Ubiquitin-conjugating Enzyme E2I Research Articles

Dynamics of Hydrogen Bonds between Water and Intrinsically Disordered and Structured Regions of Proteins.

Recent studies indicate more restricted dynamics of water around intrinsically disordered proteins (IDPs) than structured proteins. We examine here the dynamics of hydrogen bonds between water molecules and two proteins, small ubiquitin-related modifier-1 (SUMO-1) and ubiquitin-conjugating enzyme E2I (UBC9), which we compare around intrinsically disordered regions (IDRs) and structured regions of these proteins. It has been recognized since some time that excluded volume effects, which influence access of water molecules to hydrogen-bonding sites, and the strength of hydrogen bonds between water and protein affect hydrogen bond lifetimes. While we find those two properties to mediate lifetimes of hydrogen bonds between water and protein residues in this study, we also find that the lifetimes are affected by the concentration of charged groups on other nearby residues. These factors are more important in determining the hydrogen bond lifetimes than whether a residue hydrogen bonding with water belongs to an IDR or to a structured region.

Combined Evaluation of mRNA and Protein Expression, Promoter Methylation, and Immune Infiltration of UBE2I in Pan-Digestive System Tumors.

Background Digestive system tumors (DSTs) have high morbidity and mortality worldwide. This study explored the potential value of ubiquitin-conjugating enzyme E2 I (UBE2I) in pan-digestive system tumors (pan-DSTs). Methods Differential expression, tumor stages, and survival outcomes of UBE2I in pan-DSTs were determined using the GEPIA database. The TIMER database was used to confirm the correlation of UBE2I expression with pan-DSTs and immune infiltrates. Differential analyses of UBE2I promoter methylation and protein levels were performed using the UALCAN database. The underlying mechanisms of UBE2I involvement in pan-DSTs were visualized using interaction networks. The diagnostic value of UBE2I in pan-DSTs was identified using the Oncomine database. Results UBE2I was differentially and highly expressed in cholangiocarcinoma (CHOL), pancreatic adenocarcinoma (PAAD), colon adenocarcinoma (COAD), rectal adenocarcinoma (READ), liver hepatocellular carcinoma (LIHC), and stomach adenocarcinoma (STAD). According to survival analysis, upregulated UBE2I was associated with adverse overall and disease-free survival in PAAD and favorable overall survival in READ. UBE2I expression was partially linked to the purity of immune infiltration in COAD, LIHC, PAAD, READ, and STAD, as indicated by the immune infiltration analysis. Promoter methylation analysis showed differential and high methylation of UBE2I in PAAD as well as stratified analysis by gender, nodal metastasis, and race. Protein expression analysis in colon cancer revealed that UBE2I had differential and high expression in tumors as well as stratified analysis by gender, tumor histology, race, and tumor stage. Mechanism explorations demonstrated that in COAD and PAAD, UBE2I was involved in spliceosomal snRNP complex, Notch signaling pathway, etc. Diagnostic analysis indicated that UBE2I had consistent diagnostic value for COAD and PAAD. Conclusions Upregulated UBE2I may be a diagnostic and surveillance predictive signature for PAAD and COAD. The potential significance of immune infiltrates and promoter methylation in PAAD and COAD needs further exploration.

NRIP1 aggravates lung injury caused by Pseudomonas aeruginosa in mice by increasing PIAS1 ubiquitination.

Recently, evidence has shown that nuclear receptor interacting protein 1 (NRIP1) is involved in acute lung injury (ALI) progression, but the specific mechanism remains unclear. Pseudomonas aeruginosa (PA)-treated TC-1 cells were transfected with pcDNA-NRIP1 or si-NRIP1, and we found that overexpression of NRIP1 inhibited cell viability and promoted cell apoptosis and secretion of inflammatory factors, and transfection of si-NRIP1 reversed these effects. Furthermore, online bioinformatics analysis and co-immunoprecipitation assay results indicated that NRIP1 could bind to Ubiquitin Conjugating Enzyme E2I (UBE2I), and promoted UBE2I expression. Next, the PA-treated TC-1 cells were transfected with si-NRIP1 alone or together with pcDNA-UBE2I, and we observed that transfection with si-NRIP1 inhibited UBE2I expression, promoted cell viability, and reduced cell apoptosis and inflammatory factor secretion, which could be reversed by UBE2I overexpression. Moreover, UBE2I could bind to protein inhibitor of activated signal transducer and activators of transcription 1 (PIAS1). Overexpression of NRIP1 promoted UBE2I expression and inhibited PIAS1 expression, and NRIP1 promoted PIAS1 ubiquitination and degradation by UBE2I. The PA-treated TC-1 cells were transfected with si-UBE2I alone or together with si-PIAS1, and the results indicated that transfection of si-UBE2I had the same effect as transfection of si-NRIP1. Finally, our in vivo findings indicated that the expression of NRIP1 and UBE2I was decreased, and PIAS1 expression was increased, in the lung tissues of mice with NRIP1 knocked-down, and the inflammatory infiltration in the lung tissue was reduced. In conclusion, our study demonstrates that NRIP1 aggravates PA-induced lung injury in mice by promoting PIAS1 ubiquitination.

UBE2I promotes metastasis and correlates with poor prognosis in hepatocellular carcinoma

BackgroundA comprehensive investigation of ubiquitin-conjugating enzyme E2I (UBE2I) in cancer is still insufficiency. In this study, we aimed to analyze its role and mechanism in cancer by combination of bioinformatic analysis and experimental validation.MethodsThe expression profile of UBE2I in human cancers were obtained using GEPIA. Kaplan–Meier plotter was used to assess the prognostic values of UBE2I in diverse types of cancer. ROC curve analysis was employed to determine the diagnostic role of UBE2I in hepatocellular carcinoma (HCC). The expression differences based on various clinicopathological features was evaluated by UALCAN. Wound healing assay and transwell invasion assay were used to detected the effects of UBE2I on migration and invasion of HCC cells, respectively. The miRNA regulatory mechanism of UBE2I was successively investigated by binding prediction, expression analysis, survival analysis and dual-luciferase reporter assay. The correlation of UBE2I mRNA expression and UBE2I promoter methylation level was assessed using cBioPortal. STRING was finally introduced to perform co-expression analysis and enrichment analysis for UBE2I.ResultsUBE2I was upregulated in HCC, correlated with cancer progression and poor prognosis of HCC. We also found a significant diagnostic value of UBE2I in HCC. Functional experiments revealed that knockdown of UBE2I significantly inhibited HCC migration and invasion. Further research on mechanism suggested that loss of inhibition of hsa-miR-195-3p and dysregulation of UBE2I promoter methylation might account for UBE2I overexpression in HCC. Analysis of UBE2I-invovled regulatory network identified six key genes (NSMCE2, SAE1, UBA2, RANGAP1, SUMO1 and SUMO2) whose expression linked to poor prognosis in HCC.ConclusionsIn conclusion, UBE2I may be a promising therapeutic target and biomarker in cancer, especially HCC.

Dietary energy level affects adipose depot mass but does not impair in vitro subcutaneous adipose tissue response to short-term insulin and tumor necrosis factor-α challenge in nonlactating, nonpregnant Holstein cows

We assessed effects of overfeeding energy to nonlactating and nonpregnant Holstein cows during a length of time similar to a typical dry period on body lipid storage and the abundance of genes related to insulin signaling, inflammation, and ubiquitination in subcutaneous adipose tissue (SAT) in vitro challenged with insulin and recombinant bovine tumor necrosis factor-α. Fourteen cows were randomly assigned to either a high-energy (OVE; net energy for lactation = 1.60 Mcal/kg of dry matter; n = 7) or control (CON; net energy for lactation = 1.30 Mcal/kg of dry matter; n = 7) diet for 6 wk. Immediately after slaughter, liver, kidneys, and mammary gland were separated and weighed. The adipose tissue mass in the omental, mesenteric, and perirenal depots was dissected and weighed. Subcutaneous adipose tissue was collected from the tail-head region and was used as follows: control, bovine insulin (INS) at 1 µmol/L, tumor necrosis factor-α at 5 ng/mL (TNF), and their combination. Despite a lack of difference in final body condition score, OVE cows had greater energy intake and were heavier than CON cows. Furthermore, overfeeding led to greater mass of mesenteric and perirenal adipose, liver, and mammary gland. Overall, SAT incubated with INS had an upregulation of insulin receptor (INSR), interleukin-10 (IL10), small ubiquitin-like modifier 3 (SUMO3), and ubiquitin conjugating enzyme E2I (UBC9), whereas TNF upregulated peroxisome proliferator-activated receptor gamma (PPARG), diacylglycerol O-acyltransferase 2 (DGAT2), interleukin-6 (IL6), nuclear factor kappa B subunit 1 (NFKB1), small ubiquitin-like modifier 2 (SUMO2), and UBC9. Regardless of in vitro treatment, feeding OVE upregulated PPARG, fatty acid synthase (FASN), and insulin induced gene 1 (INSIG1). Abundance of PPARG was greater in SAT of OVE cows cultured individually with INS and TNF. The interaction between diet and in vitro treatment revealed that sterol regulatory element binding transcription factor 1 (SREBF1) had greater abundance in SAT from the CON group in response to culture with INS, whereas SAT from OVE cows had greater SREBF1 abundance in response to culture with TNF. The mRNA abundance of IL6 and NFKB1 was greater in response to TNF treatment and overall in CON cows. Furthermore, SAT from these cows had greater IL10 abundance when cultured with INS and TNF. Overall, data highlighted that overfeeding energy increases adipose tissue mass in part by stimulating transcription of key genes associated with insulin signaling, adipogenesis, and lipogenesis. Because SAT thickness or mass was not measured, the lack of effect of overfeeding on body condition score limits its use to predict overall body lipid storage. An overt inflammatory response in SAT after a 6-wk period of over-consumption of energy could not be discerned.

Effect of MicroRNA-30e on the Behavior of Vascular Smooth Muscle Cells via Targeting Ubiquitin-Conjugating Enzyme E2I.

Many microRNAs (miRNAs) have recently been shown to demonstrate critical roles in differentiation, proliferation and migration of vascular smooth muscle cells (VSMCs).Methods and Results:In this study, a certain amount of miRNA expression in VSMCs was evaluated by real-time polymerase chain reaction, and it was found that microRNA-30e (miR-30e) was expressed more strongly than other common vascular well-expressed miRNAs in vitro. Subsequently, both a gain and loss of function study was performed in vitro and in vivo. It was found that miR-30e in VSMCs was strongly downregulated concomitantly with stimulation, and miR-30e inhibited VSMCs proliferation and migration both in vitro and in vivo. Furthermore, ubiquitin-conjugating enzyme E2I (Ube2i) was identified as the target gene of endogenous miR-30e by luciferase reporter assay, and it was confirmed that overexpression of miR-30e significantly reduced Ube2i and inhibited the phenotypic switch of VSMCs. Knockdown of Ube2i had an influence over the proliferation and migration of cultured VSMCs, as same as the miR-30e mimic did. Overexpression of miR-30e induced the apoptosis of VSMCs and deregulated the protein expression of IkBα, which is crucial for the NFκB signal pathway. The results of this study indicated that miR-30e in VSMCs exerted an anti-atherosclerosis effect via inhibiting proliferation and migration, and promoting apoptosis of VSMCs. More specifically, it was demonstrated that miR-30e exhibited these effects on VSMCs partially through targeting Ube2i and downregulating the IκBα/NFκB signaling pathway.

The SUMO Conjugating Enzyme Ubc9 Is Required for Inducing and Maintaining Stem Cell Pluripotency

Sumoylation adds a small ubiquitin-like modifier (SUMO) polypeptide to the ε-amino group of a lysine residue. Reminiscent of ubiquitination, sumoylation is catalyzed by an enzymatic cascade composed of E1, E2, and E3. For sumoylation, this cascade uses Ubc9 (ubiquitin conjugating enzyme 9, now officially named ubiquitin conjugating enzyme E2I [UBE2I]) as the sole E2 enzyme. Here, we report that expression of endogenous Ubc9 increases during reprogramming of mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. In addition, this E2 enzyme is required for reprogramming as its suppression dramatically inhibits iPS cell induction. While Ubc9 knockdown does not affect survival of MEFs and immortalized fibroblasts, Ubc9 is essential for embryonic stem cell (ESC) survival. In addition, we have found that Ubc9 knockdown stimulates apoptosis in ESCs but not in MEFs. Furthermore, the knockdown decreases the expression of the well-known pluripotency marker Nanog and the classical reprogramming factors Klf4, Oct4, and Sox2 in ESCs. Together, these observations indicate that while dispensable for fibroblast survival, the sole SUMO E2 enzyme Ubc9 plays a critical role in reprogramming fibroblasts to iPS cells and maintaining ESC pluripotency.

UHRF2, a Ubiquitin E3 Ligase, Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.

SUMOylation of claudin‐2

The C-terminal cytoplasmic tails of claudins are likely sites for interaction with proteins that regulate their function. We performed a yeast two-hybrid screen with the tail of human claudin-2 against a human kidney cDNA library and identified interactions with the PDZ3 domain of ZO-2 as well as ubiquitin-conjugating enzyme E2I (SUMO ligase-1) and E3 SUMO-protein ligase PIAS; the first is a predicted interaction, while the latter two are novel and suggest that claudin-2 is a substrate for SUMOylation. Using an in vitro SUMOylation assay, we identified K218 as a conjugation site on claudin-2; mutation of that lysine to arginine blocked SUMOylation. Stable expression of inducible GFP-SUMO-1 in MDCK cells resulted in decreased levels of claudin-2 protein by immunoblot and decreased claudin-2 membrane expression by immunofluorescence microscopy. We conclude that the cellular levels of claudin-2 may be modulated by SUMOylation, warranting further investigation of cellular pathways that regulate this modification in vivo.

Differential effect of oxidative or excitotoxic stress on the transcriptional profile of amyotrophic lateral sclerosis‐linked mutant SOD1 cultured neurons

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal, degenerative disorder of motor neurons. The causes of most cases of ALS are as yet undefined. In a previous study, it was shown that N-methyl-D-aspartate (NMDA) and H(2)O(2) stimuli reduce neuronal survival in cortical neurons in culture (Boutahar et al., 2008). To identify variations in gene expression in response to these neurotoxins in transgenic vs. control cortical neurons cultures, both microarray and RT-PCR analysis were performed. High-density oligonucleotide microarrays showed changes in the expression of about 600 genes involved in protein degradation, neurotrophic factors pathway, cell cycle, inflammation, cytoskeleton, cell adhesion, transcription, or signalling. The most up-regulated genes following H(2)O(2) treatment were involved in cytoskeletal organization and axonal transport, such as ARAP2, KIF17, and DKK2, or in trophic factors pathways, such as insulin-like growth factor-binding protein 4 (IGFBP4), FGF17, and serpin2. The most down-regulated genes were involved in ion transport, such as TRPV1. After NMDA treatment, the most up-regulated genes were involved in protein degradation, such as ubiquitin-conjugating enzyme E2I and cathepsin H, and the most down-regulated genes were involved in ion transport, such as SCN7A. We conclude that these neurotoxins act through different transcriptional inductions, and these changes may reflect an adaptative cellular response to the cellular stress induced by the neurotoxins involved in ALS in the presence of mutant human SOD1.

Ubc9 gene polymorphisms and late-onset Alzheimer's disease in the Korean population: A genetic association study

Ubiquitin-conjugating enzyme E2I (Ubc9) ligates small ubiquitin-related modifier (SUMO) to target proteins, resulting in changes of their localization, activity, or stability. Sumoylation of amyloid precursor protein (APP) was reported to be associated with decreased levels of beta amyloid (Aβ) aggregates, suggesting that sumoylation may play a role in the pathogenesis of Alzheimer's disease (AD). We investigated the association between genetic variations of Ubc9 gene ( UBE2I) and late-onset Alzheimer's disease (AD). Five single nucleotide polymorphisms (SNPs) in UBE2I were genotyped in the DNA samples of 312 AD patients, 347 subjects with mild cognitive impairment (MCI), and 489 cognitively healthy controls. The genotype distribution of a polymorphism in intron 7 (rs761059) differed between AD cases and controls, with an adjusted odds ratio (OR) of 1.45 ( p = 0.046, 95% CI: 1.01–2.08). One haplotype (ht2 CAGAG) was found in 14.0% of the AD patients and in 11.1% of the controls ( p = 0.04, OR = 1.43. 95% CI; 1.01–2.01). Stratification by the ApoE-ɛ4 allele gave no significant difference between the groups. When the samples were stratified by gender, the genotypes of two SNPs (rs8052688, rs8063) were significantly associated with the risk of MCI among women. Our investigation suggests that UBE2I polymorphisms might be associated with a risk of AD and MCI.

Novel progerin-interactive partner proteins hnRNP E1, EGF, Mel 18, and UBC9 interact with lamin A/C

The Hutchinson–Gilford progeria syndrome (HGPS or progeria) is an apparent accelerated aging disorder of childhood. Recently, HGPS has been characterized as one of a growing group of disorders known as laminopathies, which result from genetic defects of the lamin A/C ( LMNA) gene. The majority of HGPS mutant alleles involve a silent mutation, c.2063C>T resulting in G608G, that generates a cryptic splicing site in exon 11 of LMNA and consequently truncates 50 amino acids near the C-terminus of pre-lamin A/C. To explore possible mechanisms underlying the development of HGPS, we began a search for proteins that would uniquely interact with progerin (the truncated lamin A in HGPS) using a yeast two-hybrid system. Four new progerin interactive partner proteins were identified that had not been previously found to interact with lamin A/C: hnRNP E1, UBC9 (ubiquitin conjugating enzyme E2I), Mel-18, and EGF1. However, using control and progeria fibroblasts, co-immunoprecipitation studies of endogenous proteins did not show differential binding affinity compared to normal lamin A/C. Thus, we did not find evidence for uniquely interacting partner proteins using this approach, but did identify four new lamin A/C interactive partners.

Protein partners of C/EBPε

CCAAT-enhancer binding protein-epsilon (C/EBPepsilon) is a nuclear transcription factor implicated in the regulation of terminal myeloid differentiation. Using a yeast two-hybrid screen, potential interaction partners of C/EBPepsilon involved in myeloid development were identified. C/EBPepsilon was found to associate with other C/EBP family members, including C/EBPepsilon and CHOP as well as other proteins that are known to contain a leucine-zipper protein interaction motif including CREB2, LDOC1, E6TP1, and AF-17. In addition, C/EBPepsilon demonstrated the potential for interaction with proteins that do not possess a leucine-zipper motif, including proteins that may be involved in sumoylation (protein inhibitor of activated STAT1 [PIAS1] and ubiquitin-conjugating enzyme E2I). As expected, the association of C/EBPepsilon with other C/EBP family members depends on the presence of a functional leucine-zipper motif. Mapping studies of C/EBPepsilon with PIAS1 (as an example of a nonleucine-zipper-containing protein) showed that C/EBPepsilon interacts with the amino-terminal domain of PIAS1. The function of C/EBPepsilon interacting proteins was further investigated. Co-expression of C/EBPepsilon with C/EBPdelta resulted in potent transactivation in a lactoferrin reporter system. A gel mobility shift assay suggests that C/EBPepsilon, C/EBPalpha, and C/EBPdelta proteins can bind as heterodimers to a C/EBP consensus DNA-binding site. As CHOP is known to represent a transcriptional repressor, the functional interaction between C/EBPepsilon and CHOP was investigated. Co-expression of C/EBPepsilon and c-Myb with CHOP caused marked transcriptional repression of target reporter genes. Our results suggest heterodimeric partners of C/EBPepsilon modulate the function of C/EBPepsilon in mediating gene transcription during myelopoiesis.

Identification and characterization of proteins that selectively interact with isoforms of the mRNA binding protein AUF1 (hnRNP D).

The mRNAs that encode certain cytokines and proto-oncogenes frequently contain a typical AU-rich motif that is located in their 3'-untranslated region. The protein AUF1 is the first factor identified that binds to AU-rich regions and mediates the fast degradation of the target mRNAs. AUF1 exists as four different isoforms (p37, p40, p42 and p45) that are generated by alternative splicing. The fact that AUF1 does not degrade mRNA itself had led to the suggestion that other AUF1 interacting proteins might be involved in the process of selective mRNA degradation. Here we used the yeast two-hybrid system in order to identify proteins that bind to AUF1. We detected AUF1 itself, as well as the ubiquitin-conjugating enzyme E2I and three RNA binding proteins: NSEP-1, NSAP-1 and IMP-2, as AUF1 interacting proteins. We confirmed all interactions in vitro and mapped the protein domains that are involved in the interaction with AUF1. Gel-shift assays with the recombinant purified proteins suggest that the interacting proteins and AUF1 can bind simultaneously to an AU-rich RNA oligonucleotide. Most interestingly, the AUF1 interacting protein NSEP-1 showed an endoribonuclease activity in vitro. These data suggest the possibility that the identified AUF1 interacting proteins might be involved in the regulation of mRNA stability mediated by AUF1.

Mutation screening in Rett syndrome patients

Rett syndrome (RTT) was first described in 1966. Its biological and genetic foundations were not clear until recently when Amiret al reported that mutations in the MECP2 gene were detected...