residues is remotely located from the catalytic site that is reminiscent of the location found in HECT E3 enzymes where it is used for ubiquitin catalysis. These observations provide an initial structural rationale for the RING/HECT hybrid mechanism for ubiquitination used by the RBR E3 ligases.     

The emerging role of deubiquitination in nucleotide excision repair

Nucleotide excision repair (NER) protects genome stability by eliminating DNA helix distorting lesions, such as those induced by UV radiation. The addition and removal of ubiquitin, namely, ubiquitination and deubiquitination, have recently been demonstrated as general mechanisms to regulate protein functions. Accumulating evidence shows that several NER factors are subjected to extensive regulation by ubiquitination and deubiquitination. Thus, the balance between E3 ligases and deubiquitinating enzyme activities can dynamically alter the ubiquitin landscape at DNA damage sites, thereby regulating NER efficiency. Current knowledge about XPC ubiquitination by different ubiquitin E3 ligases highlights the importance of ubiquitin linkage types in regulating XPC binding and release from damaged DNA. Here, we discuss the emerging roles of deubiquitinating enzymes and their ubiquitin linkage specificities in NER.     

Reduced cul-5 Activity Causes Aberrant Follicular Morphogenesis and Germ Cell Loss in Drosophila Oogenesis

Drosophila oogenesis is especially well suited for studying stem cell biology, cellular differentiation, and morphogenesis. The small modifier protein ubiquitin regulates many cellular pathways. Ubiquitin is conjugated to target proteins by a diverse class of enzymes called ubiquitin E3 ligases. Here we characterize the requirement of Cul-5, a key component of a subgroup of Cullin-RING-type ubiquitin E3 ligases, in Drosophila oogenesis. We find that reduced cul-5 activity causes the formation of aberrant follicles that are characterized by excess germ cells. We show that germ line cells overproliferate in cul-5 mutant females, causing the formation of abnormally large germ line cysts. Also, the follicular epithelium that normally encapsulates single germ line cysts develops aberrantly in cul-5 mutant, leading to defects in cyst formation. We additionally found that Cul-5 is required for germ cell maintenance, as germ cells are depleted in a substantial fraction of cul-5 mutant ovaries. All of these cul-5 phenotypes are strongly enhanced by reduced activity of gustavus (gus), which encodes a substrate receptor of Cul-5-based ubiquitin E3 ligases. Taken together, our results implicate Cul-5/Gus ubiquitin E3 ligases in ovarian tissue morphogenesis, germ cell proliferation and maintenance of the ovarian germ cell population.