Ub Gene Research Articles

Role of dietary isoleucine supplementation in facilitating growth performance and muscle growth in juvenile largemouth bass (Micropterus salmoides)

To improve the breeding efficient of fish, it is crucial to provided essential nutrients based on the nutritional demands. Isoleucine is an essential amino acid, and it plays a significant role in growth and metabolism of largemouth bass. Therefore, this study aimed to investigate the effects of dietary isoleucine on the growth and muscle development of largemouth bass (Micropterus salmoides) by conducting an 8-week feeding experiment. Fish (initial weight 19.97 ± 0.04 g) were randomly distributed into six treatments with three replicate groups of 20 fish and fish were fed diets with varying isoleucine levels (1.47%, 1.68%, 1.93%, 2.30%, 2.46%, and 2.72% of dry diet). Results revealed that isoleucine supplementation increased the specific growth rate (SGR) and significantly decreased the feed conversion ratio (FCR). Moreover, histological analysis of the muscle tissue revealed a significant correlation between the size and density of muscle fibers and isoleucine levels. This alteration may be attributed to the downregulation of myostatin and muscle growth-related genes, MyoD and MyoG, respectively. Additionally, fish fed an isoleucine-supplemented diet showed downregulated expression of apoptosis-related (Caspase-3, Caspase-8, and Caspase-9) and ubiquitin-proteasome pathway-related (Ub, Murf1, Mafbx, and Psmc1) genes (1.93%−2.30%). The muscle and plasma total protein contents showed similar patterns of variation to the gene described above. Furthermore, isoleucine supplementation significantly increased body protein levels and decreased body lipid levels. Quadratic regression analysis revealed that the optimal isoleucine levels for juvenile largemouth bass were 1.997% and 1.837% in the dry diet (4.21% and 3.87% of dietary protein) for SGR and FCR, respectively.

The substrate selectivity of papain-like proteases from human-infecting coronaviruses correlates with innate immune suppression.

Coronaviruses that can infect humans can cause either common colds (HCoV-NL63, HCoV-229E, HCoV-HKU1, and HCoV-OC43) or severe respiratory symptoms (SARS-CoV-2, SARS-CoV, and MERS-CoV). The papain-like proteases (PLPs) of SARS-CoV, SARS-CoV-2, MERS-CoV, and HCoV-NL63 function in viral innate immune evasion and have deubiquitinating (DUB) and deISGylating activities. We identified the PLPs of HCoV-229E, HCoV-HKU1, and HCoV-OC43 and found that their enzymatic properties correlated with their ability to suppress innate immune responses. A conserved noncatalytic aspartic acid residue was critical for both DUB and deISGylating activities, but the PLPs had differing ubiquitin (Ub) chain cleavage selectivities and binding affinities for Ub, K48-linked diUb, and interferon-stimulated gene 15 (ISG15) substrates. The crystal structure of HKU1-PLP2 in complex with Ub revealed binding interfaces that accounted for the unusually high binding affinity between this PLP and Ub. In cellular assays, the PLPs from the severe disease-causing coronaviruses strongly suppressed innate immune IFN-I and NF-κB signaling and stimulated autophagy, whereas the PLPs from the mild disease-causing coronaviruses generally showed weaker effects on immune suppression and autophagy induction. In addition, a PLP from a SARS-CoV-2 variant of concern showed increased suppression of innate immune signaling pathways. Overall, these results demonstrated that the DUB and deISGylating activities and substrate selectivities of these PLPs differentially contribute to viral innate immune evasion and may affect viral pathogenicity.

Genome-wide analysis of genes encoding core components of the ubiquitin system during cerebral cortex development

Ubiquitination involves three types of enzymes (E1, E2, and E3) that sequentially attach ubiquitin (Ub) to target proteins. This posttranslational modification controls key cellular processes, such as the degradation, endocytosis, subcellular localization and activity of proteins. Ubiquitination, which can be reversed by deubiquitinating enzymes (DUBs), plays important roles during brain development. Furthermore, deregulation of the Ub system is linked to the pathogenesis of various diseases, including neurodegenerative disorders. We used a publicly available RNA-seq database to perform an extensive genome-wide gene expression analysis of the core components of the ubiquitination machinery, covering Ub genes as well as E1, E2, E3 and DUB genes. The ubiquitination network was governed by only Uba1 and Ube2m, the predominant E1 and E2 genes, respectively; their expression was positively regulated during cortical formation. The principal genes encoding HECT (homologous to the E6-AP carboxyl terminus), RBR (RING-in-between-RING), and RING (really interesting new gene) E3 Ub ligases were also highly regulated. Pja1, Dtx3 (RING ligases) and Stub1 (U-box RING) were the most highly expressed E3 Ub ligase genes and displayed distinct developmental expression patterns. Moreover, more than 80 DUB genes were expressed during corticogenesis, with two prominent genes, Uch-l1 and Usp22, showing highly upregulated expression. Several components of the Ub system overexpressed in cancers were also highly expressed in the cerebral cortex under conditions not related to tumour formation or progression. Altogether, this work provides an in-depth overview of transcriptomic changes during embryonic formation of the cerebral cortex. The data also offer new insight into the characterization of the Ub system and may contribute to a better understanding of its involvement in the pathogenesis of neurodevelopmental disorders.

Eicosapentaenoic acid suppresses cisplatin-induced muscle atrophy by attenuating the up-regulated gene expression of ubiquitin

Previously it was shown that cisplatin causes muscle atrophy. Under this condition, cisplatin increased the expression of atorogenes, such as muscle ring finger 1 and atrogin-1 (also known as muscle atrophy F-box protein), in mouse skeletal muscle. It was reported recently that ubiquitin (Ub) and ubiquitinated protein levels in skeletal muscle were also up-regulated in cisplatin-induced muscle atrophy, and cisplatin-induced ubiquitinated proteins were degraded by the 26S proteasome pathway. Eicosapentaenoic acid (EPA) is effective against skeletal muscle atrophy in mice. However, it is unclear how EPA suppresses the Ub–proteasome pathway. In this study, the effect of EPA on cisplatin-induced muscle atrophy in mice was examined. Mice were intraperitoneally injected with cisplatin or vehicle control once daily for 4 d. EPA or its vehicle was orally administered 30 min before cisplatin administration. Cisplatin systemic administration induced decrease in muscle mass, myofiber diameter, and increase in Ub genes and ubiquitinated proteins in mouse skeletal muscle were recovered by co-treatment with EPA. However, weight loss and up-regulated atrogenes induced by cisplatin were not changed by co-treatment with EPA in skeletal muscle. In this study, EPA attenuated cisplatin-induced muscle atrophy via down-regulation of up-regulated Ub gene expression. Although further clinical studies are needed, EPA administration can be effective in the development of muscle atrophy in cisplatin-treated patients.

The Ubiquitin Gene Expression Pattern and Sensitivity to UBB and UBC Knockdown Differentiate Primary 23132/87 and Metastatic MKN45 Gastric Cancer Cells.

Gastric cancer (GC) is one of the most common and lethal cancers. Alterations in the ubiquitin (Ub) system play key roles in the carcinogenetic process and in metastasis development. Overexpression of transcription factors YY1, HSF1 and SP1, known to regulate Ub gene expression, is a predictor of poor prognosis and shorter survival in several cancers. In this study, we compared a primary (23132/87) and a metastatic (MKN45) GC cell line. We found a statistically significant higher expression of three out of four Ub coding genes, UBC, UBB and RPS27A, in MKN45 compared to 23132/87. However, while the total Ub protein content and the distribution of Ub between the conjugated and free pools were similar in these two GC cell lines, the proteasome activity was higher in MKN45. Ub gene expression was not affected upon YY1, HSF1 or SP1 small interfering RNA (siRNA) transfection, in both 23132/87 and MKN45 cell lines. Interestingly, the simultaneous knockdown of UBB and UBC mRNAs reduced the Ub content in both cell lines, but was more critical in the primary GC cell line 23132/87, causing a reduction in cell viability due to apoptosis induction and a decrease in the oncoprotein and metastatization marker β-catenin levels. Our results identify UBB and UBC as pro-survival genes in primary gastric adenocarcinoma 23132/87 cells.

Dynamic transcription of ubiquitin genes under basal and stressful conditions and new insights into the multiple UBC transcript variants

Ubiquitin (Ub) is a small 76-amino acid protein that is engaged in many different pathways within the cell, including protein turnover. During proteotoxic stress, when the demand of clearing damaged/misfolded proteins strongly increases, cells activate Ub gene transcription to face the need of extra ubiquitin. This paper shows the contribution of the four ubiquitin coding genes (UBB, UBC, UBA52, RPS27A) to the ubiquitin RNA pool under basal and stressful conditions. Our results reveal that UBC and RPS27A represent the major fraction of the Ub transcriptome in different cell lines, but when converted to the coding potential, polyubiquitin genes UBC and UBB mainly contribute to determine the intracellular ubiquitin content under basal conditions. Both the polyubiquitin genes UBB and UBC are upregulated upon proteasome inhibition and oxidative stress, with markedly higher responses from the UBC promoter. A similar output, with lower fold-inductions, is detected in heat-stressed cells, with UBC acting as the main contributor to thermotolerance. By contrast, upon these stressors, the levels of UBA52 and RPS27A mRNAs remain unchanged. Remarkably, UV irradiation fails to induce Ub gene transcription, but rather seems to act at the post-transcriptional level, by stabilizing ubiquitin mRNAs at UV doses which induce rapid degradation of other RNA molecules. Moreover, the evidence that the UBC core promoter contains multiple transcription start sites and their responsiveness to stress, is here reported for the first time.

Overexpression of TaUb2 enhances disease resistance to Pseudomonas syringae pv. tomato DC3000 in tobacco

Ubiquitin (Ub), a 76-amino acid protein, functions as a tag that can be attached to substrates for selected degradation in a Ub/26S proteasome system (UPS). In this paper, effects of the Ub gene TaUb2 from wheat expressed in tobacco plants are reported. Three TaUb2-overexpressing transgenic tobacco lines were used to study the functions of Ub in response to Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000). After infection with Pst DC3000, wild-type (WT) plants displayed more severe disease symptoms than transgenic plants. We observed a smaller stomatal aperture, slower water loss and less leaf wilt in transgenic plants after Pst DC3000 infection compared to WT plants. After Pst DC3000 treatment, electrolyte leakage and malondialdehyde levels were lower and were consistent with the higher antioxidant enzyme activities of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) that were observed in the transgenic plants compared with the WT plants; however, there was no significant difference in these parameters between the transgenic plants and WT before Pst DC3000 treatment. At the same time, a hypersensitive response (HR) was also observed in transgenic plants when infected by Pst DC3000, which may be induced by a reactive oxygen species (ROS) burst. Real-time RT-PCR analysis revealed that the transcript expressions of some pathogenesis-related genes (PR) in transgenic plants were obviously higher than those in WT plants after Pst DC3000 treatment. These results suggest that Ub is involved in plant disease resistance. Stomatal aperture, antioxidant capacity and ROS-burst-induced disease resistance may be important mechanisms underlying enhanced disease resistance in transgenic tobacco with overexpression of TaUb2.

Manipulation of monoubiquitin improves chilling tolerance in transgenic tobacco (Nicotiana tabacum)

Ubiquitin (Ub) is a multifunctional protein that mainly functions to tag proteins for selective degradation by the 26S proteasome. We cloned an Ub gene TaUb2 from wheat (Triticum aestivum L.) previously. To study the function of TaUB2 in chilling stress, sense and antisense Ub transgenic tobacco plants (Nicotiana tabacum L.), as well as wild type (WT) and vector control β-glucuronidase (T-GUS) plants, were used. Under stress, leaf wilting in sense plants was significantly less than in controls, but more severe in antisense plants. Meanwhile, the net photosynthetic rate (Pn) and the maximal photochemical efficiency of PSII (Fv/Fm) in sense plants were greater than controls, but lower in antisense plants during chilling stress and recovery. Less wilting in sense plants resulted from improved water status, which may be related to the accumulation of proline and solute sugar. Furthermore, as indicated by electrolyte leakage, membrane damage under stress was less in sense plants and more severe in antisense plants than controls. Consistent with electrolyte leakage, the malondialdehyde (MDA) content was less in sense plants, but more in antisense plants compared to controls. Meanwhile, the less accumulation of reactive oxygen species (ROS) and the greater antioxidant enzyme activity in sense plants implied the improved antioxidant competence by the overexpression of monoubiquitin gene Ta-Ub2 from wheat. We suggest that overexpressing Ub is a useful strategy to promote chilling tolerance. The improvement of ROS scavenging may be an important mechanism underlying the role of Ub in promoting plants tolerant to chilling stress.

Molecular characterization and expression profiles of Sp-Ub during gonad development in Scylla paramamosain

Scylla paramamosain is an economically important breeding crab found along the southeast coast of China.Although extensive research on reproductive physiology has been carried out for this species,description of the regulatory mechanism of the gonad development at a molecular level is limited.Within all tissues,the majority of intracellular proteins are degraded by the ubiquitin(Ub)–proteasome pathway(UPP).In this pathway,Ub is conjugated to proteins that are destined for degradation by an ATP-dependent process that involves three enzymes.To date,some studies have discovered that components of the ubiquitin system may be involved in various steps and processes during gametogenesis,modulation of steroid receptor concentrations,placental development and endometrial modification at the beginning of pregnancy.However,research on the action of UPP in gonad devel-opment in crab species remains largely unknown.In this study,the S.paramamosain Ub gene(Sp-Ub) was identi-fied and characterized using an approach which combines the expressed sequence tag(EST) method and rapid amplification of cDNA end(RACE).The expression pro?le of Sp-Ub in the various tissues and different develop-ing stages of the gonad was determined by real-time quantitative PCR.The full length cDNA of the Sp-Ub gene is of 555 bp.It encodes a protein of 154 amino acids,in which the 1-76 amino acids fall within the ubiquitin domain,and the 101–147 amino acids fall within the ribosomal_S27a domain.The second structure prediction revealed that the ubiquitin domain of the Sp-Ub protein contains five extended strands,an α-helix and some random coils.Moreover,the three-dimensional structure predicted by the SWISS-MODEL showed that it contains an α-helix and four β-pleated sheets.Alignment of the amino acid sequences within different Ub proteins indicated that the ubiq-uitin domain and the ribosomal_S27a domain among different species are highly conserved during evolution.Real-time quantitative PCR results showed that Sp-Ub was expressed by the ubiquitin domain in all tissues exam-ined(P0.05) which illustrated that the Ub gene plays a wide variety of roles in biological processes.The expres-sion profiles of Sp-Ub in the different developing stages of the ovary revealed that its expression level was the highest at stage O5 following stage O1,O4 and O3.It was the lowest level at stage O2 which was significantly lower than stage O5(P0.05).Within the developing stages of the testis,the expression level of Sp-Ub was higher in stage T2 following stage T1 and T3,and its expression level was significantly higher in stage T2 compared to stage T3(P0.05).During gametogenesis,the oogonia and spermatogonium undergo active mitosis proliferation for increasing their number at the proliferation stage.Previous research showed that cyclin/Cdks work closely with the ubiquitin-proteasome pathway during the mitotic cycle.Thus,it is reasonable to observe that the expression level of Sp-Ub was relatively high at stage O1.The reduction of the Sp-Ub expression level at stage O2 may be due to the initiation of formation of primary oocytes,and initial vitellogenesis with a low level of proliferation.Moreover,the expression level of Sp-Ub gradually increased as the yolk proteins were accumulating in the grow-ing oocytes during vitellogenesis.A mass of proteins needed to be synthesized and degraded during gametogenesis,and the evidence of ubiquitin –conjugated vitellin polypeptides in insect oocytes had been reported.These results indicated that Sp-Ub may be involved in vitellogenesis.Furthermore,the testis of crab contains germ cells of various development stages within the seminiferous tubules.Hence,the higher expression level of Sp-Ub at stage T2 of testis development may be necessary for fast increasing the number of spermatocyte cells,which indicated that UPP was more active in these cells.Conclusively,our studies are the first report of cloning and expression profiling of Ub in crustaceans.All of these results can provide basic data showing the role of Sp-Ub within the gonad development of the crab.

Increased gibberellin contents contribute to accelerated growth and development of transgenic tobacco overexpressing a wheat ubiquitin gene

Overexpressing TaUb2 promoted stem growth and resulted in early flowering in transgenic tobacco plants. Ubiquitin are involved in the production, metabolism and proper function of gibberellin. The ubiquitin-26S proteasome system (UPS), in which ubiquitin (Ub) functions as a marker, is a post-translational regulatory system that plays a prominent role in various biological processes. To investigate the impact of different Ub levels on plant growth and development, transgenic tobacco (Nicotiana tabacum L.) plants were engineered to express an Ub gene (TaUb2) from wheat (Triticum aestivum L.) under the control of cauliflower mosaic virus 35S promoter. Transgenic tobacco plants overexpressing TaUb2 demonstrated an accelerated growth rate at early stage and an early flowering phenotype in development. The preceding expression of MADS-box genes also corresponded to the accelerated developmental phenotypes of the transgenic tobacco plants compared to that of wild-type (WT). Total gibberellin (GA) and active GA contents in transgenic tobacco plants were higher than those in WT at the corresponding developmental stages, and some GA metabolism genes were upregulated. Treatment with GA(3) conferred a similarly accelerated grown rate in WT plants to that of transgenic tobacco plants, while growth was inhibited when transgenic tobacco plants were treated with a GA biosynthesis inhibitor. Thus, the results suggest that Ub are involved in the production, metabolism and proper function of GA, which is important in the regulation of plant growth and development.

MOLECULAR MARKERS ASSOCIATED WITH DROUGHT TOLERANCE IN Citrullus colocynthis

In the present study Citrullus colocynthis L. (Cucurbitaceae) plants were collected from four different sites (Red sea coast (Elba Mountain), New Valley area, North Sinai, and Saint Katreen) by Egyptian Desert Genebank .Molecular markers associated with drought tolerance were studied by fourteen preselected (ISSR) primers exhibited polymorphism obtained from the DNAs of sixteen samples of C. colocynthis (C1-C4) from the four different sites. Detection and sequencing of some drought tolerance genes in Citrullus colocynthis using the genomic DNA based on the four plants from different sites. The specific primers of those drought tolerant genes were (Dehydrin gene), (UB gene), (P5CS gene), (PEPKS gene) and (ACT gene), which were succeed to detect some drought tolerant genes among the four locations of Citrullus colocynthis.

In silico analysis of ubiquitin/ubiquitin-like modifiers and their conjugating enzymes in Entamoeba species

Covalent modification of proteins by ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) regulates many cellular functions in eukaryotes. These modifications are likely to be associated with pathogenesis, growth, and development of many protozoan parasites but molecular details about this pathway are unavailable for most protozoa. This study presents an analysis of the Ub pathway in three members of the Entamoeba species. Using bioinformatics tools we have identified all Ub and Ubl genes along with their corresponding activating, conjugating, and ligating enzymes (E1, E2s, and E3s) in three Entamoeba species, Entamoeba histolytica, Entamoeba dispar, and Entamoeba invadens. Phylogenetic trees were established for the identified E2s and RING finger E3s using maximum-likelihood method to infer the relationship among these proteins. In silico co-domain analysis of RING finger E3s implicates these proteins in a variety of functions. Several known and putative regulatory motifs were identified in the upstream regions of RING finger domain containing E3 genes. All E2 and E3 genes were analyzed in genomic context in E. histolytica and E. dispar. Most E2s and E3s were in syntenic positions in the two genomes. Association of these genes with transposable elements (TEs) was compared between E. histolytica and E. dispar. A closer association was found between RING finger E3s with TEs in E. histolytica. In summary, our analyses suggests that the complexity of the Ub pathway in Entamoeba species is close to that observed in higher eukaryotes. This study provides important data for further understanding the role of Ub pathway in the biology of these organisms.

Giardia intestinalis: Expression of ubiquitin, glucosamine-6-phosphate and cyst wall protein genes during the encystment process

We determined the relative expression of ubiquitin (ub), glucosamine-6-phosphate-isomerase (gn6pi) and cyst wall protein (cwp) genes during encystment of the Portland-1 and Portland-1R strains of Giardia intestinalis. Encystment was induced with bile for different time periods. The presence of encystment-specific vesicles (ESVs) and the relative expression of genes (log10ΔRn) were determined by transmission electron microscopy and real-time PCR, respectively. Our results demonstrated the gene expression and the presence of ESVs after 6h of encystment. Values of cwp2 gene expression increased by 591-fold in strain Portland-1 and 78.2-fold in strain Portland-1R at this time point compared to values at 0h, after which values gradually decreased until reaching basal values between 8 and 18h after the encystment started. Expression of gn6pi was 43.5- and 46.3-fold higher than basal values, in Portland-1 and Portland-1R, respectively. Ub gene expression was 82.25-fold higher than its basal levels at 4h, after which expression decreased gradually until reaching basal values after 16h. Conclusions: This work showed the relationship between the presence of ESVs and encystment gene expression at 6h, and resistance to albendazole does not inhibit the encystment process. The results revealed important knowledge with implications in the control of parasite dissemination for preventing parasite transmission.

Validation of novel promoter sequences derived from two endogenous ubiquitin genes in transgenic Aedes aegypti

To date, only a limited number of promoter sequences have been described to drive transgene expression in the disease vector Aedes aegypti. We sought to increase this repertoire by characterizing the ability of upstream sequences derived from the Ae. aegypti Ub(L40) and polyubiquitin genes to drive the expression of marker proteins. Both genomic fragments were able to drive robust expression of luciferase in cultured mosquito cells. Following Mos1-transformation, the Ub(L40) promoter drove strong expression of a fluorescent marker in early larvae and in ovaries, while the polyubiquitin promoter drove robust EGFP expression in all stages of development, including constitutive expression throughout the midgut. These promoter fragments provide two new expression profiles for future Ae. aegypti genetic experiments.

Targeting antigen to MHC Class I and Class II antigen presentation pathways for malaria DNA vaccines

An effective malaria vaccine which protects against all stages of Plasmodium infection may need to elicit robust CD8 + and CD4 + T cell and antibody responses. To achieve this, we have investigated strategies designed to improve the immunogenicity of DNA vaccines encoding the Plasmodium yoelii pre-erythrocytic stage antigens PyCSP and PyHEP17, by targeting the encoded proteins to the MHC Classes I and II processing and presentation pathways. For enhancement of CD8 + T cell responses, we targeted the antigens for degradation by the ubiquitin (Ub)/proteosome pathway following the N-terminal rule. We constructed plasmids containing PyCSP or PyHEP17 genes fused to the Ub gene: plasmids where the N-terminal antigen residues were mutated from the stabilizing amino acid methionine to destabilizing arginine, plasmids where the C-terminal residues of Ub were mutated from glycine to alanine, and plasmids in which the potential hydrophobic leader sequences of the antigens were deleted. For enhancement of CD4 + T cell and antibody responses, we targeted the antigens for degradation by the endosomal/lysosomal pathway by linking the antigen to the lysosome-associated membrane protein (LAMP). We found that immunization with DNA vaccine encoding PyHEP17 fused to Ub and bearing arginine induced higher IFN-γ, cytotoxic and proliferative T cell responses than unmodified vaccines. However, no effect was seen for PyCSP using the same targeting strategies. Regarding Class II antigen targeting, fusion to LAMP did not enhance antibody responses to either PyHEP17 or PyCSP, and resulted in a marginal increase in lymphoproliferative CD4 + T cell responses. Our data highlight the antigen dependence of immune enhancement strategies that target antigen to the MHC Class I and II pathways for vaccine development.

The mouse polyubiquitin gene UbC is essential for fetal liver development, cell-cycle progression and stress tolerance

UbC is one of two stress-inducible polyubiquitin genes in mammals and is thought to supplement the constitutive UbA genes in maintaining cellular ubiquitin (Ub) levels during episodes of cellular stress. We have generated mice harboring a targeted disruption of the UbC gene. UbC(-/-) embryos die between embryonic days 12.5 and 14.5 in utero, most likely owing to a severe defect in liver cell proliferation. Mouse embryonic fibroblasts from UbC(-/-) embryos exhibit reduced growth rates, premature senescence, increased apoptosis and delayed cell-cycle progression, with slightly, but significantly, decreased steady-state Ub levels. UbC(-/-) fibroblasts are hypersensitive to proteasome inhibitors and heat shock, and unable to adequately increase Ub levels in response to these cellular stresses. Most, but not all of the UbC(-/-) phenotypes can be rescued by providing additional Ub from a poly hemagglutinin-tagged Ub minigene expressed from the Hprt locus. We propose that UbC is regulated by a process that senses Ub pool dynamics. These data establish that UbC constitutes an essential source of Ub during cell proliferation and stress that cannot be compensated by other Ub genes.

Assembly of Lysine 63-linked Ubiquitin Conjugates by Phosphorylated α-Synuclein Implies Lewy Body Biogenesis

alpha-Synuclein (alpha-syn) and ubiquitin (Ub) are major protein components deposited in Lewy bodies (LBs) and Lewy neurites, which are pathologic hallmarks of idiopathic Parkinson disease (PD). Almost 90% of alpha-syn in LBs is phosphorylated at serine 129 (Ser(129)). However, the role of Ser(129)-phosphorylated alpha-syn in the biogenesis of LBs remains unclear. Here, we show that compared with coexpression of wild type (WT)alpha-syn and Ub, coexpression of phospho-mimic mutant alpha-syn (S129D) and Ub in neuro2a cells results in an increase of Ub-conjugates and the formation of ubiquitinated inclusions. Furthermore, S129D alpha-syn fails to increase the Ub-conjugates and form ubiquitinated inclusions in the presence of a K63R mutant Ub. In addition, as compared with WT alpha-syn, S129D alpha-syn increased cytoplasmic and neuritic aggregates of itself in neuro2a cells treated with H(2)O(2) and serum deprivation. These results suggest that the contribution of Ser(129)-phosphorylated alpha-syn to the Lys(63)-linked Ub-conjugates and aggregation of itself may be involved in the biogenesis of LBs in Parkinson disease and other related synucleinopathies.

Multidimensional Protein Identification Technology (MudPIT) Analysis of Ubiquitinated Proteins in Plants

Protein conjugation with ubiquitin, known as ubiquitination, is a key regulatory mechanism to control protein abundance, localization, and activity in eukaryotic cells. To identify ubiquitin-dependent regulatory steps in plants, we developed a robust affinity purification/identification system for ubiquitinated proteins. Using GST-tagged ubiquitin binding domains, we performed a large scale affinity purification of ubiquitinated proteins from Arabidopsis cell suspension culture. High molecular weight ubiquitinated proteins were separated by SDS-PAGE, and the trypsin-digested samples were then analyzed by a multidimensional protein identification technology (MudPIT) system. A total of 294 proteins specifically bound by the GST-tagged ubiquitin binding domains were identified. From these we determined 85 ubiquitinated lysine residues in 56 proteins, confirming the enrichment of the target class of proteins. Our data provide the first view of the ubiquitinated proteome in plants. We also provide evidence that this technique can be broadly applied to the study of protein ubiquitination in diverse plant species.

Involvement of the Ubiquitin-Mediated Proteolytic System in the Signaling Pathway Induced by Ceramide in Primary Astrocyte Cultures

The selective degradation of abnormal or short half-life proteins in eukaryotic cells proceeds through the ubiquitin-mediated proteolytic system (UbPS). The signals that tag the proteins for their ubiquitination are well known. In the present study, our aim was to investigate the relationship between the action of ceramide and the changes in the expression of certain mRNAs of the Ub pathway and in the activation of the UbPS in cultured astrocytes (ASTs). Changes in the expression of components that are known to be substrates of the UbPS and that participate in the regulation of the cell death process were also studied. Addition of different concentrations of C₂ ceramide to cultured ASTs produced an increase in the expression of the Ub gene and in the gene that encodes E₁, one of the enzymes involved in the ubiquitination process, without any changes on cell viability. Immunocytochemical studies showed an increase in the expression of Bcl-2 with no changes in cytochrome c. Also, there was an increase in the nuclear reactivity of NFĸB, suggesting a translocation of this factor towards the nucleus. Western blots showed a decrease in IĸB and its phosphorylated form as well as an increase in Bcl-2 with no changes in cytochrome c. All of these compounds appear to be acting as possible modulators of AST responses to C₂ ceramide. Our results suggest that in AST primary cultures, C₂ ceramide, at the concentrations used in this study, does not produce apoptosis. However, it induces an activation of the UbPS, probably as a consequence of an activation of phosphatases and kinases, or through the generation of reactive oxygen species, which act as triggering signals of the UbPS. The fundamental role of NFĸB and Bcl-2 as antiapoptotic factors is discussed.

Ubiquitin Binding Site of the Ubiquitin E2 Variant (UEV) Protein Mms2 Is Required for DNA Damage Tolerance in the Yeast RAD6 Pathway

Different ubiquitin modifications to proliferating cell nuclear antigen (PCNA) signal distinct modes of lesion bypass in the RAD6 pathway of DNA damage tolerance. The modification of PCNA with monoubiquitin signals an error-prone bypass, whereas the extension of this modification into a Lys-63-linked polyubiquitin chain promotes error-free bypass. Chain formation is catalyzed by the Mms2/Ubc13 conjugating enzyme variant/conjugating enzyme (UEV.E2) complex together with the Rad5 ubiquitin ligase. In vitro studies of this UEV.E2 complex have identified a ubiquitin binding site that is mainly localized on Mms2. However, the role of this site in DNA damage tolerance and the molecular features of the ubiquitin/Mms2 interaction are poorly understood. Here we identify two molecular determinants, the side chains of Mms2-Ile-57 and ubiquitin-Ile-44, that are required for chain assembly in vitro and error-free lesion bypass in vivo. Mutating either of these side chains to alanine elicits a severe 10-20-fold inhibition of chain synthesis that is caused by compromised binding of the acceptor ubiquitin to Mms2. These results suggest that the ubiquitin binding site of Mms2 is necessary for error-free lesion bypass in the RAD6 pathway and provide new insights into ubiquitin recognition by UEV proteins.