C-terminal Ubiquitin-associated Domain Research Articles

1H, 15N, 13C backbone and Cβ resonance assignments for UBQLN1 UBA and UBAA domains

UBQLN1 functions in autophagy and proteasome-mediated protein degradation. It contains an N-terminal ubiquitin-like domain (UBL), a C-terminal ubiquitin-associated domain (UBA), and a flexible central region which functions as a chaperone to prevent protein aggregation. Here, we report the 1H, 15N, and 13C resonance assignments for the backbone (NH, N, C’, Cα, and Hα) and sidechain Cβ atoms of the UBQLN1 UBA and an N-terminally adjacent segment called the UBA-adjacent domain (UBAA). We find a subset of the resonances corresponding to the UBAA to have concentration-dependent chemical shifts, likely due to self-association. We also find the backbone amide nitrogen of T572 to be shifted upfield relative to the average value for a threonine amide nitrogen, a phenomenon likely caused by T572 Hγ1 engagement in a hydrogen bond with adjacent backbone carbonyl atoms. The assignments described in this manuscript can be used to study the protein dynamics of the UBQLN1 UBA and UBAA as well as the interaction of these domains with other proteins.

HYPK coordinates degradation of polyneddylated proteins by autophagy

ABSTRACT Selective degradation of protein aggregates by macroautophagy/autophagy is an essential homeostatic process of safeguarding cells from the effects of proteotoxicity. Among the ubiquitin-like proteins, NEDD8 conjugation to misfolded proteins is prominent in stress-induced protein aggregates, albeit the function of neddylation in autophagy is unclear. Here, we report that polyneddylation functions as a post-translational modification for autophagic degradation of proteotoxic-stress induced protein aggregates. We also show that HYPK functions as an autophagy receptor in the polyneddylation-dependent aggrephagy. The scaffolding function of HYPK is facilitated by its C-terminal ubiquitin-associated domain and N-terminal tyrosine-type LC3-interacting region which bind to NEDD8 and LC3 respectively. Both NEDD8 and HYPK are positive modulators of basal and proteotoxicity-induced autophagy, leading to protection of cells from protein aggregates, such as aggregates of mutant HTT exon 1. Thus, we propose an indispensable and additive role of neddylation and HYPK in clearance of protein aggregates by autophagy, resulting in cytoprotective effect during proteotoxic stress. Abbreviations: ATG5, autophagy related 5; ATG12, autophagy related 12; ATG14, autophagy related 14; BECN1, beclin 1; CBL, casitas B-lineage lymphoma; CBLB, Cbl proto-oncogene B; GABARAP, GABA type A receptor-associated protein; GABARAPL1, GABA type A receptor associated protein like 1; GABARAPL2, GABA type A receptor associated protein like 2; GFP, green fluorescent protein; HTT, huntingtin; HTT97Q exon 1, huntingtin 97-glutamine exon 1; HUWE1, HECT, UBA and WWE domain containing E3 ubiquitin protein ligase 1; HYPK, huntingtin interacting protein K; IgG, immunoglobulin G; IMR-32, Institute for Medical Research-32; KD, knockdown; Kd, dissociation constant; LAMP1, lysosomal associated membrane protein 1; LIR, LC3 interacting region; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MAP1LC3A/LC3A, microtubule associated protein 1 light chain 3 alpha; MAP1LC3B/LC3B, microtubule associated protein 1 light chain 3 beta; MARK1, microtubule affinity regulating kinase 1; MARK2, microtubule affinity regulating kinase 2; MARK3, microtubule affinity regulating kinase 3; MARK4, microtubule affinity regulating kinase 4; MCF7, Michigan Cancer Foundation-7; MTOR, mechanistic target of rapamycin kinase; NAE1, NEDD8 activating enzyme E1 subunit 1; NBR1, NBR1 autophagy cargo receptor; NEDD8, NEDD8 ubiquitin like modifier; Ni-NTA, nickel-nitrilotriacetic acid; NUB1, negative regulator of ubiquitin like proteins 1; PIK3C3, phosphatidylinositol 3-kinase catalytic subunit type 3; PolyQ, poly-glutamine; PSMD8, proteasome 26S subunit, non-ATPase 8; RAD23A, RAD23 homolog A, nucleotide excision repair protein; RAD23B, RAD23 homolog B, nucleotide excision repair protein; RFP, red fluorescent protein; RPS27A, ribosomal protein S27a; RSC1A1, regulator of solute carriers 1; SNCA, synuclein alpha; SIK1, salt inducible kinase 1; siRNA, small interfering ribonucleic acid; SOD1, superoxide dismutase 1; SPR, surface plasmon resonance; SQSTM1, sequestosome 1; SUMO1, small ubiquitin like modifier 1; TAX1BP1, Tax1 binding protein 1; TDRD3, tudor domain containing 3; TNRC6C, trinucleotide repeat containing adaptor 6C; TOLLIP, toll interacting protein; TUBA, tubulin alpha; TUBB, tubulin beta class I; UBA, ubiquitin-associated; UBA1, ubiquitin like modifier activating enzyme 1; UBA5, ubiquitin like modifier activating enzyme 5; UBAC1, UBA domain containing 1; UBAC2, UBA domain containing 2; UBAP1, ubiquitin associated protein 1; UBAP2, ubiquitin associated protein 2; UBASH3B, ubiquitin associated and SH3 domain containing B; UBD/FAT10, ubiquitin D; UBE2K, ubiquitin conjugating enzyme E2 K; UBLs, ubiquitin-like proteins; UBL7, ubiquitin like 7; UBQLN1, ubiquilin 1; UBQLN2, ubiquilin 2; UBQLN3, ubiquilin 3; UBQLN4, ubiquilin 4; UBXN1, UBX domain protein 1; ULK1, unc-51 like autophagy activating kinase 1; URM1, ubiquitin related modifier 1; USP5, ubiquitin specific peptidase 5; USP13, ubiquitin specific peptidase 13; VPS13D, vacuolar protein sorting 13 homolog D

Toxoplasma gondii UBL-UBA Shuttle Protein DSK2s Are Important for Parasite Intracellular Replication

Toxoplasma gondii (T. gondii) is an important human and veterinary pathogen causing life-threatening disease in immunocompromised patients. The UBL-UBA shuttle protein family are important components of the ubiquitin–proteasome system. Here, we identified a novel UBL-UBA shuttle protein DSK2b that is charactered by an N-terminal ubiquitin-like domain (UBL) and a C-terminal ubiquitin-associated domain (UBA). DSK2b was localized in the cytoplasm and nucleus. The deletion of dsk2b did not affect the degradation of ubiquitinated proteins, parasite growth in vitro or virulence in mice. The double-gene knockout of dsk2b and its paralogs dsk2a (ΔΔdsk2adsk2b) results in a significant accumulation of ubiquitinated proteins and the asynchronous division of T. gondii. The growth of ΔΔdsk2adsk2b was significantly inhibited in vitro, while virulence in mice was not attenuated. In addition, autophagy occurred in the ΔΔdsk2adsk2b, which was speculated to degrade the accumulated ubiquitinated proteins in the parasites. Overall, DSK2b is a novel UBL-UBA shuttle protein contributing to the degradation of ubiquitinated proteins and is important for the synchronous cell division of T. gondii.

Molecular insights into an ancient form of Paget’s disease of bone

Paget's disease of bone (PDB) is a chronic skeletal disorder that can affect one or several bones in individuals older than 55 y of age. PDB-like changes have been reported in archaeological remains as old as Roman, although accurate diagnosis and natural history of the disease is lacking. Six skeletons from a collection of 130 excavated at Norton Priory in the North West of England, which dates to medieval times, show atypical and extensive pathological changes resembling contemporary PDB affecting as many as 75% of individual skeletons. Disease prevalence in the remaining collection is high, at least 16% of adults, with age at death estimations as low as 35 y. Despite these atypical features, paleoproteomic analysis identified sequestosome 1 (SQSTM1) or p62, a protein central to the pathological milieu of PDB, as one of the few noncollagenous human sequences preserved in skeletal samples. Targeted proteomic analysis detected >60% of the ancient p62 primary sequence, with Western blotting indicating p62 abnormalities, including in dentition. Direct sequencing of ancient DNA excluded contemporary PDB-associated SQSTM1 mutations. Our observations indicate that the ancient p62 protein is likely modified within its C-terminal ubiquitin-associated domain. Ancient miRNAs were remarkably preserved in an osteosarcoma from a skeleton with extensive disease, with miR-16 expression consistent with that reported in contemporary PDB-associated bone tumors. Our work displays the use of proteomics to inform diagnosis of ancient diseases such as atypical PDB, which has unusual features presumably potentiated by yet-unidentified environmental or genetic factors.

The STI and UBA Domains of UBQLN1 Are Critical Determinants of Substrate Interaction and Proteostasis.

There are five Ubiquilin proteins (UBQLN1-4, UBQLN-L), which are evolutionarily conserved and structurally similar. UBQLN proteins have three functional domains: N-terminal ubiquitin-like domain (UBL), C-terminal ubiquitin-associated domain (UBA), and STI chaperone-like regions in the middle. Alterations in UBQLN1 gene have been detected in a variety of disorders ranging from Alzheimer's disease to cancer. UBQLN1 has been largely studied in neurodegenerative disorders in the context of protein quality control. Several studies have hypothesized that the UBA domain of UBQLN1 binds to poly-ubiquitin chains of substrate and shuttles it to the proteasome via its UBL domain for degradation. UBQLN1 either facilitates degradation (Ataxin3, EPS15) or stabilizes (PSEN1/2, BCLb) substrates it binds to. The signal that determines this fate is unknown and there is conflicting data to support the existing working model of UBQLN1. Using BCLb as a model substrate, we characterized UBQLN1-substrate interaction. We identified the first two STI domains of UBQLN1 as critical for binding to BCLb. Interaction of UBQLN1 with BCLb is independent of ubiquitination of BCLb, but interaction with ubiquitin via UBA domain is required for stabilization of BCLb. Similarly, we showed that UBQLN1 interacts with IGF1R and ESYT2 through the STI domains and stabilizes these proteins through its UBA domain. Interactions that are not dependent on STI domains, for example, UBL mediated interaction with PSMD4 and BAG6, do not appear to be stabilized by UBQLN1. We conclude that fate of substrates that UBQLN1 associates with, is interaction domain specific. J. Cell. Biochem. 118: 2261-2270, 2017. © 2017 Wiley Periodicals, Inc.

CblY371H Transgene Combined with Hematopoietic Deletion of the Endogenous c-Cbl Gene Results in GM-CSF Hypersensitivity and Leukocytosis

Juvenile Myelomonocytic Leukemia (JMML) is a mixed myeloproliferative/myelodysplastic disease that is rapidly fatal with infiltration of myeloid cells into multiple organs. About 15% of JMML patient samples contain a mutation in c-Cbl, and germline mutation results in the predisposition for developing JMML. The c-Cbl gene encodes a multifunctional adaptor protein that contains an N-terminal tyrosine-kinase binding (TKB) domain, a RING finger motif that contains E3 ligase activity, and a C-terminal ubiquitin-associated domain. The TKB domain is involved in adaptor functions of the protein, whereas the ubiquitin ligase domain results in mono-ubiquitination of receptors which promotes lysosomal mediated degradation of activated receptors. Interestingly, a hotspot for mutations at residue 371 exists in JMML patients, where 1/3 of the detected mutations are a tyrosine to histidine substitution, Y371H. This residue belongs in the linker region of the CBL protein, and it was previously observed that Tyr-371 plays key roles in activating the ubiquitin ligase activity of the protein. In vitro, CblY371H mutation does indeed destroy its ligase function, resulting in prolonged signaling through the Ras pathway only when the endogenous c-Cbl gene is silenced. How mutant Cbl gives rise to JMML, however, and how it acts in concert with other genes in the pathogenesis of JMML requires further study.To address these questions, we overexpressed the oncogenic CblY371H mutation using transgenic mice. As expected, overexpression of CblY371H by itself in wildtype mice had no apparent phenotype. Therefore, Cbl transgenic mice were bred to Cbl heterozygous knockout mice (Cbl+/-) followed by further breeding in an attempt to generate Cbl transgenic mice with the endogenous Cbl gene inactivated (CblY371H; Cbl-/-). Surprisingly, unlike Cbl null mice, which are viable, overexpression of mutant Cbl allele in Cbl null mice caused embryonic lethality between 11.5 dpc and 12.5 dpc. In order to circumvent the developmental effects of expressing the mutant Cbl protein, we used a conditional Cbl knockout mouse to tissue specifically delete the endogenous Cbl gene. We chose the MMTV-Cre strain, which expresses Cre recombinase in only 10% of hematopoietic stem cells (CD34-; Lin-; Sca-1+; c-Kit+). With subsequent breeding with the CblY371H transgenic mice, we were able to bypass the embryonic lethality and produce mice with the correct genotype (MMTV-Cre;CblY371H;Cblfl/fl). These mice look normal but develop significant leukocytosis and show GM-CSF hypersensitivity even though only 10% of hematopoietic stem cells are affected. These mice, however, appear unaffected by the leukocytosis, and show no obvious difference with its littermates up to one year of age. We conclude that mutant CblY371H by itself is not sufficient for the development of JMML in this model and requires additional cooperating events. Whether further aging of these mice will result in JMML remains to be seen.In conclusion, we have developed a mouse model overexpressing the CblY371H protein ubiquitously, which causes deleterious development when it is the only c-Cbl protein available. This confirms the important role of c-Cbl activity during development. In hematopoietic cells, the overexpression of CblY371H results in leukocytosis and GM-CSF hypersensitivity when the endogenous gene is inactivated. We are currently investigating the cooperating events that are required for the development of JMML in this mouse model. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Overexpression Of Leukemia Associated CblY371H Mutation In Transgenic Mice Causes Dosage Dependent Embryonic Lethality

Juvenile Myelomonocytic Leukemia (JMML) is a devastating childhood cancer which is rapidly fatal with infiltration of myeloid cells into multiple organs (Hess, Zutter, Castleberry, & Emanuel, 1996). Based on the observation that uniparental disomy was found in the chromosomal region 11q in JMML patient samples, about 15% of patient samples were found to contain a mutation in c-Cbl (Loh et al., 2009). Moreover, mutant Cbl was also found to be a tumor suppressor gene where a germline mutation results in the predisposition for developing JMML (Niemeyer et al., 2010). The c-Cbl gene encodes a multifunctional adaptor protein which contains an N-terminal tyrosine-kinase binding (TKB) domain, a RING finger motif which contain E3 ligase activity, and a C-terminal ubiquitin-associated domain. Previous mutations in myeloid malignancies have been described where mutations occur in the RING finger domain or the linker domain (Caligiuri et al., 2007; Dunbar et al., 2008). Interestingly, a hotspot for mutations at residue 371 exists in JMML patients where 1/3 of the detected mutations are a tyrosine to histidine substitution, Y371H (Loh et al., 2009). This residue belongs in the linker region of the CBL protein, and it was previously observed that Y371 mediates the binding of c-Cbl to the p85 subunit of PI3 kinase (Blaydes et al., 2001). In vitro, CblY371H mutation does indeed destroy its ligase function resulting in prolonged signaling through the Ras pathway only when the endogenous c-Cbl gene was silenced (Niemeyer et al., 2010). However, how mutant Cbl gives rise to JMML and how it acts in concert with other genes in the pathogenesis of JMML requires further study.To address these questions, we tested the oncogenicity of the CblY371H mutation in transgenic mice. Of the 6 founder transgenic mice, we chose L5 line for further analysis because it had the highest level of expression. As expected, overexpression of CblY371H by itself in wild type mice had no phenotype since inactivation of wild type Cbl allele is seen in patients. We, then, generated transgenic mice which were heterozygous for both the BAC transgene and the cbl null mutation (CblY371H; Cbl+/-) and then bred them to Cbl heterozygous knockout mice (Cbl+/-). Because homozygous knockout mice have reduced fertility, we preferentially utilized heterozygous mice for the breeding (El Chami et al., 2005). Interestingly, out of the first 118 progeny genotyped where 1/8 of the pups is expected to be the desired genotype (CblY371H;Cbl-/-), no pups were of the desired genotype suggesting embryonic lethality. Because mice which lack both c-cbl and cbl-b are embryonic lethal, we reasoned that high expression that is achieved from the L5 line was causing a dominant negative affect on cbl-b function resulting in an embryonic lethal phenotype. We then tested if a lower level of the transgene expression would result in viable pups. We used the K5 line which was the second highest expressing transgenic line. We were now able to generate the desired genotype (CblY371H; Cbl-/-) although at less than expected numbers. Thus far, we have generated 4 mice which have the correct genotype which are being monitored for disease. We also monitored the heterozygous mice that carried the transgene (CblY371H; Cbl+/-) which were also asymptomatic without any obvious phenotype. Further studies are being done to characterize the hematopoietic phenotype in these mice by isolating fetal liver cells followed by transplantation into lethally irradiated mice and will be discussed. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Molecular evaluation of human Ubiquilin 2 gene PXX domain in familial frontotemporal dementia patients

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) phenotypes appear in the same pedigrees [1] and can occur in the same individuals [2] (FTDALS, OMIM 105550). In fact about 2–3 % of ALS patients develop dementia [3]. Consequently, in some individuals, there appears to be a common genetic etiology for both diseases. Genetic variation identified in GRN, FUS, TDP43, and more recently, in C9ORF72 and UBQLN2 genes has been associated to familial ALS (FALS, OMIN 104105), familial FTD (OMIM 600274) and/or FTDALS consistent with the notion that both disorders may share genetic components and, probably, functional pathogenic mechanisms [4–8]. UBQLN2 gene was recently identified by Deng et al. [7] as a novel dominant but not fully penetrant X-linked FTDALS gene. Specifically, five segregating mutations in four different proline residues within the PXX repeat domain of UBQLN2 gene were identified in families afflicted with ALS/dementia. Importantly, the authors also found a correlation of hippocampal UBQLN2 pathology with dementia in ALS cases with or without UBQLN2 mutations, suggesting that UBQLN2 gene could be involved in ALS-related dementia, even without UBQLN2 mutations [7]. UBQLN2 gene encodes an ubiquitin-like protein (ubiquilin) that shares a high degree of similarity with related proteins in multiple eukaryotic organisms. Specifically, ubiquilins contain an N-terminal ubiquitin-like domain and a C-terminal ubiquitin-associated domain; therefore, UBQLN2 could be functionally linked to ubiquitination enzymatic processes. Functional analysis showed that mutations in UBQLN2 lead to an impairment of protein degradation [7]. Of note, UBQLN2 protein has also been shown to bind the Stch protein which is also involved in aggresome malfunction [9]. The role of ubiquitination in neurodegeneration is well established in different human neurodegenerative disorders such Parkinson’s disease or the Marinesco-Sjogren syndrome [10, 11]. Furthermore, it has been suggested that there is a link between Alzheimer’s disease and ubiquitins [12]. The purpose of the present study is to further explore the role of UBQLN2 mutations in familial FTD [13]. We examined 77 FTD index patients with a positive family history of dementia who had complete clinical evaluations. Importantly, we maintained FTD patients with observed male to male transmission (which makes a dominant X-linked transmission impossible) as negative controls of this study (n = 12). For the purpose of this study, we examined subjects with FTD and its subtypes (behavioral variant FTD (bvFTD), semantic dementia, progressive nonfluent aphasia), according to Neary et al. [14], as well as patients within the FTD spectrum, such as with those with combined phenotype of FTD and ALS, corticobasal syndrome or progressive supranuclear palsy (Table 1). All individuals agreed to participate in the study and blood samples were obtained after informed consent from Electronic supplementary material The online version of this article (doi:10.1007/s00415-012-6568-5) contains supplementary material, which is available to authorized users.

The E2-25K ubiquitin-associated (UBA) domain aids in polyubiquitin chain synthesis and linkage specificity

E2-25K is an ubiquitin-conjugating enzyme with the ability to synthesize Lys48-linked polyubiquitin chains. E2-25K and its homologs 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.

Sequestosome-1/p62 Is the Key Intracellular Target of Innate Defense Regulator Peptide

Innate defense regulator-1 (IDR-1) is a synthetic peptide with no antimicrobial activity that enhances microbial infection control while suppressing inflammation. Previously, the effects of IDR-1 were postulated to impact several regulatory pathways including mitogen-activated protein kinase (MAPK) p38 and CCAAT-enhancer-binding protein, but how this was mediated was unknown. Using a combined stable isotope labeling by amino acids in cell culture-proteomics methodology, we identified the cytoplasmic scaffold protein p62 as the molecular target of IDR-1. Direct IDR-1 binding to p62 was confirmed by several biochemical binding experiments, and the p62 ZZ-type zinc finger domain was identified as the IDR-1 binding site. Co-immunoprecipitation analysis of p62 molecular complexes demonstrated that IDR-1 enhanced the tumor necrosis factor α-induced p62 receptor-interacting protein 1 (RIP1) complex formation but did not affect tumor necrosis factor α-induced p62-protein kinase ζ complex formation. In addition, IDR-1 induced p38 MAPK activity in a p62-dependent manner and increased CCAAT-enhancer-binding protein β activity, whereas NF-κB activity was unaffected. Collectively, these results demonstrate that IDR-1 binding to p62 specifically affects protein-protein interactions and subsequent downstream events. Our results implicate p62 in the molecular mechanisms governing innate immunity and identify p62 as a potential therapeutic target in both infectious and inflammatory diseases.

A Novel Connexin43-interacting Protein, CIP75, Which Belongs to the UbL-UBA Protein Family, Regulates the Turnover of Connexin43

The degradation of connexin43 (Cx43) has been reported to involve both lysosomal and proteasomal degradation pathways; however, very little is known about the mechanisms regulating these Cx43 degradation pathways. Using yeast two-hybrid, glutathione S-transferase pull-down, and co-immunoprecipitation approaches, we have identified a novel Cx43-interacting protein of approximately 75 kDa, CIP75. Laser confocal microscopy showed that CIP75 is located primarily at the endoplasmic reticulum, as indicated by the calnexin marker, with Cx43 co-localization in this perinuclear region. CIP75 belongs to the UbL (ubiquitin-like)-UBA (ubiquitin-associated) domain-containing protein family with a N-terminal UbL domain and a C-terminal UBA domain. The UBA domain of CIP75 is the main element mediating the interaction with Cx43, whereas the CIP75-interacting region in Cx43 resides in the PY motif and multiphosphorylation sites located between Lys 264 and Asn 302. Interestingly, the UbL domain interacts with the S2/RPN1 and S5a/RPN10 protein subunits of the regulatory 19 S proteasome cap subunit of the 26 S proteasome complex. Overexpression experiments suggested that CIP75 is involved in the turnover of Cx43 as measured by a significant stimulation of Cx43 degradation and reduction in its half-life with the opposite effects on Cx43 degradation observed in small interference RNA knockdown experiments.

Yeast UBL-UBA proteins have partially redundant functions in cell cycle control

BackgroundProteins containing ubiquitin-like (UBL) and ubiquitin associated (UBA) domains have been suggested to shuttle ubiquitinated substrates to the proteasome for degradation. There are three UBL-UBA containing proteins in budding yeast: Ddi1, Dsk2 and Rad23, which have been demonstrated to play regulatory roles in targeting ubiquitinated substrates to the proteasome for degradation. An involvement of these proteins in cell cycle related events has also been reported. We tested whether these three proteins act redundantly in the cell cycle.ResultsHere we show that the UBL-UBA proteins are partially redundant for cell cycle related roles. RAD23 is redundant with DDI1 and DSK2, but DDI1 and DSK2 are not redundant with each other and the triple deletion shows a synthetic effect, suggesting the existence of at least two roles for RAD23 in cell cycle control. The rad23Δddi1Δdsk2Δ triple deletion strain delays both in G2/M-phase and in mid-anaphase at high temperatures with duplicated spindle pole bodies. Cell cycle progression in the triple deletion strain can only be partially rescued by a rad23 allele lacking the c-terminal UBA domain, suggesting that RAD23 requires its c-terminal UBA domain for full function. In addition to their ability to bind ubiquitin and the proteasome, the UBL-UBA proteins also share the ability to homodimerize. Rad23 and Dsk2 dimerization requires their UBL and/or UBA domains whereas Ddi1 dimerization does not. Here we show that Ddi1 homodimerization is necessary for its cell cycle related functions.ConclusionThe three yeast UBL-UBA proteins have partially redundant roles required for progression through mitosis.

Structures of the Dsk2 UBL and UBA domains and their complex

The yeast proteins Dsk2 and Rad23 belong to a family of proteins that contain an N-terminal ubiquitin-like domain (UBL) and a C-terminal ubiquitin-associated domain (UBA). Both Dsk2 and Rad23 function as adaptors to target ubiquitin-labelled proteins to the proteasome through recognition of polyubiquitin (four or more K48-linked ubiquitins) by their UBA domains and to the yeast proteasomal subunit Rpn1 by their UBL domains. The crystal structures of the Dsk2 UBL domain, the Dsk2 UBA domain and the Dsk2 UBA-UBL complex are reported. In the crystal, the Dsk2 UBA domains associate through electrostatic interactions to form ninefold helical ribbons that leave the ubiquitin-binding surface exposed. The UBA-UBL complex explains the reduced affinity of the UBA domain for UBL compared with ubiquitin and has implications for the regulation of Dsk2 adaptor function during ubiquitin-mediated proteasomal targeting. A model is discussed in which two or more Dsk2 UBA molecules may selectively bind to K48-linked polyubiquitin.

Ubiquitin Manipulation by an E2 Conjugating Enzyme Using a Novel Covalent Intermediate

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.

The UBA2 Domain Functions as an Intrinsic Stabilization Signal that Protects Rad23 from Proteasomal Degradation

The proteasome-interacting protein Rad23 is a long-lived protein. Interaction between Rad23 and the proteasome is required for Rad23's functions in nucleotide excision repair and ubiquitin-dependent degradation. Here, we show that the ubiquitin-associated (UBA)-2 domain of yeast Rad23 is a cis-acting, transferable stabilization signal that protects Rad23 from proteasomal degradation. Disruption of the UBA2 domain converts Rad23 into a short-lived protein that is targeted for degradation through its N-terminal ubiquitin-like domain. UBA2-dependent stabilization is required for Rad23 function because a yeast strain expressing a mutant Rad23 that lacks a functional UBA2 domain shows increased sensitivity to UV light and, in the absence of Rpn10, severe growth defects. The C-terminal UBA domains of Dsk2, Ddi1, Ede1, and the human Rad23 homolog hHR23A have similar protective activities. Thus, the UBA2 domain of Rad23 is an evolutionarily conserved stabilization signal that allows Rad23 to interact with the proteasome without facing destruction.

Structure of the Ubiquitin-associated Domain of p62 (SQSTM1) and Implications for Mutations That Cause Paget's Disease of Bone

The p62 protein (also known as SQSTM1) mediates diverse cellular functions including control of NFkappaB signaling and transcriptional activation. p62 binds non-covalently to ubiquitin and co-localizes with ubiquitylated inclusions in a number of human protein aggregation diseases. Mutations in the gene encoding p62 cause Paget's disease of bone (PDB), a common disorder of the elderly characterized by excessive bone resorption and formation. All of the p62 PDB mutations identified to date cluster within the C-terminal region of the protein, which shows low sequence identity to previously characterized ubiquitin-associated (UBA) domains. We report the first NMR structure of a recombinant polypeptide that contains the C-terminal UBA domain of the human p62 protein (residues 387-436). This sequence, which confers multiubiquitin chain binding, forms a compact three-helix bundle with a structure analogous to the UBA domains of HHR23A but with differences in the loop regions connecting helices that may be involved in binding accessory proteins. We show that the Pro392 --> Leu PDB substitution mutation modifies the structure of the UBA domain by extending the N terminus of helix 1. In contrast to the p62 PDB deletion mutations that remove the UBA domain and ablate multiubiquitin chain binding, the Pro392 --> Leu substitution does not affect interaction of the UBA domain with multiubiquitin chains. Thus, phenotypically identical substitution and deletion mutations do not appear to predispose to PDB through a mechanism dependent on a common loss of ubiquitin chain binding by p62.

Rad23 Ubiquitin-associated Domains (UBA) Inhibit 26 S Proteasome-catalyzed Proteolysis by Sequestering Lysine 48-linked Polyubiquitin Chains

Most substrates of the 26 S proteasome are recognized only following conjugation to a Lys48-linked polyubiquitin chain. Rad23 is one member of a family of proteins that possesses an N-terminal ubiquitin-like domain (UbL) and a C-terminal ubiquitin-associated domain(s) (UBA). Recent studies have shown that UbLs interact with 26 S proteasomes, whereas UBAs bind polyubiquitin chains. These biochemical properties suggest that UbL-UBA proteins may shuttle polyubiquitinated substrates to proteasomes. Here we show that contrary to prediction from this model, the effect of human Rad23A on the degradation of polyubiquitinated substrates catalyzed by purified proteasomes is exclusively inhibitory. Strong inhibition is dependent on the presence of both UBAs, independent of the UbL, and can be explained by competition between the UBA domains and the proteasome for binding to substrate-linked polyubiquitin chains. The UBA domains bind Lys48-linked polyubiquitin chains in strong preference to Lys63 or Lys29-linked chains, leading to selective inhibition of the assembly and disassembly of Lys48-linked chains. These results place constraints on the mechanism(s) by which UbL-UBA proteins promote proteasome-catalyzed proteolysis and reveal new properties of UBA domains.

Budding yeast Dsk2p is a polyubiquitin-binding protein that can interact with the proteasome.

Dsk2p from Saccharomyces cerevisiae belongs to the class of proteins that contain a ubiquitin-like (UbL) domain at the N terminus together with a ubiquitin-associated (UBA) domain at the C terminus. We show here that the C-terminal UBA domain of Dsk2p binds to K48-linked polyubiquitin chains, and the N-terminal UbL domain of Dsk2p interacts with the proteasome. Overexpression of Dsk2p caused the accumulation of large amounts of polyubiquitin, and extragenic suppressors of the Dsk2p-mediated lethality proved to be temperature-sensitive mutations in two proteasome subunits, rpn1 and pre2. K48-linked ubiquitin-dependent degradation was impaired by disruption of the DSK2 gene. These results indicate that Dsk2p is K48-linked polyubiquitin-binding protein and also interacts with the proteasome. We discuss a possible role of adaptor function of Dsk2p via its UbL and UBA domains in the ubiquitin-proteasome pathway.