E1 ubiquitin activating enzyme catalyzes the initial step in all ubiquitin-dependent processes. We report the isolation of uba1-204, a temperature-sensitive allele of the essential Saccharomyces cerevisiae E1 gene, UBA1. Uba1-204 cells exhibit dramatic inhibition of the ubiquitin–proteasome system, resulting in rapid depletion of cellular ubiquitin conjugates and stabilization of multiple substrates. We have employed the tight phenotype of this mutant to investigate the role ubiquitin conjugates play in the dynamic interaction of the UbL/UBA adaptor proteins Rad23 and Dsk2 with the proteasome. Although proteasomes purified from mutant cells are intact and proteolytically active, they are depleted of ubiquitin conjugates, Rad23, and Dsk2. Binding of Rad23 to these proteasomes in vitro is enhanced by addition of either free or substrate-linked ubiquitin chains. Moreover, association of Rad23 with proteasomes in mutant and wild-type cells is improved upon stabilizing ubiquitin conjugates with proteasome inhibitor. We propose that recognition of polyubiquitin chains by Rad23 promotes its shuttling to the proteasome in vivo.
Post-translational protein modifications are systems designed to expand restricted genomic information through functional conversion of target molecules. Ubiquitin-like post-translational modifiers regulate numerous cellular events through their covalent linkages to target protein(s) by an enzymatic cascade analogous to ubiquitylation consisting of E1 (activating), E2 (conjugating) and E3 (ligating) enzymes. In this study, we report the essential role of Uba5, a specific activating enzyme for the ubiquitin-like modifier, Ufm1, in erythroid development. Mice lacking Uba5 exhibited severe anaemia, followed by death in utero. Although Uba5 was dispensable for the production of erythropoietin, its genetic loss led to impaired development of megakaryocyte and erythroid progenitors from common myeloid progenitors. Intriguingly, transgenic expression of Uba5 in the erythroid lineage rescued the Uba5-deficient embryos from anaemia and prolonged their survival, demonstrating the importance of Uba5 in cell-autonomous erythroid differentiation. Our results suggest that one of the ubiquitin-like protein modification systems, the Ufm1 system, is involved in the regulation of haematopoiesis.
Post-translational modifications are important in regulating protein function and turnover, and Ufm1 is part of a recently identified protein modification system. In this study, the authors show that Uba5, a component of the Ufm1 system, is important for regulating haematopoiesis and the differentiation of erythroid cells.
Poly-ubiquitination of target proteins typically marks them for destruction via the proteasome and provides an essential mechanism for the dynamic control of protein levels. The E1 ubiquitin-activating enzyme lies at the apex of the ubiquitination cascade, and its activity is necessary for all subsequent steps in the reaction. We have isolated a temperature-sensitive mutation in the Caenorhabditis elegans uba-1 gene, which encodes the sole E1 enzyme in this organism. Manipulation of UBA-1 activity at different developmental stages reveals a variety of functions for ubiquitination, including novel roles in sperm fertility, control of body size, and sex-specific development. Levels of ubiquitin conjugates are substantially reduced in the mutant, consistent with reduced E1 activity. The uba-1 mutation causes delays in meiotic progression in the early embryo, a process that is known to be regulated by ubiquitin-mediated proteolysis. The uba-1 mutation also demonstrates synthetic lethal interactions with alleles of the anaphase-promoting complex, an E3 ubiquitin ligase. The uba-1 mutation provides a sensitized genetic background for identifying new in vivo functions for downstream components of the ubiquitin enzyme cascade, and it is one of the first conditional mutations reported for the essential E1 enzyme in a metazoan animal model.
Proteins that control an organism's development must first be turned on at the proper time and place, and then turned off when they are no longer needed. One of the “off” signals occurs through the attachment of a small protein, known as ubiquitin, to the target protein, which typically leads to the destruction of the target. Attachment of ubiquitin is controlled by a series of enzymes, the first of which is known as E1. Most organisms have a single gene for the E1 enzyme, and its activity is crucial for the degradation of a wide range of target proteins throughout development. We have identified a temperature-sensitive mutation in the E1 enzyme of the nematode Caenorhabditis elegans. By manipulating the growth temperature, we have determined the various functions of E1 at different stages of development. We find that this enzyme controls embryonic and larval development, sperm fertility, and body size. We also characterized sex-specific roles for E1; males exhibit progressive paralysis and defects in the tail, which is used for mating. In addition to the knowledge gained, this mutation provides a means of identifying both the functions of other ubiquitin enzymes during development as well as the target proteins that are marked for destruction.
The covalent attachment of ubiquitin to target proteins influences various cellular processes, including DNA repair, NF-κB signalling and cell survival1. The most common mode of regulation by ubiquitin-conjugation involves specialized ubiquitin-binding proteins that bind to ubiquitylated proteins and link them to downstream biochemical processes. Unravelling how the ubiquitin-message is recognized is essential because aberrant ubiquitin-mediated signalling contributes to tumour formation2. Recent evidence indicates that inhibitor of apoptosis (IAP) proteins are frequently overexpressed in cancer and their expression level is implicated in contributing to tumorigenesis, chemoresistance, disease progression and poor patient-survival3. Here, we have identified an evolutionarily conserved ubiquitin-associated (UBA) domain in IAPs, which enables them to bind to Lys 63-linked polyubiquitin. We found that the UBA domain is essential for the oncogenic potential of cIAP1, to maintain endothelial cell survival and to protect cells from TNF-α-induced apoptosis. Moreover, the UBA domain is required for XIAP and cIAP2–MALT1 to activate NF-κB. Our data suggest that the UBA domain of cIAP2–MALT1 stimulates NF-κB signalling by binding to polyubiquitylated NEMO. Significantly, 98% of all cIAP2–MALT1 fusion proteins retain the UBA domain, suggesting that ubiquitin-binding contributes to the oncogenic potential of cIAP2–MALT1 in MALT lymphoma. Our data identify IAPs as ubiquitin-binding proteins that contribute to ubiquitin-mediated cell survival, NF-κB signalling and oncogenesis.
E2-25K is an ubiquitin-conjugating enzyme with the ability to synthesize Lys48-linked polyubiquitin chains. E2-25K and its homologues represent the only known E2 enzymes which contain a C-terminal ubiquitin-associated (UBA) domain as well as the conserved catalytic ubiquitin-conjugating (UBC) domain. As an additional non-covalent binding surface for ubiquitin, the UBA domain must provide some functional specialization. We mapped the protein-protein interface involved in the E2-25K UBA/ubiquitin complex by solution nuclear magnetic resonance (NMR) spectroscopy and subsequently modeled the structure of the complex. Domain-domain interactions between the E2-25K catalytic UBC domain and the UBA domain do not induce significant structural changes in the UBA domain or alter the affinity of the UBA domain for ubiquitin. We determined that one of the roles of the C-terminal UBA domain, in the context of E2-25K, is to increase processivity in Lys48-linked polyubiquitin chain synthesis, possibly through increased binding to the ubiquitinated substrate. Additionally, we see evidence that the UBA domain directs specificity in polyubiquitin chain linkage.
Ubiquitin; Ubiquitin-conjugating enzyme; E2-25K; UBA domain
Vertebrates express two enzymes for activation of ubiquitin – UBA1, which is responsible for activation of the vast majority of E2 conjugating enzymes, and UBA6, which uses the dedicated E2, USE1. However, targets and E3s for UBA6-USE1 are unknown. Here, we demonstrate that UBA6-USE1 functions with the UBR1-3 subfamily of N-recognin E3s to degrade the N-end rule substrates RGS4, RGS5, and Arg(R)-GFP. This pathway functions in the cytoplasm in parallel with the UBA1-UBE2A/B-UBR2 cascade, which promotes turnover of nuclear RGS4/5 proteins and an apparently phenotypically distinct pool of cytoplasmic RGS4/5. UBR2 promotes Lys48 (K48)-specific ubiquitin discharge from, and RGS4 ubiquitylation by, both USE1 and UBE2A in vitro. This work provides insight into the machinery employed by the UBA6-USE1 cascade to promote protein turnover, and suggests that the UBA6 and UBA1 pathways can function in parallel with the same E3 to degrade the same targets in a spatially distinct manner.
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.
Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo, NEDD8, and ISG15 are ligated to targets by E1-E2-E3 multienzyme cascades. The Sumo cascade, conserved among all eukaryotes, regulates numerous biological processes including protein localization, transcription, DNA replication, and mitosis. Sumo conjugation is initiated by the heterodimeric Aos1-Uba2 E1 enzyme (in humans called Sae1-Uba2), which activates Sumo's C-terminus, binds the dedicated E2 enzyme Ubc9, and promotes Sumo C-terminal transfer between the Uba2 and Ubc9 catalytic cysteines. To gain insights into details of E1-E2 interactions in the Sumo pathway, we determined crystal structures of the C-terminal ubiquitin fold domain (ufd) from yeast Uba2 (Uba2ufd), alone and in complex with Ubc9. The overall structures of both yeast Uba2ufd and Ubc9 superimpose well on their individual human counterparts, suggesting conservation of fundamental features of Sumo conjugation. Docking the Uba2ufd-Ubc9 and prior full-length human Uba2 structures allows generation of models for steps in Sumo transfer from Uba2 to Ubc9, and supports the notion that Uba2 undergoes remarkable conformational changes during the reaction. Comparisons to previous structures from the NEDD8 cascade demonstrate that UBL cascades generally utilize some parallel E1-E2 interaction surfaces. In addition, the structure of the Uba2ufd-Ubc9 complex reveals interactions unique to Sumo E1 and E2. Comparison with a previous Ubc9-E3 complex structure demonstrates overlap between Uba2 and E3 binding sites on Ubc9, indicating that loading with Sumo and E3-catalyzed transfer to substrates are strictly separate steps. The results suggest mechanisms establishing specificity and order in Sumo conjugation cascades.
Genetic immunization is a promising new technology for developing vaccines against tuberculosis that are more effective. In the present study, we evaluated the effects of intracellular turnover of antigens expressed by DNA vaccines on the immune response induced by these vaccines in a mouse model of pulmonary tuberculosis. The mycobacterial culture filtrate protein MPT64 was expressed as a chimeric protein fused to one of three variants of the ubiquitin protein (UbG, UbA, and UbGR) known to differentially affect the intracellular processing of the coexpressed antigens. Immunoblot analysis of cell lysates of in vitro-transfected cells showed substantial differences in the degradation rate of ubiquinated MPT64 (i.e., UbG64 < UbA64 < UbGR64). The specific immune response generated in mice correlated with the stability of the ubiquitin-conjugated antigen. The UbA64 DNA vaccine induced a weak humoral response compared to UbG64, and a mixed population of interleukin-4 (IL-4)- and gamma interferon (IFN-γ)-secreting cells. Vaccination with the UbGR64 plasmid generated a strong Th1 cell response (high IFN-γ, low IL-4) in the absence of a detectable humoral response. Aerogenic challenge of vaccinated mice with Mycobacterium tuberculosis indicated that immunization with both the UbA64- and UbGR64-expressing plasmids evoked an enhanced protective response compared to the vector control. The expression of mycobacterial antigens from DNA vaccines as fusion proteins with a destabilizing ubiquitin molecule (UbA or UbGR) shifted the host response toward a stronger Th1-type immunity which was characterized by low specific antibody levels, high numbers of IFN-γ-secreting cells, and significant resistance to a tuberculous challenge.
Ubiquitin-dependent protein degradation within malarial parasites is a burgeoning field of interest due to several encouraging reports of proteasome inhibitors that were able to confer antimalarial activity. Despite the growing interest in the Plasmodium proteasome system, relatively little investigation has been done to actually characterize the parasite degradation machinery. In this report, we provide an initial biological investigation of the ubiquitylating components of the endoplasmic reticulum-associated degradation (ERAD) system, which is a major pathway in targeting misfolded proteins from the ER to the cytosol for proteasome degradation. We are able to show that the ERAD system is essential for parasite survival and that the putative Plasmodium HRD1 (E3 ubiquitin ligase), UBC (E2 ubiquitin conjugating enzyme) and UBA1 (E1 ubiquitin activating enzyme) are able to mediate in vitro ubiquitylation. Furthermore, by using immunofluorescence, we report that Plasmodium HRD1 localizes to the ER membranes, while the Plasmodium UBC and UBA1 localize to the cytosol. In addition, our gene disruption experiments indicate that the Plasmodium HRD1 is likely essential. We have conducted an initial characterization of the ubiquitylating components of the Plasmodium ERAD system, a major pathway for protein degradation and parasite maintenance. In conjunction with promising proteasome inhibitor studies, we explore the possibility of targeting the Plasmodium ERAD system for future bottom-up drug development approaches.
Rad23 contains a ubiquitin-like domain (UbLR23) that interacts with catalytically active proteasomes and two ubiquitin (Ub)-associated (UBA) sequences that bind Ub. The UBA domains can bind Ub in vitro, although the significance of this interaction in vivo is poorly understood. Rad23 can interfere with the assembly of multi-Ub chains in vitro, and high-level expression caused stabilization of proteolytic substrates in vivo. We report here that Rad23 interacts with ubiquitinated cellular proteins through the synergistic action of its UBA domains. Rad23 plays an overlapping role with Rpn10, a proteasome-associated multi-Ub chain binding protein. Mutations in the UBA domains prevent efficient interaction with ubiquitinated proteins and result in poor suppression of the growth and proteolytic defects of a rad23Δ rpn10Δ mutant. High-level expression of Rad23 revealed, for the first time, an interaction between ubiquitinated proteins and the proteasome. This increase was not observed in rpn10Δ mutants, suggesting that Rpn10 participates in the recognition of proteolytic substrates that are delivered by Rad23. Overexpression of UbLR23 caused stabilization of a model substrate, indicating that an unregulated UbLR23-proteasome interaction can interfere with the efficient delivery of proteolytic substrates by Rad23. Because the suppression of a rad23Δ rpn10Δ mutant phenotype required both UbLR23 and UBA domains, our findings support the hypothesis that Rad23 encodes a novel regulatory factor that translocates ubiquitinated substrates to the proteasome.
Extensive genome-wide remodeling occurs during the formation of the somatic macronuclei from the germ line micronuclei in ciliated protozoa. This process is limited to sexual reproduction and includes DNA amplification, chromosome fragmentation, and the elimination of internal segments of DNA. Our efforts to define the pathways regulating these events revealed a gene encoding a homologue of ubiquitin activating enzyme 2 (UBA2) that is upregulated at the onset of macronuclear development in Paramecium tetraurelia. Uba2 enzymes are known to activate the protein called small ubiquitin-related modifier (SUMO) that is covalently attached to target proteins. Consistent with this relationship, Northern analysis showed increased abundance of SUMO transcripts during sexual reproduction in Paramecium. RNA interference (RNAi) against UBA2 or SUMO during vegetative growth had little effect on cell survival or fission rates. In contrast, RNAi of mating cells resulted in failure to form a functional macronucleus. Despite normal amplification of the genome, excision of internal eliminated sequences was completely blocked. Additional experiments showed that the homologous UBA2 and SUMO genes in Tetrahymena thermophila are also upregulated during conjugation. These results provide evidence for the developmental regulation of the SUMO pathway in ciliates and suggest a key role for the pathway in controlling genome remodeling.
In this report, we demonstrate the existence of the ubiquitin fold modifier-1 (Ufm1) and its conjugation pathway in trypanosomatid parasite Leishmania donovani. LdUfm1 is activated by E1-like enzyme LdUba5. LdUfc1 (E2) specifically interacted with LdUfm1 and LdUba5 to conjugate LdUfm1 to proteinaceous targets. Mass spectrometry analysis revealed that LdUfm1 is conjugated to Leishmania protein targets that are associated with mitochondria. Immunofluorescence experiments showed that Leishmania Ufm1, Uba5 and Ufc1 are associated with the mitochondria. The demonstration that all the components of this system as well as the substrates are associated with mitochondrion suggests it may have physiological roles not yet described in any other organism. Overexpression of a non-conjugatable form of LdUfm1 and an active site mutant of LdUba5 resulted in reduced survival of Leishmania in the macrophage. Since mitochondrial activities are developmentally regulated in the life cycle of trypanosomatids, Ufm1 mediated modifications of mitochondrial proteins may be important in such regulation. Thus, Ufm1 conjugation pathway in Leishmania could be explored as a potential drug target in the control of Leishmaniasis.
For cell regulation, E2-like ubiquitin-fold modifier conjugating enzyme 1 (Ufc1) is involved in the transfer of ubiquitin-fold modifier 1 (Ufm1), a ubiquitin like protein which is activated by E1-like enzyme Uba5, to various target proteins. Thereby, Ufc1 participates in the very recently discovered Ufm1-Uba5-Ufc1 ubiquination pathway which is found in metazoan organisms. The structure of human Ufc1 was solved by using both NMR spectroscopy and X-ray crystallography. The complementary insights obtained with the two techniques provided a unique basis for understanding the function of Ufc1 at atomic resolution. The Ufc1 structure consists of the catalytic core domain conserved in all E2-like enzymes and an additional N-terminal helix. The active site Cys116, which forms a thio-ester bond with Ufm1, is located in a flexible loop that is highly solvent accessible. Based on the Ufc1 and Ufm1 NMR structures, a model could be derived for the Ufc1-Ufm1 complex in which the C-terminal Gly83 of Ufm1 may well form the expected thio-ester with Cys116, suggesting that Ufm1-Ufc1 functions as described for other E1-E2-E3 machineries. α-helix 1 of Ufc1 adopts different conformations in the crystal and in solution, suggesting that this helix plays a key role to mediate specificity.
Ufc1; Ufm1; Ubiquitin; E2; Ubiquitin Conjugating Enzyme
The ubiquitination–proteasome and degradation system is an essential process that regulates protein homeostasis. This system is involved in the regulation of cell proliferation, differentiation and survival, and dysregulations in this system lead to pathologies including cancers. The ubiquitination system is an enzymatic cascade that mediates the marking of target proteins by an ubiquitin label and thereby directs their degradation through the proteasome pathway. The ubiquitination of proteins occurs through a three-step process involving ubiquitin activation by the E1 enzyme, allowing for the transfer to a ubiquitin-conjugated enzyme E2 and to the targeted protein via ubiquitin-protein ligases (E3), the most abundant group of enzymes involved in ubiquitination. Significant advances have been made in our understanding of the role of E3 ubiquitin ligases in the control of bone turnover and tumorigenesis. These ligases are implicated in the regulation of bone cells through the degradation of receptor tyrosine kinases, signaling molecules and transcription factors. Initial studies showed that the E3 ubiquitin ligase c-Cbl, a multi-domain scaffold protein, regulates bone resorption by interacting with several molecules in osteoclasts. Further studies showed that c-Cbl controls the ubiquitination of signaling molecules in osteoblasts and in turn regulates osteoblast proliferation, differentiation and survival. Recent data indicate that c-Cbl expression is decreased in primary bone tumors, resulting in excessive receptor tyrosine kinase signaling. Consistently, c-Cbl ectopic expression reduces bone tumorigenesis by promoting tyrosine kinase receptor degradation. Here, we review the mechanisms of action of E3 ubiquitin ligases in the regulation of normal and pathologic bone formation, and we discuss how targeting the interactions of c-Cbl with some substrates may be a potential therapeutic strategy to promote osteogenesis and to reduce tumorigenesis.
ubiquitin ligases; proteasome; receptor tyrosine kinases; bone tumors; Cbl proteins; ubiquitination
NEDD8/Rub1 is a ubiquitin (Ub)-like molecule that covalently ligates to target proteins through an enzymatic cascade analogous to ubiquitylation. This modifier is known to target all cullin (Cul) family proteins. The latter are essential components of Skp1/Cul-1/F-box protein (SCF)–like Ub ligase complexes, which play critical roles in Ub-mediated proteolysis. To determine the role of the NEDD8 system in mammals, we generated mice deficient in Uba3 gene that encodes a catalytic subunit of NEDD8-activating enzyme. Uba3−/− mice died in utero at the periimplantation stage. Mutant embryos showed selective apoptosis of the inner cell mass but not of trophoblastic cells. However, the mutant trophoblastic cells could not enter the S phase of the endoreduplication cycle. This cell cycle arrest was accompanied with aberrant expression of cyclin E and p57Kip2. These results suggested that the NEDD8 system is essential for both mitotic and the endoreduplicative cell cycle progression. β-Catenin, a mediator of the Wnt/wingless signaling pathway, which degrades continuously in the cytoplasm through SCF Ub ligase, was also accumulated in the Uba3−/− cytoplasm and nucleus. Thus, the NEDD8 system is essential for the regulation of protein degradation pathways involved in cell cycle progression and morphogenesis, possibly through the function of the Cul family proteins.
NEDD8; ubiquitin; cullin; knock-out; cell cycle
E3 ubiquitin ligases are a large family of proteins that are engaged in the regulation of the turnover and activity of many target proteins. Together with ubiquitin-activating enzyme E1 and ubiquitin-conjugating enzyme E2, E3 ubiquitin ligases catalyze the ubiquitination of a variety of biologically significant protein substrates for targeted degradation through the 26S proteasome, as well as for nonproteolytic regulation of their functions or subcellular localizations. E3 ubiquitin ligases, therefore, play an essential role in the regulation of many biologic processes. Increasing amounts of evidence strongly suggest that the abnormal regulation of some E3 ligases is involved in cancer development. Furthermore, some E3 ubiquitin ligases are frequently overexpressed in human cancers, which correlates well with increased chemoresistance and poor clinic prognosis. In this review, E3 ubiquitin ligases (such as murine double minute 2, inhibitor of apoptosis protein, and Skp1-Cullin-F-box protein) will be evaluated as potential cancer drug targets and prognostic biomarkers. Extensive study in this field would lead to a better understanding of the molecular mechanism by which E3 ligases regulate cellular processes and of how their deregulations contribute to carcinogenesis. This would eventually lead to the development of a novel class of anticancer drugs targeting specific E3 ubiquitin ligases, as well as the development of sensitive biomarkers for cancer treatment, diagnosis, and prognosis.
Apoptosis; biomarkers; cancer targets; E3 ubiquitin ligases; protein degradation
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.
The ubiquitin–proteasome pathway of protein degradation is one of the major mechanisms that are involved in the maintenance of the proper levels of cellular proteins. The regulation of proteasomal degradation thus ensures proper cell functions. The family of proteins containing ubiquitin-like (UbL) and ubiquitin-associated (UBA) domains has been implicated in proteasomal degradation. UbL–UBA domain containing proteins associate with substrates destined for degradation as well as with subunits of the proteasome, thus regulating the proper turnover of proteins.
UbL; UBA; Proteasome; Degradation; Ubiquitin
The ubiquitin-associated (UBA) domain of the mRNA nuclear export receptor Mex67 helps in coordinating transcription elongation and nuclear export by interacting both with ubiquitin conjugates and specific targets, such as Hpr1, a component of the THO complex. Here, we analyzed substrate specificity and ubiquitin selectivity of the Mex67 UBA domain. UBA-Mex67 is formed by three helices arranged in a classical UBA fold plus a fourth helix, H4. Deletion or mutation of helix H4 strengthens the interaction between UBA-Mex67 and ubiquitin, but it decreases its affinity for Hpr1. Interaction with Hpr1 is required for Mex67 UBA domain to bind polyubiquitin, possibly by inducing an H4-dependent conformational change. In vivo, deletion of helix H4 reduces cotranscriptional recruitment of Mex67 on activated genes, and it also shows an mRNA export defect. Based on these results, we propose that H4 functions as a molecular switch that coordinates the interaction of Mex67 with ubiquitin bound to specific substrates, defines the selectivity of the Mex67 UBA domain for polyubiquitin, and prevents its binding to nonspecific substrates.
Homeostasis of the endoplasmic reticulum (ER) is essential for normal cellular functions. Disturbance of this homeostasis causes ER stress and activates the Unfolded Protein Response (UPR). The Ufm1 conjugation system is a novel Ubiquitin-like (Ubl) system whose physiological target(s) and biological functions remain largely undefined. Genetic study has demonstrated that the Ufm1-activating enzyme Uba5 is indispensible for erythroid differentiation in mice, highlighting the importance of this novel system in animal development. In this report we present the evidence for involvement of RCAD/Ufl1, a putative Ufm1-specific E3 ligase, and its binding partner C53/LZAP protein in ufmylation of endogenous Ufm1 targets. Moreover, we found that the Ufm1 system was transcriptionally up-regulated by disturbance of the ER homeostasis and inhibition of vesicle trafficking. Using luciferase reporter and ChIP assays, we dissected the Ufm1 promoter and found that Ufm1 was a potential target of Xbp-1, one of crucial transcription factors in UPR. We further examined the effect of Xbp-1 deficiency on the expression of the Ufm1 components. Interestingly, the expression of Ufm1, Uba5, RCAD/Ufl1 and C53/LZAP in wild-type mouse embryonic fibroblasts (MEFs) was significantly induced by inhibition of vesicle trafficking, but the induction was negated by Xbp-1 deficiency. Finally, we found that knockdown of the Ufm1 system in U2OS cells triggered UPR and amplification of the ER network. Taken together, our study provided critical insight into the regulatory mechanism of the Ufm1 system and established a direct link between this novel Ubl system and the ER network.
Inhibitor of apoptosis proteins (IAPs) belong to a pivotal antiapoptotic protein family that plays a crucial role in tumorigenesis, cancer progression, chemoresistance and poor patient-survival. X-linked inhibitor of apoptosis protein (XIAP) is a prominent member of IAPs attracting intense research because it has been demonstrated to be a physiological inhibitor of caspases and apoptosis. Recently, an evolutionarily conserved ubiquitin-associated (UBA) domain was identified in XIAP and a number of RING domain-bearing IAPs. This has placed the IAPs in the group of ubiquitin binding proteins. Here, we explore the three-dimensional structure of the XIAP UBA domain (XIAP-UBA) and how it interacts with mono-ubiquitin and diubiquitin conjugates.
The solution structure of the XIAP-UBA domain was determined by NMR spectroscopy. XIAP-UBA adopts a typical UBA domain fold of three tightly packed α-helices but with an additional N-terminal 310 helix. The XIAP-UBA binds mono-ubiquitin as well as Lys48-linked and linear-linked diubiquitins at low-micromolar affinities. NMR analysis of the XIAP-UBA–ubiquitin interaction reveals that it involves the classical hydrophobic patches surrounding Ile44 of ubiquitin and the conserved MGF/LV motif surfaces on XIAP-UBA. Furthermore, dimerization of XIAP-UBA was observed. Mapping of the self-association surface of XIAP-UBA reveals that the dimerization interface is formed by residues in the N-terminal 310 helix, helix α1 and helix α2, separate from the ubiquitin-binding surface.
Our results provide the first structural information of XIAP-UBA and map its interaction with mono-ubiquitin, Lys48-linked and linear-linked diubiquitins. The notion that XIAP-UBA uses different surfaces for ubiquitin-binding and self-association provides a plausible model to explain the reported selectivity of XIAP in binding polyubiquitin chains with different linkages.
Ubiquitination is a post-translational modification that generally directs proteins for degradation by the proteasome or by lysosomes. However, ubiquitination has been implicated in many other cellular processes, including transcriptional regulation, DNA repair, regulation of protein-protein interactions and association with ubiquitin-binding scaffolds. Ubiquitination is a dynamic process. Ubiquitin is added to proteins by E3 ubiquitin ligases as a covalent modification to one or multiple lysine residues as well as non-lysine amino acids. Ubiquitin itself contains seven lysines, each of which can also be ubiquitinated, leading to polyubiquitin chains that are best characterized for linkages occurring through K48 and K63. Ubiquitination can also be reversed by the action of deubiquitination enzymes (DUbs). Like E3 ligases, DUbs play diverse and critical roles in cells.1 Ubiquitin is expressed as a fusion protein, as a linear repeat or as a fusion to ribosomal subunits, and DUbs are necessary to liberate free ubiquitin, making them the first enzyme of the ubiquitin cascade. Proteins destined for degradation by the proteasome or by lysosomes are deubiquitinated prior to their degradation, which allows ubiquitin to be recycled by the cell, contributing to the steady-state pool of free ubiquitin. Proteins destined for degradation by lysosomes are also acted upon by both ligases and DUbs. Deubiquitination can also act as a means to prevent protein degradation, and many proteins are thought to undergo rounds of ubiquitination and deubiquitination, ultimately resulting in either the degradation or stabilization of those proteins. Despite years of study, examining the effects of the ubiquitination of proteins remains quite challenging. This is because the methods that are currently being employed to study ubiquitination are limiting. Here, we briefly examine current strategies to study the effects of ubiquitination and describe an additional novel approach that we have developed.
ubiquitin; endosome; ligase; lysosome; degradation
Hedgehog transduces signal by promoting cell surface expression of the seven-transmembrane protein Smoothened (Smo) in Drosophila, but the underlying mechanism remains unknown. Here we demonstrate that Smo is downregulated by ubiquitin-mediated endocytosis and degradation, and that Hh increases Smo cell surface expression by inhibiting its ubiquitination. We find that Smo is ubiquitinated at multiple Lysine residues including those in its autoinhibitory domain (SAID), leading to endocytosis and degradation of Smo by both lysosome- and proteasome-dependent mechanisms. Hh inhibits Smo ubiquitination via PKA/CK1-mediated phosphorylation of SAID, leading to Smo cell surface accumulation. Inactivation of the ubiquitin activating enzyme Uba1 or perturbation of multiple components of the endocytic machinery leads to Smo accumulation and Hh pathway activation. In addition, we find that the non-visual β-arrestin Kurtz (Krz) interacts with Smo and acts in parallel with ubiquitination to downregulate Smo. Finally, we show that Smo ubiquitination is counteracted by the deubiquitinating enzyme UBPY/USP8. Gain and loss of UBPY lead to reciprocal changes in Smo cell surface expression. Taken together, our results suggest that ubiquitination plays a key role in the downregulation of Smo to keep Hh pathway activity off in the absence of the ligand, and that Hh-induced phosphorylation promotes Smo cell surface accumulation by inhibiting its ubiquitination, which contributes to Hh pathway activation.
The Hedgehog (Hh) family of secreted proteins governs cell growth and patterning in diverse species ranging from Drosophila to human. Hh signals across the cell surface membrane by regulating the subcellular location and conformation of a membrane protein called Smoothened (Smo). In Drosophila, Smo accumulates on the cell surface in response to Hh, whereas in the absence of Hh it is internalized and degraded. The molecular mechanisms that control this intracellular trafficking and degradation of Smo were unknown, but here we show that Smo is modified by attachment of several molecules of a small protein called ubiquitin, which tags it for internalization and degradation within the cell. Hh inhibits this ubiquitination of Smo by inducing another modification, phosphorylation, of its intracellular tail by two types of protein kinase enzymes. This loss of ubiquitination and gain of phosphorylation causes the accumulation of Smo at the cell surface. What's more, we find that another protein called Kurtz interacts with Smo and acts in parallel with the ubiquitination process to promote internalization of Smo, and that the deubiquitinating enzyme UBPY/USP8 counteracts ubiquitination of Smo to promote its cell surface accumulation. Our study demonstrates that reversible ubiquitination plays a key role in regulating Smo trafficking to and from the cell surface and thus it provides novel insights into the mechanism of Hh signaling from the outside to the inside of the cell.
The ubiquitin–proteasome pathway is the major nonlysosomal proteolytic system in eukaryotic cells responsible for regulating the level of many key regulatory molecules within the cells. Modification of cellular proteins by ubiquitin and ubiquitin-like proteins, such as small ubiquitin-like modifying protein (SUMO), plays an essential role in a number of biological schemes, and ubiquitin pathway enzymes have become important therapeutic targets. Ubiquitination is a dynamic reversible process; a multitude of ubiquitin ligases and deubiquitinases (DUBs) are responsible for the wide-ranging influence of this pathway as well as its selectivity. The DUB enzymes serve to maintain adequate pools of free ubiquitin and regulate the ubiquitination status of cellular proteins. Using SUMO fusions, a novel assay system, based on poliovirus RNA-dependent RNA polymerase activity, is described here. The method simplifies the isopeptidase assay and facilitates high-throughput analysis of these enzymes. The principle of the assay is the dependence of the viral polymerase on a free N terminus for activity; accordingly, the polymerase is inactive when fused at its N terminus to SUMO or any other ubiquitin-like protein. The assay is sensitive, reproducible, and adaptable to a high-throughput format for use in screens for inhibitors/activators of clinically relevant SUMO proteases and deubiquitinases.
Isopeptidases; Protein degradation; N terminus; 3D polymerase