泛素/26S蛋白酶体途径调节作物种子大小的研究进展

泛素/26S蛋白酶体系统（ubiquitin/26S proteasome system, UPS）是蛋白质泛素化的重要途径,作为蛋白质翻译后重要的修饰系统,在作物生长发育的各个阶段发挥重要作用。大量研究表明,UPS中泛素受体蛋白、E3泛素连接酶、去泛素化酶等能相互协调,通过在目标蛋白上连接或去除不同数量的泛素来介导目标蛋白的降解、改变亚细胞定位、蛋白活性等,从而调控种子的大小。对不同作物中泛素/26S蛋白酶体系统调节种子大小方面的研究进展进行了综述,并对其未来的发展进行了展望,为研究种子大小的调控机制及育种改良提供参考。     

A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein

The ubiquitin-dependent degradation of a test protein beta-galactosidase (beta gal) is preceded by ubiquitination of beta gal. The many (from 1 to more than 20) ubiquitin moieties attached to a molecule of beta gal occur as an ordered chain of branched ubiquitin-ubiquitin conjugates in which the carboxyl-terminal Gly76 of one ubiquitin is jointed to the internal Lys48 of an adjacent ubiquitin. This multiubiquitin chain is linked to one of two specific Lys residues in beta gal. These same Lys residues have been identified by molecular genetic analysis as components of the aminoterminal degradation signal in beta gal. The experiments with ubiquitin mutated at its Lys48 residue indicate that the multiubiquitin chain in a targeted protein is essential for the degradation of the protein.     

栗疫病菌泛素核糖体L40融合蛋白基因的克隆与分析

用酿酒酵母(Saccharyomyces cerevisae)泛素融合蛋白基因从COGEME(Consortium for the functional genomics of microbial eukaryotes)植物病原菌EST(Expressed sequence tag)库中BLAST(Basic local alignment search tool)得到栗疫病菌的泛素融合蛋白EST,设计引物从栗疫病菌cDNA中克隆到该基因并测序,得到在进化上高度保守的基因,该基因开放阅读框为387 bp,编码128个氨基酸残基组成的泛素融合蛋白前体;由cDNA序列推定的氨基酸序列分析结果表明,泛素融合蛋白前体包括氮末端的泛素结构域(76个氨基酸残基)和碳末端的核糖体蛋白L40结构域(52个氨基酸残基).该蛋白为高碱性蛋白,碳末端含有一个"锌指"模式结构.与19个物种比较的结果表明,栗疫病菌与真菌泛素融合蛋白氨基酸序列相似度较高,具有高度的保守性.     

白羽王鸽泛素(UB)基因的生物信息学分析

[目的]对白羽王鸽泛素基因的cDNA序列进行相关分析。[方法]运用相关生物信息学软件对该基因eDNA序列进行分析和预测,编码蛋白的理化性质及二级结构。[结果]生物信息学分析结果表明,白羽王鸽泛素基因编码区长度为1037bp,编码的多肽由76个氨基酸残基组成,相对分子质量为34．368kD,理论等电点为6．94,二级结构中α-螺旋占24．6％,无规则卷曲占43．3％。B延伸链占32．1％。[结论]通过白羽王鸽泛素基因相关生物学信息分析,为进一步研究泛素基因在白羽王鸽机体内的作用奠定了基础。     

The neuron-specific protein PGP 9.5 is a ubiquitin carboxyl-terminal hydrolase

A complementary DNA (cDNA) for ubiquitin carboxyl-terminal hydrolase isozyme L3 was cloned from human B cells. The cDNA encodes a protein of 230 amino acids with a molecular mass of 26.182 daltons. The human protein is very similar to the bovine homolog, with only three amino acids differing in over 100 residues compared. The amino acid sequence deduced from the cDNA was 54% identical to that of the neuron-specific protein PGP 9.5. Purification of bovine PGP 9.5 confirmed that it is also a ubiquitin carboxyl-terminal hydrolase. These results suggest that a family of such related proteins exists and that their expression is tissue-specific.     

泛素/26S蛋白酶体途径在水稻中的生物学功能研究进展

生物体内的蛋白质降解方式有两种,一种不需要能量,一种需要能量。而泛素/26S蛋白酶体途径便是目前已知的依赖ATP、高效、有高度选择性的蛋白降解途径。它介导了真核生物中80%~85%的蛋白质降解,几乎参与到植物生长发育的各个环节,是植物体内蛋白高效专一降解最重要、最精细的调控机制之一。概述了泛素蛋白酶体途径,重点阐述了泛素结合酶E2和泛素连接酶E3的蛋白结构及其在水稻生长发育、激素信号传导、生物和非生物胁迫响应中的生物学功能及机制,并对进一步研究进行了展望,将有助于揭示泛素/26S蛋白酶体途径在水稻生长发育中的精细调控过程,并为水稻抗逆育种提供了指导和借鉴。     

泛素与融合表达

泛素广泛存在于真核生物中,是高度保守的低分子量蛋白.近年来,外源基因与泛素融合以增强表达效率的研究,成为解决生物反应器目标产物含量低问题的热点之一.本文简述泛素及其结构,综述近年泛素与融合表达的研究进展.     

Recognition dynamics up to microseconds revealed from an RDC-derived ubiquitin ensemble in solution

Protein dynamics are essential for protein function, and yet it has been challenging to access the underlying atomic motions in solution on nanosecond-to-microsecond time scales. We present a structural ensemble of ubiquitin, refined against residual dipolar couplings (RDCs), comprising solution dynamics up to microseconds. The ensemble covers the complete structural heterogeneity observed in 46 ubiquitin crystal structures, most of which are complexes with other proteins. Conformational selection, rather than induced-fit motion, thus suffices to explain the molecular recognition dynamics of ubiquitin. Marked correlations are seen between the flexibility of the ensemble and contacts formed in ubiquitin complexes. A large part of the solution dynamics is concentrated in one concerted mode, which accounts for most of ubiquitin's molecular recognition heterogeneity and ensures a low entropic complex formation cost.     

Regulation of receptor fate by ubiquitination of activated beta 2-adrenergic receptor and beta-arrestin

Although trafficking and degradation of several membrane proteins are regulated by ubiquitination catalyzed by E3 ubiquitin ligases, there has been little evidence connecting ubiquitination with regulation of mammalian G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR) function. Agonist stimulation of endogenous or transfected beta2-adrenergic receptors (beta2ARs) led to rapid ubiquitination of both the receptors and the receptor regulatory protein, beta-arrestin. Moreover, proteasome inhibitors reduced receptor internalization and degradation, thus implicating a role for the ubiquitination machinery in the trafficking of the beta2AR. Receptor ubiquitination required beta-arrestin, which bound to the E3 ubiquitin ligase Mdm2. Abrogation of beta-arrestin ubiquitination, either by expression in Mdm2-null cells or by dominant-negative forms of Mdm2 lacking E3 ligase activity, inhibited receptor internalization with marginal effects on receptor degradation. However, a beta2AR mutant lacking lysine residues, which was not ubiquitinated, was internalized normally but was degraded ineffectively. These findings delineate an adapter role of beta-arrestin in mediating the ubiquitination of the beta2AR and indicate that ubiquitination of the receptor and of beta-arrestin have distinct and obligatory roles in the trafficking and degradation of this prototypic GPCR.     

U-box蛋白质的结构及其功能研究进展

泛素系统是选择性降解细胞内蛋白质的重要系统之一,U-box蛋白质是此系统中决定底物特异性识别的一种新型E3蛋白质,部分U-box蛋白质属于泛素链聚集因子-E4。U-box结构域大约由70个氨基酸残基构成,在酵母、植物和动物等真核生物中保守存在,但植物中的数目远多于动物中。该蛋白质在细胞内异常蛋白质的降解及质量控制方面具有重要的作用,了解U-box蛋白质的功能对疾病的发生控制机理有重要意义。     

RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites

Mutations affecting the BRCT domains of the breast cancer-associated tumor suppressor BRCA1 disrupt the recruitment of this protein to DNA double-strand breaks (DSBs). The molecular structures at DSBs recognized by BRCA1 are presently unknown. We report the interaction of the BRCA1 BRCT domain with RAP80, a ubiquitin-binding protein. RAP80 targets a complex containing the BRCA1-BARD1 (BRCA1-associated ring domain protein 1) E3 ligase and the deubiquitinating enzyme (DUB) BRCC36 to MDC1-gammaH2AX-dependent lysine(6)- and lysine(63)-linked ubiquitin polymers at DSBs. These events are required for cell cycle checkpoint and repair responses to ionizing radiation, implicating ubiquitin chain recognition and turnover in the BRCA1-mediated repair of DSBs.     

Detecting and measuring cotranslational protein degradation in vivo

Nascent polypeptides emerging from the ribosome and not yet folded may at least transiently present degradation signals similar to those recognized by the ubiquitin system in misfolded proteins. The ubiquitin sandwich technique was used to detect and measure cotranslational protein degradation in living cells. More than 50 percent of nascent protein molecules bearing an amino-terminal degradation signal can be degraded cotranslationally, never reaching their mature size before their destruction by processive proteolysis. Thus, the folding of nascent proteins, including abnormal ones, may be in kinetic competition with pathways that target these proteins for degradation cotranslationally.     

铁观音茶树RING型E3泛素连接酶基因克隆及其干旱胁迫下的表达分析

以铁观音茶树为试材,对其中E3泛素连接酶基因进行克隆和生物信息学分析,并采用qPCR进行不同干旱条件下的定量表达分析。研究结果表明,该序列全长为1 138bp,开放阅读框(ORF)为810bp,编码269个氨基酸(GenBank登录号KR819177)。生物信息学分析发现该铁观音茶树E3泛素连接酶基因不含跨膜结构以及信号肽,具有多个磷酸化位点,亚细胞定位于叶绿体中。经BLAST比对,该基因编码的氨基酸序列与烟草、亚麻荠、葡萄、醉蝶花、芜菁中的E3泛素连接酶基因编码的氨基酸序列分别有51%、50%、50%、50%和49%的同源性,且相关保守功能结构域翻译的蛋白质序列具有RING-finger结构,初步确定该基因为铁观音茶树的E3泛素连接酶基因。qPCR分析结果显示在不同干旱胁迫处理下铁观音茶树的E3泛素连接酶基因的表达量,与对照组相比显著增加。本研究认为铁观音茶树RING型E3泛素连接酶基因参与茶树抗旱响应机制。     

A bacterial inhibitor of host programmed cell death defenses is an E3 ubiquitin ligase

The Pseudomonas syringae protein AvrPtoB is translocated into plant cells, where it inhibits immunity-associated programmed cell death (PCD). The structure of a C-terminal domain of AvrPtoB that is essential for anti-PCD activity reveals an unexpected homology to the U-box and RING-finger components of eukaryotic E3 ubiquitin ligases, and we show that AvrPtoB has ubiquitin ligase activity. Mutation of conserved residues involved in the binding of E2 ubiquitin-conjugating enzymes abolishes this activity in vitro, as well as anti-PCD activity in tomato leaves, which dramatically decreases virulence. These results show that Pseudomonas syringae uses a mimic of host E3 ubiquitin ligases to inactivate plant defenses.     

人泛素结合酶UbcH5c活性突变体的构建·纯化以及催化活性研究

[目的]构建人泛素结合酶UbcH5c的活性位点突变体S22R和F62A,同时分析这2种突变体蛋白与野生型蛋白在泛素化反应中的活性差异。[方法]通过点突变(Site-Directed Mutagenesis)的方法构建2种突变体质粒,利用0.5 mmol/L IPTG分别诱导2种突变体蛋白在大肠杆菌中进行表达;将菌体超声破碎后,依次利用阳离子交换层析法对UbcH5c突变蛋白进行分离纯化,最后利用体外泛素化酶促反应结合蛋白免疫印迹杂交的方法分析野生型UbcH5c与其活性突变体UbcH5c S22R和UbcH5c F62A在泛素化修饰上的差异。[结果]人泛素结合酶UbcH5c的2个突变体蛋白S22R、F62A的泛素化修饰程度明显弱于野生型蛋白。[结论]突变体S22R和F62A突变均不同程度的改变了人泛素结合酶UbcH5c与泛素分子之间的结合活性,即2个突变的位点中第62位苯丙氨酸残基及第22位丝氨酸残基均是UbcH5c结合泛素的关键位点,而且后者对于UbcH5c结合泛素活性的影响更大。     

Ubiquitin is a component of paired helical filaments in Alzheimer's disease

Paired helical filaments (PHF), which constitute a distinct type of pathological neuronal fiber, are the principal constituent of neurofibrillary tangles that occur in the brain of patients with Alzheimer's disease. Their insolubility in sodium dodecyl sulfate and urea has prevented the analysis of their subunit composition by gel electrophoresis. A monoclonal antibody (DF2) was isolated that specifically labeled PHF at both the light and electron microscopic levels. It labeled a small polypeptide (5 kilodaltons) that was shown to be ubiquitin in immunoblots of the soluble fraction of brain homogenates. To obtain direct evidence that ubiquitin is a component of PHF, PHF were treated with concentrated formic acid and digested with lysylendopeptidase; ubiquitin-derived peptides were then identified by reversed-phase high-performance liquid chromatography. Two fragments in the PHF digest were identified as derived from ubiquitin by protein sequencing. This procedure should make possible definitive identification of other PHF components.     

Function of WW domains as phosphoserine- or phosphothreonine-binding modules

Protein-interacting modules help determine the specificity of signal transduction events, and protein phosphorylation can modulate the assembly of such modules into specific signaling complexes. Although phosphotyrosine-binding modules have been well-characterized, phosphoserine- or phosphothreonine-binding modules have not been described. WW domains are small protein modules found in various proteins that participate in cell signaling or regulation. WW domains of the essential mitotic prolyl isomerase Pin1 and the ubiquitin ligase Nedd4 bound to phosphoproteins, including physiological substrates of enzymes, in a phosphorylation-dependent manner. The Pin1 WW domain functioned as a phosphoserine- or phosphothreonine-binding module, with properties similar to those of SRC homology 2 domains. Phosphoserine- or phosphothreonine-binding activity was required for Pin1 to interact with its substrates in vitro and to perform its essential function in vivo.     

水稻OsWrky基因的细胞核定位研究

绿色荧光蛋白（GFP）基因是一种被广泛应用的报告基因。为了了解克五笔型的水稻WRKY（OsWrky）基因的核定位性质，将OsWRKY与GFP基因融合，并构建在Ubiquitin启动子控制的pCambia130载体上（pCU-oSwrkyGFP)。利用基因枪介导的方法将pCU-OsWrkyGFP导入洋葱内表皮后，荧光显微镜观察GFP的发光部位仅在细胞核内，而作为对照的没有融合OsWRKY基因的载体（pCU-GFP)轰击后细胞质和细胞核中都有荧光。充分说明OsWRKY能作为一个转录因子在细胞核内起作用。