FORMATION OF ISOASPARTATE IN PEPTIDES AND PROTEINS

最近的研究结果表明，形成异天冬氨酸（isoAsp），通过 不稳定Asn-X序列的脱酰胺化或在某些条件下的直接异构化 Asp-X序列是自发性蛋白质损伤的主要来源， 生理pH值和温度，这一过程可能具有重要的 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形成 异天冬氨酸，并探讨异构化对蛋白质的影响 功能 提出了四种实验方法。 一是 确定突触蛋白1、组织纤溶酶原 激活剂，磷酸丙糖异构酶和主要的内源性蛋白， 眼睛的透镜。 所有这四种蛋白质似乎都能产生isoAsp， 在生理pH和温度下显著速率。 二是 合成了一系列同源含天冬酰胺和天冬氨酸五肽， 其中与Asn或Asp相邻的氨基酸以 系统的方式。 替代品将被设计为填补几个 我们对邻近氨基酸如何影响 天冬酰胺或天冬氨酸异构化的倾向。 对网站的了解 上述蛋白质和合成肽中的isoAsp形成将是 用来加强或反驳我们目前的范式，它强调 与Asn/Asp-羧基连接的氨基酸的性质，以及 区域灵活性，以预测isoAsp形成的位点。 我们的第三 目的是确定isoAsp在多大程度上影响几种功能性 突触蛋白1的属性，包括它被磷酸化的能力， cAMP和II型钙/钙调蛋白依赖性蛋白激酶，其 结合小突触囊泡的能力，以及结合 肌动蛋白 最后，我们将研究蛋白质 易于形成异天冬氨酸的序列也可能易于 交联 这些研究将提供重要的新信息， 控制蛋白质稳定性和完整性的基本机制， 体外和体内。

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.