泛肽系统的组成和功能(一)—系统组成、底物识别与蛋白质泛肽化

泛肽（Ｕｂｉｑｕｉｔｉｎ,简称Ｕｂ）是一个由７６个氨基酸残基组成的非常保守的小蛋白质。泛肽依赖性的蛋白质降解途径（Ｕｂｉｑｕｉｔｉｎ＿ｄｅｐｅｎｄｅｎｔｐｒｏｔｅｏｌｙｔｉｃｐａｔｈｗａｙ）是目前已知的最重要的、有高度选择性的蛋白质降解途径。泛肽系统由Ｕｂ、Ｕｂ活化酶、Ｕｂ结合酶、Ｕｂ＿蛋白质连接酶、Ｕｂ＿Ｃ末端水解酶和２６Ｓ蛋白酶体组成。本文详细地介绍了泛肽系统各个组成部分的种类、结构与功能,蛋白质泛肽化及其降解机制和底物识别模式。     

泛肽与植物逆境响应

泛肽（ubiquitin）是存在于真核生物中的,含76个氨基酸残基的高度保守的小肽。泛肽途径是真核细胞选择性降解蛋白质的一种途径,它需要E1、E2、E3及26S的蛋白降解体来完成。植物在多种逆境下,都有泛肽途径中某些组分的上调或下调表达。     

泛肽、核糖体蛋白及泛肽-核糖体蛋白S27a与肿瘤的关系

泛肽-核糖体蛋白S27a(Ubiquitin-ribosomal protein S27a,UBRS27a)是泛肽和核糖体蛋白的融合蛋白,N端为泛肽,C端由含C2-C2型锌指结构域的高度保守核糖体蛋白S27a构成。在真核细胞中表达时,被酶解成泛肽和核糖体蛋白。该多功能核糖体蛋白在各种活性增殖细胞和瘤组织中高度表达,在多种类型的肿瘤细胞中,该基因的过量表达是一个典型特征。本实验室对该蛋白在家蚕中的作了初步研究,也发现RPS27a在活性增殖细胞中表达量很高。大多数核糖体蛋白的功能还没有完全探明,它们不仅仅在组装成核糖体时起作用,往往还有核糖体外的功能。回顾了最近几年有关该融合蛋白以及与它相关的泛肽途径、核糖体蛋白与肿瘤之间的关系。通过对它们的研究,有可能预示肿瘤的发生和发展,并为肿瘤临床诊断提供依据,为恶性肿瘤的治疗提供靶点。     

泛肽系统的组成和功能(二)——生物学功能及在生物技术中的应用前景

本文讨论了泛肽系统的生物学功能及其在生物技术中的应用前景。介绍了泛肽系统参与调节的多种生理和发育过程     

泛肽系统的组成和功能(二) ——生物学功能及在生物技术中的应用前景

本文讨论了泛肽系统的生物学功能及其在生物技术中的应用前景。介绍了泛肽系统参与调节的多种生理和发育过程。     

泛肽、核糖体蛋白及泛肽-核糖体蛋白S27a与肿瘤的关系

泛肽-核糖体蛋白S27a（Ubiquitin-ribosomal protein S27a,UBRS27a）是泛肽和核糖体蛋白的融合蛋白,N端为泛肽,C端由含C2-C2型锌指结构域的高度保守核糖体蛋白S27a构成。在真核细胞中表达时,被酶解成泛肽和核糖体蛋白。该多功能核糖体蛋白在各种活性增殖细胞和瘤组织中高度表达,在多种类型的肿瘤细胞中,该基因的过量表达是一个典型特征。本实验室对该蛋白在家蚕中的作了初步研究,也发现RPS27a在活性增殖细胞中表达量很高。大多数核糖体蛋白的功能还没有完全探明,它们不仅仅在组装成核糖体时起作用,往往还有核糖体外的功能。回顾了最近几年有关该融合蛋白以及与它相关的泛肽途径、核糖体蛋白与肿瘤之间的关系。通过对它们的研究,有可能预示肿瘤的发生和发展,并为肿瘤临床诊断提供依据,为恶性肿瘤的治疗提供靶点。     

菜豆多聚泛肽基因在重金属胁迫下的表达

差别筛选HgCl2胁迫的菜豆(PhaseolusvulgarisL.)幼苗叶片cDNA库,分离出两个重金属胁迫相应基因PvSR5和PvSR51(Phaseolusvulgarisstress_relatedgene)片段。cDNA和氨基酸序列分析表明PvSR5和PvSR51分别编码一种多聚泛肽。Northernblot分析表明多聚泛肽是组成性表达蛋白,主要在根中表达,叶片和茎中表达较少;Hg、Cd、Cu和Zn等重金属、高温和水杨酸能强烈地刺激其在叶片中的表达,而受伤几乎没有影响。推测多聚泛肽在抵抗重金属胁迫和提高植物的抗逆性方面有重要作用。     

泛肽交联酶（E2）通过苯丙氨酸解氨酶基因启动子参与水稻对紫外光的防护

迄今为止,诱导苯丙类化合物和黄酮类化合物合成并造成类黄酮的积累被认为是植物抵御紫外光的惟一主要的途径。苯丙氨酸解氨酶(PAL)是苯丙类化合物合成的关键酶。通过体内足印试验表明,在pal-1基因启动子中有一段序列CTCCAACAACCCCCTTC可以作为顺式元件参与紫外信号的传递。为了克隆与其相对应的反式因子,我们使用了酵母单杂交体系进行筛选。在鱼饵质粒中,我们将3个拷贝的顺式元件序列串联并与酵母异细胞血红素C(CYC)基本启动子连接。将鱼饵质粒和水稻cDNA表达质粒库共同转化到同一酵母中,通过筛选鉴定,克隆到一些编码泛肽交联酶(E2)基因。氨基酸同源性分析表明,该酶E2(RE2)与其他物种的泛肽交联酶具有很高的同源性。水稻泛肽交联酶受紫外光诱导加速合成。当RE2与水稻核蛋白进行交联反应后能与pal-1基因中顺式元件CTCCAACAACCCCTTC专一结合。从这些结果可以推测水稻泛肽交联酶RE2可能通过苯丙类化合物的合成代谢参与对紫外光的防护。     

一种新的人泛肽交联酶的cDNA的克隆

ｐ５３可以同细胞内的许多种蛋白质形成复合物从而参与细胞周期调节、基因表达调控、细胞分化、细胞程序化死亡和抑制肿瘤的发生等各项生理过程。为了筛选与ｐ５３作用的蛋白质因子,利用酵母双杂交（ｔｗｏ－ｈｙｂｒｉｄ）系统,筛选了ＨｅＬａ细胞的ｃＤＮＡ文库。在１．５×１０６个转化子中筛选到６个阳性克隆。经ＤＮＡ全序列分析,结果表明５个阳性克隆中的ｃＤＮＡ序列编码产物均由１５８个氨基酸构成,分子量为１７ｋＤ的人泛肽交联酶。同源性比较表明,该泛肽交联酶与人的ＵＢＣ９（９７％）、线虫的ＵＢＣ（７６％）、裂殖酵母的ＨＵＳ５（６６％）、拟南芥菜的ＵＢＣ（６６％）和啤酒酵母的ＵＢＣ９（５６％）有较高的同源性。进行了１６种正常人组织和７种肿瘤细胞株的Ｎｏｒｔｈｅｒｎ杂交分析,结果显示：它在心脏、胎盘、胸腺、睾丸、卵巢、结肠和外周血白细胞中的表达较在肝脏、骨骼肌、肾脏、胰腺、脾脏、前列腺和小肠中为高,而在脑和肺中则检测不到表达。它在宫颈癌细胞株、乳腺癌细胞株、淋巴瘤细胞株和畸胎瘤细胞株中的表达较高,而在肺癌细胞株、肝癌细胞株和神经胶质瘤细胞株中则无明显表达。     

泛肽交联酶(E2)通过苯丙氨酸解氨酶基因启动子参与水稻对紫外光的防护

迄今为止,诱导苯丙类化合物和黄酮类化合物合成并造成类黄酮的积累被认为是植物抵御紫外光的惟一主要的途径.苯丙氨酸解氨酶(PAL)是苯丙类化合物合成的关键酶.通过体内足印试验表明,在pal-1基因启动子中有一段序列CTCCAAC从CCCCTTC可以作为顺式元件参与紫外信号的传递.为了克隆与其相对应的反式因子,我们使用了酵母单杂交体系进行筛选.在鱼饵质粒中,我们将3个拷贝的顺式元件序列串联并与酵母异细胞血红素C(CYC)基本启动子连接.将鱼饵质粒和水稻cDNA表达质粒库共同转化到同一酵母中,通过筛选鉴定,克隆到一些编码泛肽交联酶(E2)基因.氨基酸同源性分析表明,该酶E2(RE2)与其他物种的泛肽交联酶具有很高的同源性.水稻泛肽交联酶受紫外光诱导加速合成.当RE2与水稻核蛋白进行交联反应后能与pal-1基因中顺式元件CTCCAACAACCCCTTC专一结合.从这些结果可以推测水稻泛肽交联酶RE2可能通过苯丙类化合物的合成代谢参与对紫外光的防护.     

Ubiquitin conjugation to endogenous proteins in the dormant tuber of Helianthus tuberosus and during the first cell cycle

Ubiquitin is a small protein involved in an ATP-dependent proteolytic pathway in all eukaryotes. This pathway has been demonstrated to be required for both the bulk degradation of cellular proteins and the targeted proteolysis of specific regulatory proteins. We have investigated the presence of ubiquitin (Ub) and the ubiquitin-conjugating system in dormant and activated tubers of Helianthus tuberosus L. cv. OB 1 that represent a widely used model system for studies on the cell cycle in plants. Immunoblot experiments revealed the presence of free ubiquitin and ubiquitin conjugates. Furthermore, the presence of an active ubiquitin-conjugating system, both time- and ATP-dependent, was demonstrated by incubation with ¹²⁵I-labeled ubiquitin. A few proteins able to form thiol esters with ¹²⁵I-Ub and probably corresponding to ubiquitin-conjugating enzymes, E1 and E2s, were also found. During the first cell cycle, several proteins become ubiquitinated. In particular a large amount of protein conjugates was present at 6 h when the lowest content of free ubiquitin was found. Subsequently, a dramatic decrease in ubiquitin conjugates occurred. It is well known that cell cycle progression in eukaryotes depends on cyclin levels and cyclin B degradation is ubiquitin- and ATP-dependent. By immunoblot experiments we showed that cyclin B in H. tuberosus is present as at least two protein bands of 50 and 54 kDa and that their amounts undergo profound changes during the cell cycle. The 54-kDa band was also recognized by an anti-ubiquitin antibody. These data seem to indicate that in H. tuberosus activated tuber slices, the ATP-dependent ubiquitin proteolytic pathway is involved in the dedifferentiation process occurring after the artificial break of dormancy when the cells acquire the characteristics linked to the meristematic state.     

Ubiquitin: not just for proteasomes anymore

Ubiquitin is a small protein that can be covalently linked to itself or other proteins, either as single ubiquitin molecules or as chains of polyubiquitin. Addition of ubiquitin to a target protein requires a series of enzymatic activities (by ubiquitin-activating, -conjugating and -ligating enzymes). The first function attributed to ubiquitin was the covalent modification of misfolded cytoplasmic proteins, thereby directing proteasome-dependent proteolysis. More recently, additional functions have been ascribed to ubiquitin and ubiquitin-related proteins. Ubiquitin directs specific proteins through the endocytic pathway by modifying cargo proteins, and possibly also components of the cytoplasmic protein trafficking machinery.     

Characterization of a temperature-sensitive mutant of a ubiquitin-conjugating enzyme and its use as a heat-inducible degradation signal.

The ubiquitin/proteasome pathway is a highly conserved mechanism of proteolysis in all eukaryotes. Ubiquitin (Ub) is conjugated to proteolytic substrates through the sequential action of ubiquitin-activating (E1/Uba) and ubiquitin-conjugating (E2/Ubc) enzymes. The mechanism of substrate recognition and ubiquitination is an area of active investigation, and we have begun a site-directed mutagenesis approach to define the biochemical and biophysical properties of ubiquitin-conjugating enzymes. We have characterized a specific mutation in Ubc4 (Ubc4(P62S)) which was previously shown to cause a temperature-sensitive growth defect in several other Ubc's. Ubc4(P62S) was rapidly degraded in vivo, contributing to the loss of function. However, reconstitution experiments revealed that the catalytic activity of Ubc4(P62S) was reversibly inactivated at 37 degrees C, demonstrating that the primary defect of Ubc4(P62S) is its inability to form a ubiquitin thioester bond at high temperature. The in vivo defect is compounded by increased susceptibility of Ubc4(P62S) to degradation by the ubiquitin/proteasome pathway. We have exploited the temperature-dependent degradation of the P62S mutant to destabilize an otherwise stable test protein (glutathione S-transferase). The use of this mutant may provide a useful cis-acting temperature-inducible degradation signal.     

Transfer RNA is required for conjugation of ubiquitin to selective substrates of the ubiquitin- and ATP-dependent proteolytic system

Degradation of intracellular proteins via the ubiquitin- and ATP-dependent proteolytic pathway involves several steps. In the initial event, ubiquitin, an abundant 76-residue polypeptide is covalently linked to the protein substrate in an ATP-requiring reaction. Proteins marked by ubiquitin are selectively proteolyzed in a reaction that also requires ATP. Ubiquitin conjugation to proteins appears also to be involved in regulation of cell cycle and cell division, and probably in the regulation of gene expression at the level of chromatin structure. We have previously shown (Ciechanover, A., Wolin, S. L., Steitz, J. A., and Lodish, H. F. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1341-1345) that transfer RNA is an essential component of the ubiquitin pathway. Ribonucleases strongly and specifically inhibited the degradation of 125I-labeled bovine serum albumin, while tRNA purified from reticulocyte extract could restore the proteolytic activity. Specifically, pure tRNAHis isolated by immunoprecipitation with human autoimmune serum could restore the proteolytic activity. Here we demonstrate that tRNA is required for conjugation of ubiquitin to some but not all proteolytic substrates of the ubiquitin mediated pathway. Conjugation of 125I-labeled ubiquitin to reduced carboxymethylated bovine serum albumin, alpha-lactalbumin, and soybean trypsin inhibitor was strongly and specifically inhibited by ribonucleases. Consequently, the ATP-dependent degradation of these substrates in the cell-free ubiquitin-dependent reticulocyte system was inhibited as well. Addition of tRNA to the ribonuclease inhibited system (following inhibition of the ribonuclease) restored both the conjugation activity and the ubiquitin- and ATP-dependent degradation of these substrates. Conjugation of ubiquitin to some endogenous reticulocyte proteins was also inhibited by ribonucleases and could be restored by the addition of tRNA. In striking contrast, the conjugation of radiolabeled ubiquitin to lysozyme, oxidized RNase A, alpha-casein, and beta-lactoglobulin was not affected by the ribonuclease treatment, and the degradation of these substrates was significantly accelerated by the ribonucleases. These findings indicate that there are at least two distinct ubiquitin conjugation systems. One requires tRNA, and the other is tRNA independent. These pathways, however, must share some common component(s) of the system, since the inhibition of one system accelerates the other. The possible function of tRNA in the selective conjugation reaction and the possible role of the two distinct ubiquitin marking mechanisms are discussed.     

Regulation of the ubiquitin-mediated proteolytic pathway: role of the substrate alpha-NH2 group and of transfer RNA

Degradation of intracellular proteins via the ubiquitin pathway involves several steps. In the initial event, ubiquitin becomes covalently linked to the protein substrate in an ATP-requiring reaction. Following ubiquitin conjugation, the protein moiety of the adduct is selectively degraded with the release of free and reusable ubiquitin. Ubiquitin modification of a variety of protein targets in the cell plays a role in basic cellular functions. Modification of core nucleosomal histones is probably involved in regulation of gene expression at the level of chromatin structure. Ubiquitin attachment to cell surface proteins may play roles in processes of cell-cell interaction and adhesion, and conjugation of ubiquitin to other yet to be identified protein(s) could be involved in the progression of cells through the cell cycle. Despite the considerable progress that has been made in the elucidation of the mode of action and cellular roles of the ubiquitin pathway, many major problems remain unsolved. A problem f central importance is the specificity in the ubiquitin ligation system. Why are certain proteins conjugated and committed for degradation, whereas other proteins are not? A free α-NH₂ group is an important feature of the protein structure recognized by the ubiquitin conjugation system, and tRNA is required for the conjugation of ubiquitin to selective proteo-lytic substrates and for their subsequent degradation. These findings can shed light on some of the features of a substrate that render it susceptile to ubiquitin-mediated degradation.     

NEDD8 overexpression results in neddylation of ubiquitin substrates by the ubiquitin pathway

Ubiquitin and ubiquitin-like proteins use unique E1, E2, and E3 enzymes for conjugation to their substrates. We and others have recently reported that increases in the relative concentration of the ubiquitin-like protein NEDD8 over ubiquitin lead to activation of NEDD8 by the ubiquitin E1 enzyme. We now show that this results in erroneous conjugation of NEDD8 to ubiquitin substrates, such as p53, Caspase 7, and Hif1α, demonstrating that overexpression of NEDD8 is not appropriate for identification of substrates of the NEDD8 pathway.     

Protein quality control in the ER: balancing the ubiquitin checkbook

Protein maturation in the endoplasmic reticulum (ER) is subject to stringent quality control. Terminally misfolded polypeptides are usually ejected into the cytoplasm and targeted for destruction by the proteasome. Ubiquitin conjugation is essential for both extraction and proteolysis. We discuss the role of the ubiquitin conjugation machinery in this pathway and focus on the role of ubiquitin ligase complexes as gatekeepers for membrane passage. We then examine the type of ubiquitin modification applied to the misfolded ER protein and the role of de-ubiquitylating enzymes in the extraction of proteins from the ER.