Ubiquitination of HLA‐DO by MARCH family E3 ligases

HLA‐DO (DO) is a nonclassical MHC class II (MHCII) molecule that negatively regulates the ability of HLA‐DM to catalyse the removal of invariant chain‐derived CLIP peptides from classical MHCII molecules. Here, we show that DO is posttranslationally modified by ubiquitination. The location of the modified lysine residue is shared with all classical MHCII beta chains, suggesting a conserved function. Three membrane‐associated RING‐CH (MARCH1, 8 and 9) family E3 ligases that polyubiquitinate MHCII induce similar profiles of polyubiquitination on DOβ. All three MARCH proteins also influenced trafficking of DO indirectly by a mechanism that required the DOβ encoded di‐leucine and tyrosine‐based endocytosis motifs. This may be the result of MARCH‐induced ubiquitination of components of the endocytic machinery. MARCH9 was by far the most efficient at inducing intracellular redistribution of DO but did not target molecules for lysosomal degradation. The specificity of MARCH9 for HLA‐DQ and HLA‐DO suggests a need for common regulation of these two MHC‐encoded molecules.     

蛋白酶体活性抑制对大鼠海马tau蛋白的泛素化影响

目的:探讨蛋白酶体活性抑制对大鼠海马tau蛋白的泛素化影响.方法:利用双侧海马注射蛋白酶体的特异性抑制剂--乳胞素分别进行蛋白酶体活性检测、免疫印迹、免疫共沉淀,以检测大鼠海马内tau蛋白的泛素化改变情况.结果:(1)乳胞素引起蛋白酶体活性在24和48 h较对照组明显下降;(2)免疫共沉淀结果显示,蛋白酶体活性抑制使t...     

Syk‐dependent regulation of Hrs phosphorylation and ubiquitination upon FcεRI engagement: Impact on Hrs membrane/cytosol localization

Several lines of evidence suggest that Syk controls immune receptor endocytic trafficking. However, the Syk substrates that regulate this process are not currently known. Here, we demonstrate that Syk knockdown prevents the trafficking of engaged high affinity IgE receptor (FcεRI) to a degradative compartment in mast cells. We then concentrate our attention on hepatocyte growth factor‐regulated tyrosine kinase substrate (Hrs) as potential Syk substrate, since it serves as critical regulator for FcεRI entry into lysosomes. We show that Hrs undergoes antigen‐dependent tyrosine phosphorylation and ubiquitination, and identify Syk as the kinase responsible for Hrs phosphorylation. Syk was also required for Hrs ubiquitination catalyzed by c‐Cbl E3 ligase. Syk‐dependent regulation of Hrs covalent modifications, without affecting protein stability, controlled Hrs localization. The majority of phosphorylated Hrs forms were observed only in membrane compartments, whereas ubiquitinated Hrs was predominantly cytosolic, suggesting that both modifications might impact on Hrs function. Together, these findings provide a major step forward in understanding how Syk orchestrates endocytosis of engaged immune receptors.     

卵巢相关F-box蛋白的功能及研究进展

F-box蛋白广泛存在于真核生物和原核生物中，可以通过泛素-蛋白酶体途径参与调控细胞周期、细胞凋亡、细胞信号转导和肿瘤发生等多种生物学过程。近年来研究提示卵巢中存在多种F-box蛋白的表达，F-box蛋白功能紊乱可导致卵巢生殖和内分泌功能发生障碍及卵巢疾病，出现卵泡发育不良、卵巢早衰（POF）及卵巢癌等病理反应。随着对细胞内泛素化作用研究的不断深入，越来越多F-box蛋白通过调控细胞周期和信号传导通路等途径影响卵巢功能的作用机制逐渐被揭示，这也为相关卵巢疾病的临床诊治提供了理论基础和新思路。现就与卵巢相关的F-box蛋白的功能及研究进展进行综述。     

含pyrin结构域NOD样受体家族3炎性小体的翻译后修饰及其在脓毒症发生发展中的作用

翻译后修饰(post-translational modification, PTM)主要包括磷酸化、泛素化、烷基化、S-亚硝基化和ADP-核糖基化等,能够通过多分子多位点调控含pyrin结构域NOD样受体家族3(NOD-like receptors family pyrin domain containing 3, NLRP3)炎性小体。NLRP3炎性小体激活将造成炎症级联反应不断扩大。而这种炎症反应紊乱是推动脓毒症进展的重要因素。文章详细阐述了NLRP3炎性小体的PTM,并介绍了NLRP3的PTM在脓毒症中的作用,以期干预NLRP3炎性小体的活化,进而调控炎症,为未来脓毒症治疗提供新思路。     

Drosophila-Cdh1 (Rap/Fzr) a regulatory subunit of APC/C is required for synaptic morphology,synaptic transmission and locomotion

The assembly of functional synapses requires the orchestration of the synthesis and degradation of a multitude of proteins. Protein degradation and modification by the conserved ubiquitination pathway has emerged as a key cellular regulatory mechanism during nervous system development and function (Kwabe and Brose, 2011). The anaphase promoting complex/cyclosome (APC/C) is a multi-subunit ubiquitin ligase complex primarily characterized for its role in the regulation of mitosis (Peters, 2002). In recent years, a role for APC/C in nervous system development and function has been rapidly emerging (0180 and 0135). In the mammalian central nervous system the activator subunit, APC/C-Cdh1, has been shown to be a regulator of axon growth and dendrite morphogenesis (Konishi et al., 2004). In the Drosophila peripheral nervous system (PNS), APC2, a ligase subunit of the APC/C complex has been shown to regulate synaptic bouton size and activity (van Roessel et al., 2004). To investigate the role of APC/C-Cdh1 at the synapse we examined loss-of-function mutants of Rap/Fzr (Retina aberrant in pattern/Fizzy related), a Drosophila homolog of the mammalian Cdh1 during the development of the larval neuromuscular junction in Drosophila. Our cell biological, ultrastructural, electrophysiological, and behavioral data showed that rap/fzr loss-of-function mutations lead to changes in synaptic structure and function as well as locomotion defects. Data presented here show changes in size and morphology of synaptic boutons, and, muscle tissue organization. Electrophysiological experiments show that loss-of-function mutants exhibit increased frequency of spontaneous miniature synaptic potentials, indicating a higher rate of spontaneous synaptic vesicle fusion events. In addition, larval locomotion and peristaltic movement were also impaired. These findings suggest a role for Drosophila APC/C-Cdh1 mediated ubiquitination in regulating synaptic morphology, function and integrity of muscle structure in the peripheral nervous system.     

Androgen regulated HN1 leads proteosomal degradation of androgen receptor (AR) and negatively influences AR mediated transactivation in prostate cells

We recently reported that hematological and neurological expressed 1 (HN1) is a ubiquitously expressed, EGF-regulated gene. Expression of HN1 in prostate cell lines down-regulates PI3 K-dependent Akt activation. Here, we investigate whether HN1 is regulated by androgens through the putative androgen response elements (AREs) found in its promoter. Knockdown of HN1 expression by siRNA silencing leads to an increase in Akt^(S473) phosphorylation, resulting in the translocation of androgen receptor (AR) to the nucleus; these effects can be abrogated by the non-specific Akt inhibitor LY294002 but not by the ERK inhibitor PD98059. Furthermore, HN1 overexpression correlates with an increase in ubiquitination-mediated degradation (a consequence of the decrease in S213/210 phosphorylation of AR), ultimately resulting in the down-regulation of AR-mediated expression of the KLK3, KLK4, NKX3.1 and STAMP2 genes. We also found that HN1 overexpression suppresses colony formation as well as R1881-mediated growth in LNCaP cells, while it has the opposite effect (increasing colony formation but not proliferation) in PC-3 and DU145 cells. Therefore, we suggest that HN1 maintains a balance between the androgen-regulated nuclear translocation of AR and steady-state Akt phosphorylation, predominantly in the absence of androgens. If so, the balance between cell growth and EGF- and AR-signaling must be tightly regulated by HN1. This work has important implications for prostate cancer research, as AR, EGFR and HN1 are known to be highly expressed in prostate adenocarcinomas.     

泛素化调控与中药抗炎

炎症性疾病是一类极为常见且危害极大的疾病,以中药抗炎来减轻或治疗炎症性疾病在临床上已得到广泛应用,但其分子机制研究尚处于起步阶段。泛素是一类极为保守的小分子功能蛋白,其通过对关键信号分子蛋白的泛素化修饰过程,而对炎症等生理和病理活动发挥重要调控作用。从泛素/去泛素角度来探究中药对炎症信号途径的调控作用,将为中药抗炎机制研究提供崭新思路。     

Protein stability regulators screening assay (Pro-SRSA):protein degradation meets the CRISPR-Cas9 librar y

The regulation of protein stability is a fundamental issue for biophysical processes, but there has not previously been a convenient and unbiased method of identifying regulators of protein stability. However, as reported in the article entitled“A genome-scale CRISPR–Cas9 screening method for protein stability reveals novel regulators of Cdc25A,”recently published in Cell Discovery, our team developed a protein stability regulators screening assay (Pro-SRSA) by combining the whole-genome clustered regularly interspaced short palindromic repeats Cas9 (CRISPR–Cas9) library with a dual-lfuorescence-based protein stability reporter and high-throughput sequencing to screen for regulators of protein stability. Based on our ifndings, we are conifdent that this effcient and unbiased screening method at the genome scale will be used by researchers worldwide to identify regulators of protein stability.