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FORMATION OF ISOASPARTATE IN PEPTIDES AND PROTEINS

肽和蛋白质中异天冬氨酸的形成

基本信息

批准号：
2267594
负责人：
DANA WILLIAM ASWAD
金额：
$ 10.9万
依托单位：
UNIVERSITY OF CALIFORNIA-IRVINE
依托单位国家：
美国
项目类别：
财政年份：
1991
资助国家：
美国
起止时间：
1991-05-01 至 1995-04-30
项目状态：
已结题

项目摘要

Recent findings indicate that formation of isoaspartate (isoAsp) via deamidation of labile Asn-X sequences or direct isomerization at certain Asp-X sequences is a major source of spontaneous protein damage at physiological pH and temperature and that this process may have important consequences for protein function and turnover. This proposal seeks to understand the role of protein sequence and structure on formation of isoaspartate and to explore the effects of isomerization on protein function. Four experimental approaches are proposed. First, we will determine the sites of isoAsp formation in synapsin 1, tissue plasminogen activator, triosephosphate isomerase and the major intrinsic protein of the eye lens. All four of these proteins appear to generate isoAsp at significant rates at physiological pH and temperature. Second, we will synthesize a series homologous Asn- and Asp-containing pentapeptides in which the amino acids neighboring the Asn or Asp are varied in a systematic way. Substitutions will be designed to fill in several critical gaps in our knowledge of how neighboring amino acids influence the propensity of Asn or Asp to isomerize. Knowledge of the sites of isoAsp formation in the above proteins and synthetic peptides will be used to strengthen or refute our current paradigm, which emphasizes the nature of the amino acid linked to the Asn/Asp-carboxyl, together with regional flexibility, to predict sites of isoAsp formation. Our third aim is to determine to what extent isoAsp effects several functional attributes of synapsin 1, including its ability to be phosphorylated by the cAMP- and type II calcium/calmodulin-dependent protein kinases, its ability to bind to small synaptic vesicles, and its ability to bind F-actin. Finally, we will investigate the possibility that protein sequences which are prone to forming isoaspartate may also be prone to crosslinking. These studies should provide important new information on basic mechanisms which govern the stability and integrity of proteins in vitro and in vivo.

最近的发现表明，异天冬氨酸(IsAsp)的形成通过不稳定的ASN-X序列的脱酰胺化或某些情况下的直接异构化 ASP-X序列是自发性蛋白质损伤的主要来源生理pH和温度，这一过程可能有重要的对蛋白质功能和周转的影响。这项建议旨在了解蛋白质序列和结构在蛋白质形成中的作用异门冬氨酸和异构化对蛋白质的影响功能。提出了四种实验方法。首先，我们将确定组织纤溶酶原突触素1中异天冬氨酸的形成部位激活剂，磷酸丙糖异构酶和主要的内在蛋白眼镜片。这四种蛋白质似乎都能产生等天冬氨酸在生理pH和温度下有显著的速率。第二，我们将合成一系列含天冬氨酸和天冬氨酸的同源五肽其中与天冬氨酸或天冬氨酸相邻的氨基酸在系统化的方式。替换将被设计为填充几个我们对邻近氨基酸如何影响的知识存在严重差距天冬氨酸或天冬氨酸的异构化倾向。对遗址的了解在上述蛋白质和合成肽中形成等天冬氨酸用来加强或驳斥我们目前的范式，该范式强调与天冬氨酸/天冬氨酸-羧基相连的氨基酸的性质以及区域灵活性，以预测等天冬氨酸形成的地点。我们的第三个目的是确定isAsp在多大程度上影响几个功能突触素1的特性，包括它被 CAMP和II型钙/钙调素依赖性蛋白激酶，其结合小突触小泡的能力，以及它结合的能力 F-肌动蛋白。最后，我们将研究蛋白质容易形成异天冬氨酸的序列也可能倾向于交联剂。这些研究应该为以下方面提供重要的新信息控制蛋白质稳定性和完整性的基本机制体外和体内。