蛋白质泛素化降解途径

泛素系统(UPS)广泛存在于真核生物中，是精细的特异性的蛋白质降解系统。它由泛素、26S蛋白酶体、多种酶（如E1、E2、E3、去泛素酶）构成。在泛素系统中，泛素（Ubiquitin Ub）是一种序列保守的小分子蛋白，蛋白质与泛素结合后，被蛋白酶体以ATP依赖的方式降解。E1、E2酶分别称为泛素活化酶和泛素载体酶，使泛素通过Ub-腺苷酸中间产物形成E2-Ub巯基酯。泛素连接酶E3负责连接泛素和特异性的底物，这样泛素化的底物可以被26S蛋白酶体降解为若干肽段。泛素系统通过特异性的降解蛋白质，调节细胞分化、免疫反应，参与转录、离子通道、分泌的调控及神经元网络、细胞器的形成等等，泛素系统还与人类某些疾病有关。本文对泛素系统的成员、作用机制和功能的研究进展作了介绍。     

去泛素化酶的研究及其进展

泛素-蛋白酶体途径（ubiquitin—proteasome pathway）是细胞内一个重要的蛋白质降解调节系统。通过对底物蛋白的多聚泛素化并经蛋白酶体降解,可以影响或调节多种细胞活动,包括：基因转录、细胞周期调节、免疫反应、细胞受体功能及肿瘤生长、炎症过程等。该途径也是一个被严格调控的可逆过程,其中去泛素化酶的调节就是一个重要的环节。目前研究证实,细胞内广泛存在许多去泛素化酶（deubiquitinating enzymes,DUBs）,主要分为以泛素羧基末端水解酶家族和泛素特异性加工酶家族为主的5种类型。     

泛素-蛋白酶体途径降解胰腺癌细胞系中凝溶胶蛋白

目的 探讨胰腺癌中泛素-蛋白酶体途径对凝溶胶蛋白(gelsolin)的降解作用.方法 用特异性蛋白酶体抑制剂lactacystin处理胰腺癌细胞系BxPC-3和PANC-1,经Westem blot检测凝溶胶蛋白的表达,免疫沉淀细胞内凝溶胶蛋白,分析沉淀蛋白的泛素化.结果 BxPC-3细胞系经lactacystin作用12 h后,细胞内凝溶胶蛋白含量较对照组和处理前明显升高(P＜0.05),而且细胞内的凝溶胶蛋白表现出与泛素分子的相互作用.结论 泛素-蛋白酶体途径对凝溶胶蛋白的降解作用,可能是胰腺癌中凝溶胶蛋白表达降低的原因之一.     

泛素羧基末端水解酶-1的研究进展

泛素羧基末端水解酶-1（UCH-L1），又名蛋白基因产物9.5，是泛素-蛋白酶体系统中的重要成员，它除了去泛素化作用外，还具有泛素连接酶和稳定细胞内泛素单体的功能。UCH-L1通过泛素相关途径调节细胞的增生、分化和凋亡，并可能参与与之相关的神经退行性疾病、肿瘤等的发生发展过程。最近UCH-L1的特异性底物的发现为探讨其作用的具体机制开辟了新的途径。     

泛素连接酶MDM2促进外源NOLC1的泛素化和降解

目的研究泛素连接酶MDM2对其调节因子NOLC1的泛素化和降解。方法克隆原核表达了NOLC1全长蛋白和其核定位信号区域,在体外泛素化体系中利用有泛素连接酶活性的重组MDM2研究其对NOLC1的泛素化。在哺乳动物细胞中,研究MDM2对外源转染的NOLC1的降解。结果在体外泛素化体系中MDM2泛素化NOLC1全长和其核定位信号区域;在哺乳动物细胞中,MDM2促进外源NOLC1降解超过70%,且NOLC1的降解通过蛋白酶体途经实现。结论 MDM2促进外源NOLC1的泛素化和降解,为研究MDM2-TP53-NOLC1之间的相互调节提供了新的线索。     

泛素-蛋白酶体途径及意义

泛素-蛋白酶体途径介导的蛋白降解是机体调节细胞内蛋白水平与功能的一个重要机制。负责执行这个调控过程的组成成分包括泛素及其启动酶系统和蛋白酶体系统。泛素启动酶系统负责活化泛素,并将其结合到待降解的蛋白上,形成靶蛋白多聚泛素链,即泛素化。蛋白酶体系统可以识别已泛素化的蛋白并将其降解。此外,细胞内还有另一类解离泛素链分子的去泛素化蛋白酶形成反向调节。泛素-蛋白酶体途径涉及许多细胞的生理过程,其调节异常与多种疾病的发生有关。     

E3泛素连接酶在核转录共抑制因子SnoN蛋白降解中的作用

目前已知泛素-蛋白酶体途径是真核细胞内蛋白降解的主要途径,能选择性降解细胞内多种蛋白（如细胞周期蛋白、蛋白激酶、信号分子等）.其中,E3泛素连接酶因决定了靶蛋白底物的特异性,而成为该途径中起决定作用的关键酶[1].近年研究发现,核转录共抑制因子Ski相关活性蛋白N（SnoN）蛋白的泛素化降解在调控SnoN蛋白表达中起着重要作用[2].表现为多种水平多个环节上的调控,而且这种调控与肿瘤、发育障碍、中枢神经系统疾病、纤维化疾病等密切相关.本文拟对E3泛素连接酶介导SnoN蛋白泛素化的研究进展作一综述.     

泛素连接酶LNX1促进外源PBK的泛素化和降解

 目的 研究LNX1对其相互作用蛋白PBK的泛素化和降解。方法 克隆、原核表达、纯化了一系列重组人LNX1截断体蛋白和LNX1全长蛋白;在体外泛素化体系中研究其对PBK的泛素化,哺乳动物细胞内研究其对外源PBK的泛素化和降解。结果 在体外泛素化体系中LNX1泛素化PBK,并研究了不同LNX1截断体对PBK泛素化的影响;发现在哺乳动物细胞内外源LNX1促进外源PBK的泛素化,进而导致其通过蛋白酶体降解。结论 研究发现了LNX1对外源PBK的泛素化和降解,为研究LNX1的生理功能提供了重要线索。     

枸杞多糖通过泛素蛋白酶体途径降解突变亨廷顿蛋白

目的　探讨枸杞多糖（Lycium barbarum polysaccharide,LBP）降解突变亨廷顿蛋白（mutant huntingtin,mHtt）的途径。 方法　在稳定表达mHtt 160Q的HEK293细胞中使用不同浓度LBP,CCK8法检测细胞活力,caspase-3活性酶标法检测caspase-3活性;使用荧光显微镜检测、Image Pro Plus 6.0分析LBP对HEK293-160Q细胞中mHtt的影响并同时使用RT-PCR法检测LBP是否影响其mRNA水平;使用LBP、MG132及氯喹,分组处理HEK293-160Q细胞,通过Western Blot法检测不同组细胞中mHtt的变化。 结果　LBP能提高HEK293-160Q细胞活力,降低caspase-3活性;LBP能减少细胞中mHtt且不改变其mRNA;不同药物处理HEK293-160Q细胞后,发现与只使用LBP相比,同时使用LBP与MG132会显著降低mHtt的降解,而同时使用LBP与氯喹则对mHtt的降解没有影响。 结论　LBP能通过泛素蛋白酶体途径降解mHtt,减轻mHtt所引起的细胞毒性继而提高细胞活力、抑制细胞凋亡。     

UPS-E3泛素连接酶ITCH与胰岛素抵抗的关系

<正>糖尿病已成为严重的世界性公共卫生问题,预计至2045年全球糖尿病患者将超过6亿人^[1]。胰岛素抵抗是2型糖尿病的重要表型,是指各种原因导致胰岛素促进葡萄糖摄取和利用率下降,机体代偿性地分泌过多胰岛素,产生高胰岛素血症。胰岛素是一种蛋白类激素,促进糖原、脂质和蛋白质合成等多种生物代谢过程,其作用的发挥依赖于胰岛素信号正常转导;胰岛素重要靶器官如肝脏、脂肪、肌肉、胰腺和脑中胰岛素信号通路阻断将导致胰岛素抵     

PPARα ligands cause lymphocyte depletion and cell cycle block and this is associated with augmented TRB3 and reduced Cyclin B1 expression

PPARα ligands are medications used clinically to prevent cardiovascular events, however studies have shown that these agents are also anti-inflammatory. Our previous studies have shown that PPARα ligands induce lymphocyte depletion. PPARα ligands also potently upregulate TRB3, a protein that has been associated with cell cycle arrest. Therefore the following studies were undertaken to determine the mechanisms associated with lymphocyte depletion. Our studies demonstrate that WY14,643, a PPARα ligand, decreases the amount of lymphocytes recovered after stimulation and reduces cellular divisions. Cells treated with WY14,643 also accumulate in the G2/S phase of the cell cycle. TRB3 has been shown to inhibit the phosphorylation of AKT/Protein Kinase B, and reduced activation of AKT has been associated with decreased cellular divisions and survival. However in lymphocytes, TRB3 did not reduce the phosphorylation of AKT, and WY14,643 treatment was associated with enhanced activation of AKT. Drosophila tribbles (TRB3 homolog) causes G2 arrest by decreasing the expression of a Cdc25c homolog. Lymphocytes stimulated and treated with WY14,643 have reduced expression of Cdc25c, however this is not associated with enhanced expression of phosphorylated-Cdc2 which induces G2 arrest. Instead we observed that WY14,643 consistently reduces the protein and mRNA expression of Cyclin B1. Moreover, TRB3 inhibits activation of a Cyclin B1 promoter construct. In summary, we propose that PPARα ligands may reduce cellular number by augmenting TRB3 expression, which in turn induces cell cycle arrest by reducing the expression of Cyclin B1. Reduced cellular divisions and cell cycle arrest may be responsible for some of the immunomodulatory effects of these agents that have been consistently observed in human trials.     

Emodin-induced microglial apoptosis is associated with TRB3 induction

Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a natural anthraquinone compound isolated from the rhizome of rhubarb, has been reported to treat brain injury after intracerebral hemorrhage. Treatment of neurons with emodin is able to decrease glutamate excitotoxicity, modulate calcium homeostasis, and induce Bcl-2 expression. However, the effects of emodin on the brain-resident innate immune cells are unclear. In the present study, the mouse microglial cell line, BV-2, was selected to investigate the effects of emodin on microglial activation and apoptosis. Cell viability and apoptosis were sequentially measured with the CellTiter-Glo Luminescent Cell Viability Assay, YOPRO-1 and Caspase-Glo 3/7 Assay Systems. The degree of microglial activation was evaluated using quantitative RT-PCR to measure expression of inflammatory markers. Treatment of BV-2 cells with emodin caused caspase-mediated apoptosis in a dose-dependent manner, and emodin augmented LPS-induced microglial apoptosis to repress inflammatory activation. In response to emodin treatment, reactive oxygen species (ROS) production was increased, and TRB3 was markedly activated. siRNA knockdown of TRB3 attenuated emodin-induced microglial apoptosis. Ectopic overexpression of TRB3 decreased cell viability and was associated with dysregulation of the prosurvival Akt/FOXO3 pathway. These results demonstrate that emodin induces BV-2 cell apoptosis through TRB3 and consequently eliminates inflammatory microglia. Our findings provide a novel molecular basis through which emodin exerts neuroprotective effects, treating brain injury after intracerebral hemorrhage.     

TRB3 Q84R基因多态性与不同表型多囊卵巢综合征的相关性

目的探讨TRB3 Q84R基因多态性与不同表型多囊卵巢综合征（Ⅰ型：O＋H＋P;Ⅱ型：O＋H;Ⅲ型：H＋P;Ⅳ型：O＋P）的相关性。方法采用聚合酶链反应-限制性片段长度多态性（polymerase chain reaction-restriction frag-ment length polymorphism,PCR-RFLP）方法检测TRB3 Q84R的基因多态性。结果 TRB3 Q84R基因型频率分布在Ⅰ型P-COS组（O＋H＋P）与对照组之间有显著性差异（P〈0.05）;其余PCOS表型与对照组之间基因型频率分布无显著性差异（P〉0.05）。结论 TRB3 Q84R基因多态性与Ⅰ型PCOS相关。     

Profiling the pattern of human TRB/IGH-CDR3 repertoire in liver transplantation patients via high-throughput sequencing analysis

Immune processes in liver transplantation remain poorly understood. Acute allograft rejection in liver transplantation is a kind of T cell–mediated inflammatory disease accompanied by inflammatory cell infiltration. However, the effect of acute allograft rejection on the immunological characteristics of TCRs in peripheral blood mononuclear cell is unknown. In this study, we characterized the pattern of the human T cell receptor beta chain (TRB) and immunoglobulin heavy chain (IGH) complementarity-determining region 3 (CDR3) repertoires via high-throughput sequencing in 11 acute allograft rejection (AG) cases, 23 patients with stable allograft liver function (ST) who had liver transplantation performed and 20 healthy controls (HC). The diversity of TRB-CDR3 was significantly reduced in the AG group compared with the ST group and healthy controls (HC). The CDR3 and N-addition length distribution were not significantly different between the AG and ST groups. However, N-addition length distribution was significantly changed compared to HC. It seemed that AG used more short N-additions and healthy people used more long N-additions in TRB-CDR3 repertoire. Our findings suggested that the TRB-CDR3 region of AG had distinctive V gene use compared with that of HC. The characteristics of ST seemed to be in between those of AG and HC although the difference is not significant. Cluster analysis showed that the TRB repertoire could not effectively distinguish AG from ST. This research might give to a better understanding of the immune process of liver transplantation.     

Acute exercise reverses TRB3 expression in the skeletal muscle and ameliorates whole body insulin sensitivity in diabetic mice

Aim: TRB3 became of major interest in diabetes research when it was shown to interact with and inhibit the activity of Akt. Conversely, physical exercise has been linked to improved glucose homeostasis. Thus, the current study was designed to investigate the effects of acute exercise on TRB3 expression and whole body insulin sensitivity in obese diabetic mice. Methods: Male leptin-deficient (ob/ob) mice swam for two 3-h-long bouts, separated by a 45-min rest period. After the second bout of exercise, food was withdrawn 6 h before antibody analysis. Eight hours after the exercise protocol, the mice were submitted to an insulin tolerance test (ITT). Gastrocnemius muscle samples were evaluated for insulin receptor (IR) and IRS-1 tyrosine phosphorylation, Akt serine phosphorylation, TRB3/Akt association and membrane GLUT4 expression. Results: Western blot analysis showed that TRB3 expression was reduced in the gastrocnemius of leptin-deficient (ob/ob) mice submitted to exercise when compared with respective ob/ob mice at rest. In parallel, there was an increase in the insulin-signalling pathway in skeletal muscle from leptin-deficient mice after exercise. Furthermore, the GLUT4 membrane expression was increased in the muscle after the exercise protocol. Finally, a single session of exercise improved the glucose disappearance (K_ITT) rate in ob/ob mice. Conclusion: Our results demonstrate that acute exercise reverses TRB3 expression and insulin signalling restoration in muscle. Thus, these results provide new insights into the mechanism by which physical activity ameliorates whole body insulin sensitivity in type 2 diabetes.     

肝细胞癌组织中PEG10、SIAH2的表达及其意义

目的探讨遗传印记基因PEG10(paternally expressed gene 10,PEG10)与泛素连接酶SIAH2(seven in absentia homologs 2,SIAH2)在原发性肝细胞癌(hepatocellular carcinoma,HCC)组织中的表达及其临床意义。方法采用免疫组化SP法检测50例HCC和相应癌旁组织中PEG10、SIAH2蛋白的表达。结果 (1)PEG10和SIAH2在肝癌组织中的阳性率分别为84%和78%,高于相应癌旁组织中的阳性率8%和14%,差异有统计学意义(P<0.01)。(2)PEG10和SIAH2表达与肿瘤TNM分期有相关性(P<0.01),但与患者的年龄、性别、肿瘤大小、血清HbsAg、AFP水平、是否伴肝硬化无相关性(P>0.05)。(3)HCC中PEG10、SIAH2蛋白表达呈正相关(r=0.42,P<0.01)。结论 PEG10、SIAH2表达与HCC的发生、发展具有明显相关性。     

Inhibition of hepatic glycogen synthesis by hyperhomocysteinemia mediated by TRB3

Recently, epidemiological and experimental studies have linked hyperhomocysteinemia (HHcy) to insulin resistance. However, whether HHcy impairs glucose homeostasis by affecting glycogenesis in the liver is not clear. In the present study, we investigated the effect of HHcy on hepatic glycogen synthesis. Hyperhomocysteinemia was induced in mice by drinking water containing two percent methionine. Mice with HHcy showed an increase in the phosphorylation of glycogen synthase and a significant decrease in hepatic glycogen content and the rate of glycogen synthesis. The expression of TRB3 (tribbles-related protein 3) was up-regulated in the liver of mice with HHcy, concomitantly with the dephosphorylation of glycogen synthase kinase-3β and Akt. The knockdown of TRB3 by short hairpin RNA suppressed the dephosphorylation of these two kinases. Homocysteine induced an increase in the levels of hepatic cAMP and cAMP response element-binding protein phosphorylation, which in turn up-regulated the expression of peroxisome proliferator-activated receptor (PPAR)-γ coactivator-1α and TRB3. The inhibition of PPAR-α by its inhibitor, MK886, or knockdown of PPAR-α by small interfering RNA significantly inhibited the expression of TRB3 induced by homocysteine. The current study demonstrates that HHcy impairs hepatic glycogen synthesis by inducing the expression of TRB3. These results provide a novel explanation for the development and progression of insulin resistance in HHcy.     

Sox12 enhances Fbw7-mediated ubiquitination and degradation of GATA3 in Th2 cells

Allergic asthma that is caused by inhalation of house dust mites (HDMs) is mainly mediated by Th2 cells. Recently, the roles of Sox (SRY-related high-mobility-group (HMG)-box) family members in various immune responses have been investigated. However, the roles of Sox12, a member of the SoxC group, in Th2 cell differentiation and allergic airway inflammation, remain unknown. We showed that Sox12 mRNA was significantly increased during Th2 cell differentiation. In vivo, HDM-induced eosinophil infiltration into the lung and Th2 cell differentiation were exacerbated in Sox12^−/− mice compared with those in control Sox12^+/− mice. In vitro, Sox12^−/− CD4⁺ T cells that were cultured under Th2 conditions had increased production of Th2 cytokines and GATA3 protein compared with those of control Sox12^+/− CD4⁺ T cells. Importantly, forced expression of Sox12 decreased the protein levels of GATA3 in CD4⁺ T cells under Th2 conditions without affecting mRNA expression. Furthermore, Sox12 induced degradation of GATA3 through the proteasome pathway in CD4⁺ T cells. Consistently, Sox12 enhanced ubiquitination of GATA3, which was mediated by the E3 ligase Fbw7. Finally, we found that Fbw7 knockdown partly abrogated Sox12-mediated GATA3 suppression in CD4⁺ T cells. Taken together, these results suggest that Sox12 suppresses Th2 cell differentiation by accelerating Fbw7-mediated GATA3 degradation, and attenuates HDM-induced allergic inflammation.     

Reversal of α1-receptor mediated relaxation in intestinal smooth muscle

The α₁-receptor agonist phenylephrine relaxed longitudinal rabbit jejunal muscle contracted in vitro by low concentrations of barium ions (1 mM). When the Ba²⁺ concentration was increased to 10–15 mM the response to phenylephrine was a contraction, and at Ba²⁺ concentrations in between the high and low range this response was biphasic—a relaxation followed by a contractile phase. The α₂-receptor agonist clonidine did not affect the tone of the Ba²⁺ contracted preparation. When the muscle preparation was contracted by Sr²⁺ (1–20 mM) in the presence of Ca²⁺ (2.5 mM), phenylephrine relaxed it, and no contractile response to phenylephrine was observed. In the absence of extracellular Ca²⁺, 5 mM Ba²⁺ caused a contraction. Under these conditions phenylephrine had no effect on the tissue tone. When Ca²⁺ was added in a low concentration (0.2-2 mM), phenylephrine elicited a gradually increasing contractile response. At 5 mM Ca²⁺ the contractile response was replaced by the normal relaxation. The contractile response to phenylephrine in the presence of 5 mM Ba²⁺ and 2.5 mM Ca²⁺ was partially blocked by low concentrations of verapamil. In higher concentrations verapamil abolished the tissue tonus completely. The contractile response to phenylephrine in the presence of 5 mM Ba²⁺ and 2.5 mM Ca²⁺ could be reverted to the normal relaxation by the addition of 20 mM Mg²⁺. Increasing the K⁺ concentration from the normal 5.9 to 62.9 mM blocked the phenylephrine-induced relaxation. No contractile response to phenylephrine occurred. It is concluded that Ba²⁺ could reverse the response of α₁ receptor stimulation in rabbit jejunum from a relaxation to a contraction and that this contractile response was dependent on the presence of Ca²⁺.