Proper ubiquitination effect on the fertilisation outcome post‐ICSI

Ubiquitin is an 8.5‐kDa protein that tags outlived proteins for degradation by the proteasome. It also marks defective spermatozoa during epididymal passage and has been proposed as a biomarker of sperm quality. This study evaluates the relationship between sperm ubiquitination, protamine deficiency, semen parameters and fertilisation rate in infertile individuals undergoing the intracytoplasmic sperm insemination (ICSI) procedure. Semen samples from 73 ICSI candidates were collected and analysed according to World Health Organization criteria. A portion of each sample was evaluated for sperm ubiquitination using the sperm ubiquitin tag immunoassay (SUTI) with flow cytometry, and protamine deficiency by chromomycin A3 (CMA3) staining. In addition, the relationship between the fertilisation rate and sperm ubiquitination was calculated in ICSI candidates. The intensity of ubiquitination showed a significant negative correlation with sperm concentration (r = ?0.255, P = 0.032) and a positive correlation with fertilisation rate (r = 0.384, P = 0.013) post‐ICSI. No correlation was observed between protamine deficiency and the percentage of ubiquitination or ubiquitination intensity. The results of this study suggest that sperm ubiquitination prior to capacitation may be considered as a marker of defective spermatozoon. Spermatozoa that undergo proper ubiquitination may have a higher chance for fertilisation, because they are made redundant by the ubiquitin–proteasome pathway in the epididymis compared to hypo‐ubiquitinated spermatozoa.     

Novel reno-protective mechanism of Aspirin involves H2AK119 monoubiquitination and Set7 in preventing type 1 diabetic nephropathy

Background

Even after several novel therapeutic approaches, the number of people with diabetic nephropathy (DN) still continues to increase globally, this suggest to find novel therapeutic strategies to prevent it completely. Recent reports, are indicating the ubiquitin proteasome system alterations in DN. Recently, we also showed that, histone H2AK119 mono-ubiquitination (H2AK119-Ub) found to regulate Set7, a key epigenetic enzyme in the development of renal fibrosis under type 1 diabetic condition. Hence, we aimed to study the role of a known 20 s proteasome inhibitor Aspirin, on histone ubiquitination in the progression of DN.

THAP11通过抑制p53的泛素化影响食管癌细胞的增殖和凋亡

目的：探讨死亡相关蛋白(thanatos-associated protein,THAP)11对食管癌细胞增殖和凋亡的影响及其潜在 的机制。方法：采用蛋白质印迹法检测人食管上皮细胞Het-1A和人食管癌细胞(Eca109,TE-1和Ec 9706)中THAP11的 表达。将食管癌TE-1细胞分为空白对照(NC)组、阴性对照(LV-LC)组、TRA P11(LV-TRA P11)组,按分组处理细胞后, 采用MTT 法检测细胞活力,流式细胞术检测细胞凋亡,caspases试剂盒检测caspase-3和caspase-9的活性。采用体外泛素 化实验检测TE-1细胞的p53泛素化水平。结果：与Het-1A细胞相比,食管癌细胞中THAP11的表达显著下降(P<0.05)。 LV-THAP11转染食管癌细胞后,细胞活力降低(P<0.05),凋亡率升高(P<0.05),caspase-3和caspase-9活性升高(P<0.05)。 THAP11能够提高食管癌细胞中p53蛋白的表达(P<0.05)。与LV-LC组相比,转染THAP11后食管癌细胞中p53的表达上调 (P<0.05),MDM2调节的p53的泛素化也被抑制。结论：THAP11通过抑制p53的泛素化抑制食管癌细胞的增殖,促进食管 癌细胞的凋亡。     

Emerging Role of Ubiquitination in the Regulation of PD-1/PD-L1 in Cancer Immunotherapy

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Manganese‐induced downregulation of astroglial glutamine transporter SNAT3 involves ubiquitin‐mediated proteolytic system

SNAT3 is a major facilitator of glutamine (Gln) efflux from astrocytes, supplying Gln to neurons for neurotransmitter synthesis. Our previous investigations have shown that, in primary cortical astrocyte cultures, SNAT3 protein is degraded after exposure to manganese (Mn²⁺). The present studies were performed to identify the processes responsible for this effect. One of the well‐established mechanisms for protein‐level regulation is posttranslational modification via ubiquitination, which leads to the rapid degradation of proteins by the 26S proteasome pathway. Here, we show that astrocytic SNAT3 directly interacts with the ubiquitin ligase, Nedd4‐2 (neural precursor cells expressed developmentally downregulated 4‐2), and that Mn²⁺ increases both Nedd4‐2 mRNA and protein levels. Additionally, we have found that Mn²⁺ exposure elevates astrocytic ubiquitin B mRNA expression, free ubiquitin protein levels, and total protein ubiquitination. Furthermore, Mn²⁺ effectively decreases astrocytic mRNA expression and the phosphorylation of serum and glucocorticoid‐inducible kinase, a regulatory protein, which, in the active phosphorylated form, is responsible for the phosphorylation and subsequent inactivation of Nedd4‐2. Additional findings establish that Mn²⁺ increases astrocytic caspase‐like proteolytic proteasome activity and that the Mn²⁺‐dependent degradation of SNAT3 protein is blocked by the proteasome inhibitors, N‐acetyl‐leu‐leu‐norleucinal and lactacystin. Combined, these results demonstrate that Mn²⁺‐induced SNAT3 protein degradation and the dysregulation of Gln homeostasis in primary astrocyte cultures proceeds through the ubiquitin‐mediated proteolytic system. © 2010 Wiley‐Liss, Inc.     

The E3 ubiquitin ligase RNF186 and RNF186 risk variants regulate innate receptor-induced outcomes

Balancing microbial-induced cytokines and microbial clearance is critical at mucosal sites such as the intestine. How the inflammatory bowel disease (IBD)–associated gene RNF186 regulates this balance is unclear. We found that macrophages from IBD-risk rs6426833 carriers in the RNF186 region showed reduced cytokines to stimulation through multiple pattern recognition receptors (PRRs). Upon stimulation of PRRs, the E3-ubiquitin ligase RNF186 promoted ubiquitination of signaling complex molecules shared across PRRs and those unique to select PRRs. Furthermore, RNF186 was required for PRR-initiated signaling complex assembly and downstream signaling. RNF186, along with its intact E3-ubiquitin ligase activity, was required for optimal PRR-induced antimicrobial reactive oxygen species, reactive nitrogen species, and autophagy pathways and intracellular bacterial clearance in human macrophages and for bacterial clearance in intestinal myeloid cells. Cells transfected with the rare RNF186-A64T IBD-risk variant and macrophages from common rs6426833 RNF186 IBD-risk carriers demonstrated a reduction in these RNF186-dependent outcomes. These studies identify mechanisms through which RNF186 regulates innate immunity and show that RNF186 IBD-risk variants demonstrate a loss of function in PRR-initiated outcomes.

Inflammatory bowel disease (IBD) is characterized by dysregulated host–microbial responses and is composed of two subtypes: Crohn’s disease and ulcerative colitis (UC). Pattern recognition receptors (PRRs) are critical for recognition and responses to microbes. Both loss of function and gain of function in PRR-initiated outcomes can be associated with intestinal inflammation (1), thereby highlighting the importance of the balance in innate immune regulation in intestinal tissues. Despite the tremendous success in genetic studies identifying loci associated with susceptibility to IBD (2), altered functions for most of these loci are not well defined. One such region is on chromosome 1, encompassing the RNF186 gene (2, 3).The UC rs6426833 A risk variant in the RNF186 region is observed at a 0.395 to 0.548 frequency in European ancestry healthy individuals (per the Single Nucleotide Polymorphism Database; accessed July 2019), and this common variant confers a 1.265 increased risk of developing UC (2). Importantly, a rare Ring Finger Protein 186 (RNF186) Ala64Thr mutation confers a 1.49-fold increased risk for developing UC (4). RNF186 is a member of the RING family E3 ubiquitin ligases and has a conserved C₃HC₄ type zinc finger (ZnF) motif in the RING domain consistent with its ability to catalyze ubiquitination of select downstream substrates (5, 6). E3 ubiquitin ligases are key mediators for posttranslational modifications of PRR-initiated signaling intermediates (7). Only a few reports have examined roles for RNF186, and these have focused on its functions in epithelial cells (4–6, 8). In HeLa cells, RNF186 can localize to the endoplasmic reticulum (ER) and enhance ER stress-associated apoptotic signaling (5). RNF186-deficient and rare variant RNF186 A64T knockin mice demonstrated more severe dextran sodium sulfate–induced colitis associated with intestinal epithelial dysregulation (6). RNF186 can regulate nutrient sensing in epithelial cells (8). Therefore, while some functions for RNF186 have been identified in epithelial cells, roles for RNF186 in mediating outcomes in innate immunity, including downstream of PRRs, have not been examined. Moreover, the consequences of UC-associated common genetic variants in the RNF186 region have not been defined.We identify that RNF186 is expressed in human monocyte-derived and intestinal myeloid-derived cells. We establish that RNF186 is required for optimal PRR-induced signaling, cytokine secretion, and induction of a range of antimicrobial pathways. We define mechanisms and structural regions through which RNF186 regulates these PRR-initiated outcomes and identify that the rare and common RNF186 IBD-risk variants lead to a loss of function in these outcomes.     

Recent developments on transition of lung myofibroblasts北大核心CSCD

肺肌成纤维细胞是驱动特发性肺纤维化发生发展的重要效应细胞,但肺肌成纤维细胞转化的分子机制至今仍未阐明。该文将从机械转导、代谢、氧化应激、泛素化、细胞衰老等方面总结近5年关于肺肌成纤维细胞转化分子机制的研究新进展。     

ROMK1 channel activity is regulated by monoubiquitination

The ubiquitination of proteins can signal their degradation, modify their activity or target them to specific membranes or cellular organelles. Here, we show that monoubiquitination regulates the plasma membrane abundance and function of the potassium channel, ROMK. Immunoprecipitation of proteins obtained from renal cortex and outer medulla with ROMK antibody revealed that this channel was monoubiquitinated. To determine the ubiquitin binding site on ROMK1, all intracellular lysine (Lys) residues of ROMK1 were individually mutated to arginine (Arg), and a two-electrode voltage clamp was used to measure the ROMK1 channel activity in Xenopus oocytes. ROMK1 channel activity increased from 8.1 to 27.2 microA only when Lys-22 was mutated to Arg. Furthermore, Western blotting failed to detect the ubiquitinated ROMK1 in oocytes injected with R1K22R. Patch-clamp experiments showed that biophysical properties of R1K22R were identical to those of wild-type ROMK1. Although total protein expression levels of GFP-ROMK1 and GFP-R1K22R in oocytes were similar, confocal microscopy showed that the surface fluorescence intensity in oocytes injected with GFP-R1K22R was higher than that of GFP-ROMK1. In addition, biotin labeling of ROMK1 and R1K22R proteins expressed in HEK293 cells showed increased surface expression of the Lys-22 mutant channel. Finally, expression of R1K22R in COS7 cells significantly stimulated the surface expression of ROMK1. We conclude that ROMK1 can be monoubiquitinated and that Lys-22 is an ubiquitin-binding site. Thus, monoubiquitination of ROMK1 regulates channel activity by reducing the surface expression of channel protein. This finding implicates the linking of a single ubiquitin molecule to channels as an important posttranslational regulatory signal.