蟾蜍泛素羧基末端水解酶（tUCH）以不依赖其UCH活性的方式参与卵母细胞成熟调控

本实验室巳报道中华大蟾蜍卵母细胞的p28蛋白具泛素羧基末端水解酶活性,称作tUCH,它和哺乳类中发现的UCH Ll的氨基酸序列具高度同源性,二级结构同源性比较发现,二者可能具类似的功能。本文实验表明：未成熟卵母细胞和成熟卵母细胞的可溶性蛋白中均含有tUCH,约占提取物中蛋白质总量的2％。根据测定所得到的GST—tUCH和GST—UCH Ll对底物Ub—AMC的酶动力学参数,说明卵母细胞中tUCH可能与小鼠UCH L1有类似的生物学功能;anti—tUCH单抗可以与原核细胞表达的tUCH和显性失活突变类型tUCH C(90)S特异结合,但不识别小鼠的UCH Ll。Anti-tUCH单抗能够和tUCH结合但不能封闭它的UCH活性;当anti-tUCH单抗注入卵母细胞内,则孕酮诱导的生发泡破裂(germinal vesicle breakdown,GVBD)过程受到抑制,足见tUCH参与GVBD调节并不依赖其UCH活性。     

水牛BCL2L10基因的克隆、序列分析及其在卵母细胞中的表达

本研究克隆了水牛BCL2-like 10(BCL2L10)基因序列,并用生物信息学技术分析了该序列的同源性、系统进化树、蛋白的理化性质以及高级结构。同时利用免疫荧光染色的方法检测了BCL2L10蛋白在水牛卵母细胞成熟前后的表达情况。结果表明,应用RT-PCR技术克隆得到的水牛BCL2L10基因序列全长600 bp,其中编码区长度为558 bp,共编码185个氨基酸。对该序列进行对比分析发现,水牛BCL2L10基因的核苷酸序列与野牛、牛、绵羊、小鼠、大鼠、野猪的同源性分别为99%、98%、96%、84%、81%、75%。生物进化树分析显示,水牛BCL2L10基因与牦牛、野牛的亲缘关系最近。生物信息学分析得到BCL2L10蛋白的分子量为21.6 k D,理论等电点为9.54。功能结构域预测显示BCL2L10蛋白属于BCL-2家族成员,参与细胞凋亡调控过程。免疫荧光染色结果发现,BCL2L10蛋白在水牛卵母细胞成熟过程中均有表达。     

Spindlin在银鲫卵母细胞中磷酸化修饰分析

Spindlin最早在小鼠中被发现和命名,是MOS/MAP激酶通路的底物。实验室之前在银鲫卵母细胞中克隆并分离鉴定出具有同小鼠相似序列特征和表达特性的Spindlin,命名为CagSpin(Carassius auratus gibelio Spindlin)。CagSpin是一个母源表达的蛋白,存在于卵母细胞生长、减数成熟以及早期胚胎发育过程中。研究结合去磷酸化和Western blot分析,检测到CagSpin在卵母细胞中发生磷酸化。进一步通过毛细管电泳(Capillary Electrophoresis,CE)对蛋白水解后的氨基酸残基进行分析,确定在银鲫卵母细胞中CagSpin蛋白的苏氨酸残基(threonine,Thr)被磷酸化,这一结果为CagSpin在卵母细胞中的磷酸化修饰提供了证据,表明磷酸化的CagSpin在银鲫卵子发生、卵母细胞成熟和卵—胚转换中可能起了重要作用。       

新生小鼠兴奋性氨基酸转运蛋白家族成员cDNA的克隆和分析

兴奋性氨基酸转运蛋白家族包含几种结构相似的膜蛋白,它们在终止谷氨酸的突触传递作用,维持神经系统正常的递质水平起重要作用.为了在同一物种中研究这些蛋白和基因的功能,本工作对新生小鼠脑的兴奋性氨基酸转运蛋白家族成员进行了克隆,获得了3个谷氨酸转运蛋白亚型(mGLAST-1,mGLT-1,mEAAC1)和一个中性氨基酸转运蛋白(mASCT1)的cDNA,其中在小鼠中mASCT1序列为首次发表.序列分析表明,mASCT1cDNA的长度为3787bp,编码一个532个氨基酸箴基的蛋白,和人的ASCT1蛋白序列有89.3%的同源性,用非洲爪蟾卵母细胞表达系统证实了它具有转运丝氨酸的活性.同时,我们的研究表明,兴奋性氨基酸转运蛋白mRNA的5'UTR和3'UTR普遍存在组成和长度的不均一性,这种现象可能提示该家族成员的基因表达具有转录后调控机制.     

小鼠卵母细胞体外成熟不同阶段对钙的依赖性

本研究旨在探讨小鼠卵母细胞成熟与钙和钙调素的关系。研究发现,20μmol/L W7、50μM BAPTA/AM对GVBD发生没有影响,但阻断了中期Ⅰ的卵母细胞进入中期Ⅱ。通过测定成熟不同阶段细胞内钙的分布,发现GVBD后染色体周围区域有较高水平的钙分布,并且该现象能被加BAPTA/AM而消除。GVBD发生后6h左右高钙分布现象消失。我们还测定了成熟过程中MPF活性的变化,20μmol/L W7、50μmol/L BAPTA/AM对卵母细胞成熟过程中MPF活性的升高没有影响。结果表明:小鼠卵母细胞GVBD的发生不依赖钙和钙调素;钙和钙调素对中期Ⅰ的发育是必需的,并且核周区钙分布可能起着重要作用。     

以酵母单杂交体系克隆水稻RAPB基因cDNA及其序列测定

在一些真核基因的 5′上游区中存在核心序列为CCAAT的顺式元件 ,CCAAT结合蛋白以异源多聚体的方式结合于该顺式元件并行使转录调控功能 .CCAAT结合复合物至少存在 3个不同的亚基 ,且结合复合物的单个亚基都不具备DNA结合活性 .首次报道以酵母单杂交体系筛选方法 ,结合酵母功能互补法鉴定 ,从水稻中克隆了定名为RAPB的cDNA ,它编码与酵母CCAAT结合复合物中HAP2亚基具类似功能的蛋白 .RAPB蛋白的C端同样存在与HAP2功能域高度保守的区域 ,但其N端与其他HAP2类似蛋白间无明显的顺序同源性 ,且不存在谷氨酰胺丰富区 .根据Southern杂交结果推测 ,在水稻 (OrizasativaL .)基因组中仅存在一个拷贝的RAPB基因 .     

天花粉蛋白及其与抗体的复合物对蛋白质合成的抑制作用(简报)(英文)

天花粉蛋白在无细胞系统中显示强烈的抑制蛋白质生物合成的能力,其活性与经巯基试剂还原开裂的蓖麻毒蛋白相当。天花粉蛋白对BEL-7402人肝癌细胞的毒性甚低,但当它通过二硫键与抗肝癌单抗Hepama-1偶联后,其毒性增强五十倍。张学军和王家槐不久前发现天花粉蛋白与蓖麻毒蛋白A链的一级结构有很大的相似性~[5]。我们的实验结果与他们的发现互相印证。天花粉蛋白与抗体偶联后,可能通过抗体与细胞表面抗原的结合而内化,从而增强了它的细胞毒性。考虑到天花粉蛋白与其他植物单链致核糖体失活蛋白(RIPs)有不少特性十分相似,我们推断天花粉蛋白是RIP家族中的一员。天花粉蛋白与抗体偶联过程中,双功能试剂SPDP的修饰使天花粉蛋白的活性降低一个数量级,似乎意味着天花粉蛋白上的某些氨基可能对其活性至关紧要。发展新的偶联方法的工作正在进行。如能在双功能试剂修饰后保留其大部分活性,天花粉蛋白作为免疫毒素的“弹头”药物可能颇具潜力。     

褪黑素抑制小鼠卵母细胞的体外成熟

目的:探讨褪黑素(MT)对小鼠卵母细胞的体外成熟的影响.方法:通过卵母细胞自发、次黄嘌呤(HX)阻滞和激素诱导成熟三种体外培养模型研究了褪黑素(MT)对小鼠卵母细胞体外成熟的影响.结果:①0.1 g/L、0.02g/L、0.004 g/L及0.0008 g/L浓度的MT均能显著抑制小鼠卵丘卵母细胞复合体(CEOs)自发成熟过程中第一极体(PB1)的释放(P＜0.01);②动力曲线分析表明,MT对自发成熟的CEOs的GVBD和PB1有显著的推后作用,与对照组相比,处理组的GVBD和PB1分别被推后8～10 h和3～4 h;③0.1 g/L和0.02 g/L两有效浓度的MT还能显著抑制促性腺激素(FSH)诱导的HX阻滞的CEOsGVBD的发生(P＜0.05),对PB1的排出虽有一定的抑制作用,但没有统计学意义;④MT和次黄嘌呤(HX)对CEOs的自发成熟有协同抑制作用(P＜0.01),但在裸卵(DO)自发成熟的阻滞中没有协同效应.结论:MT是调节哺乳动物卵母细胞成熟的重要激素之一,其作用机制可能是通过卵丘细胞实现的.     

光裸星虫Cdc25基因的克隆及在卵母细胞中的表达分析

细胞分裂周期蛋白25(cell division cycle 25,Cdc25)是一种重要的双特异性磷酸酶,对卵母细胞减数分裂进程和胚胎发育具有重要的调控作用.本研究利用RACE技术克隆获得了光裸星虫(Sipunculus nudus)Cdc25(Sn-Cdc25)cDNA 全长.Sn-Cdc25全长为4 130 bp,其中3'UTR 为 1 849 bp,5'UTR 为427 bp,开放阅读框为1 854 bp,编码617个氨基酸.序列分析显示,Sn-Cdc25蛋白分子量为69.58kD,具有M相诱导磷酸酶结构域(M-phase inducer phosphatase domain)和硫氰酸酶同源结构域(rhodanese-like domain)两个典型的Cdc25蛋白结构域,以及能催化去磷酸化过程的活性位点序列HCX5R.多序列比对发现Cdc25同源蛋白的C端同源性较N端的高.三级结构预测表明Cdc25同源蛋白及其活性位点的三级结构构象高度相似.motif分析中共发现5个motifs,其中motif 1和motif 2分别为Paxillin LD基序和MYND结构域结合基序.系统进化树分析显示,无脊椎动物和脊椎动物的Cdc25分别聚为两大支.RT-qPCR结果显示,在不同发育时期卵母细胞中,Sni-Cdc25的表达差异显著,具有两个峰值,从卵黄形成初期至卵黄旺盛合成后期(O1～O3)Sn-Cdc25表达量上升可能与Cdc25促进DNA的复制过程相关;而从体腔液中进入到肾管后(O4～O5),表达量的迅速上升可能有利于成熟促进因子(maturation promoting factor,MPF)的活化.研究结果为深入认识星虫动物卵母细胞发育调控机制和优化人工繁育技术积累基础资料.     

A novel ubiquitin carboxyl terminal hydrolase is involved in toad oocyte maturation

p28, a 28kD protein from toad (Bufo bufo gargarizans) oocytes, was identified by using p13suc1-agaroseaffinity chromatography. Sequence homology analysis of the full-length cDNA of p28 (Gene Bank accessionnumber: AF 314091) indicated that it encodes a protein containing 224 amino-acids with about 55% iden-tities and more than 70% positives to human, rat or mouse UCH-L1, and contains homological functionaldomains of UCH family. Anti-p28 monoclonal antibody, on injecting into the oocytes, could inhibit theprogesterone-induced resumption of meiotic division in a dose-dependent manner. The recombinant proteinp28 showed similar SDS/PAGE behaviors to the native one, and promoted ubiquitin ethyl ester hydrolysis,a classical catalytic reaction for ubiquitin carboxyl terminai hydrolases (UCHs). The results in this paperreveal that a novel protein, p28, exists in the toad oocytes, is a UCH L1 homolog, was engaged in theprocess of progesterone-induced oocyte maturation possibly through an involvement in protein turnover anddegradation.     

A novel ubiquitin carboxyl terminal hydrolase is involved in toad oocyte maturation

p28, a 28kD protein from toad (Bufo bufo gargarizans) oocytes, was identified by using p13sucl-agarose affinity chromatography. Sequence homology analysis of the full-length cDNA of p28 (Gene Bank accession number: AF 314091) indicated that it encodes a protein containing 224 amino-acids with about 55% identities and more than 70% positives to human, rat or mouse UCH-L1, and contains homological functional domains of UCH family. Anti-p28 monoclonal antibody, on injecting into the oocytes, could inhibit the progesterone-induced resumption of meiotic division in a dose-dependent manner. The recombinant protein p28 showed similar SDS/PAGE behaviors to the native one, and promoted ubiquitin ethyl ester hydrolysis, a classical catalytic reaction for ubiquitin carboxyl terminal hydrolases (UCHs). The results in this paper reveal that a novel protein, p28, exists in the toad oocytes, is a UCH L1 homolog, was engaged in the process of progesterone-induced oocyte maturation possibly through an involvement in prot     

A novel ubiquitin carboxyl terminal hydrolase is involved in toad oocyte maturation

p28, a 28kD protein from toad (Bufo bufo gargarizans) oocytes, was identified by using p13(suc1)-agarose affinity chromatography. Sequence homology analysis of the full-length cDNA of p28 (Gene Bank accession number: AF 314091) indicated that it encodes a protein containing 224 amino-acids with about 55% identities and more than 70% positives to human, rat or mouse UCH-L1, and contains homological functional domains of UCH family. Anti-p28 monoclonal antibody, on injecting into the oocytes, could inhibit the progesterone-induced resumption of meiotic division in a dose-dependent manner. The recombinant protein p28 showed similar SDS/PAGE behaviors to the native one, and promoted ubiquitin ethyl ester hydrolysis, a classical catalytic reaction for ubiquitin carboxyl terminal hydrolases (UCHs). The results in this paper reveal that a novel protein, p28, exists in the toad oocytes, is a UCH L1 homolog, was engaged in the process of progesterone-induced oocyte maturation possibly through an involvement in protein turnover and degradation.     

The ubiquitin C‐terminal hydrolase UCH‐L1 promotes bacterial invasion by altering the dynamics of the actin cytoskeleton

Invasion of eukaryotic target cells by pathogenic bacteria requires extensive remodelling of the membrane and actin cytoskeleton. Here we show that the remodelling process is regulated by the ubiquitin C‐terminal hydrolase UCH‐L1 that promotes the invasion of epithelial cells by Listeria monocytogenes and Salmonella enterica. Knockdown of UCH‐L1 reduced the uptake of both bacteria, while expression of the catalytically active enzyme promoted efficient internalization in the UCH‐L1‐negative HeLa cell line. The entry of L. monocytogenes involves binding to the receptor tyrosine kinase Met, which leads to receptor phosphorylation and ubiquitination. UCH‐L1 controls the early membrane‐associated events of this triggering cascade since knockdown was associated with altered phosphorylation of the c‐cbl docking site on Tyr1003, reduced ubiquitination of the receptor and altered activation of downstream ERK1/2‐ and AKT‐dependent signalling in response to the natural ligand Hepatocyte Growth Factor (HGF). The regulation of cytoskeleton dynamics was further confirmed by the induction of actin stress fibres in HeLa expressing the active enzyme but not the catalytic mutant UCH‐L1_C90S. These findings highlight a previously unrecognized involvement of the ubiquitin cycle in bacterial entry. UCH‐L1 is highly expressed in malignant cells that may therefore be particularly susceptible to invasion by bacteria‐based drug delivery systems.     

Mechanistic studies of ubiquitin C-terminal hydrolase L1

Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.     

Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 A resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.     

Ubiquitin editing enzyme UCH L1 and microtubule dynamics: Implication in mitosis

Microtubules are essential components of the cytoskeleton and are involved in many aspects of cell responses including cell division, migration, and intracellular signal transduction. Among other factors, post-translational modifications play a significant role in the regulation of microtubule dynamics. Here, we demonstrate that the ubiquitin-editing enzyme UCH L1, abundant expression of which is normally restricted to brain tissue, is also a part of the microtubule network in a variety of transformed cells. Moreover, during mitosis, endogenous UCH L1 is expressed and tightly associated with the mitotic spindle through all stages of M phase, suggesting that UCH L1 is involved in regulation of microtubule dynamics. Indeed, addition of recombinant UCH L1 to the reaction of tubulin polymerization in vitro had an inhibitory effect on microtubule formation. Unexpectedly, Western blot analysis of tubulin fractions after polymerization revealed the presence of a specific ~50 kDa band of UCH L1 (not the normal ~25 kDa) in association with microtubules, but not with free tubulin. In addition, we show that along with 25 kDa UCH L1, endogenous high molecular weight UCH L1 complexes exist in cells, and that levels of 50 kDa UCH L1 complexes are increasing in cells during mitosis. Finally, we provide evidence that ubiquitination is involved in tubulin polymerization: the presence of ubiquitin during polymerization in vitro by itself inhibited microtubule formation and enhanced the inhibitory effect of added UCH L1. The inhibitory effects of UCH L1 correlate with an increase in ubiquitination of microtubule components. Since besides being a deubiquitinating enzyme, UCH L1 as a dimer has also been shown to exhibit ubiquitin ligase activity, we discuss the possibility that the ~50 kDa UCH L1 observed is a dimer which prevents microtubule formation through ubiquitination of tubulins and/or microtubule-associated proteins.     

Winter Hibernation and UCHL1-p34cdc2 Association in Toad Oocyte Maturation Competence

Currently, it is believed that toad oocyte maturation is dependent on the physiological conditions of winter hibernation. Previous antibody-blocking experiments have demonstrated that toad ubiquitin carboxyl-terminal hydrolase L1 (tUCHL1) is necessary for germinal vesicle breakdown during toad oocyte maturation. In this paper, we first supply evidence that tUCHL1 is highly evolutionarily conserved. Then, we exclude protein availability and ubiquitin carboxyl-terminal hydrolase enzyme activity as factors in the response of oocytes to winter hibernation. In the context of MPF (maturation promoting factor) controlling oocyte maturation and to further understand the role of UCHL1 in oocyte maturation, we performed adsorption and co-immunoprecipitation experiments using toad oocyte protein extracts and determined that tUCHL1 is associated with MPF in toad oocytes. Recombinant tUCHL1 absorbed p34^cdc2, a component of MPF, in obviously larger quantities from mature oocytes than from immature oocytes, and p13^suc1 was isolated from tUCHL1 with a dependence on the ATP regeneration system, suggesting that still other functions may be involved in their association that require phosphorylation. In oocytes from hibernation-interrupted toads, the p34^cdc2 protein level was significantly lower than in oocytes from toads in artificial hibernation, providing an explanation for the different quantities isolated by recombinant tUCHL1 pull-down and, more importantly, identifying a mechanism involved in the toad oocyte’s dependence on a low environmental temperature during winter hibernation. Therefore, in toads, tUCHL1 binds p34^cdc2 and plays a role in oocyte maturation. However, neither tUCHL1 nor cyclin B1 respond to low temperatures to facilitate oocyte maturation competence during winter hibernation.     

Characterization of the testis in congenitally ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) defective (gad) mice.

The gracile axonal dystrophy (gad) mice are known to have a deletion within the gene encoding ubiquitin carboxy-terminal hydrolase-1 (Uch-L1) and show hereditary sensory deterioration and motor paresis. Expression of Uch-L1 is reported to be almost limited to the nervous system and testis. To understand whether Uch-L1, one of the major ubiquitin carboxy-terminal hydrolase (UCH) isozymes in the testis, affects spermatogenesis and other UCH isozymes (Uch-L3, L4 and L5) expression in the testis, we compared the testis between gad, hetero and wild type mice by histological, immunohistochemical analyses and RT-PCR. Histological analysis in 25-week-old gad mice showed shrinking of seminiferous tubules, decreasing total number of cells and enlargement of remaining cells in seminiferous tubules. By immunohistochemistry, a significant decrease (p < 0.05) in the number of proliferating cell nuclear antigen (PCNA) positive cells was observed. Expression of other UCH isozyme mRNAs was not apparently affected by Uch-L1 deficiency in 25-week-old gad mice. This study is the first report on the testis of gad mutant mouse.