权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

METHYLATION OF ATYPICAL PROTEIN ASPARTYL RESIDUES

非典型蛋白天冬酰残基的甲基化

基本信息

批准号：
2732502
负责人：
CLARE M O'CONNOR
金额：
$ 26.63万
依托单位：
BOSTON COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
1985
资助国家：
美国
起止时间：
1985-08-30 至 2002-06-30
项目状态：
已结题

项目摘要

The mammalian protein isoaspartyl methyltransferase (PIMT) specifically modifies abnormal protein aspartyl residues that have arisen spontaneously in aging proteins. The experiments of this project test the hypothesis that mammalian PIMT plays a role in repairing or degrading damaged cellular proteins that this function is particularly important for terminally differentiated cells and for cells approaching senescence. In situ hybridization will be used to identify cellular patterns of PIMT gene expression in brain and testis, tissues with large numbers of postmitotic cells and high levels of PIMT activity. The experiments will identify stages of sperm differentiation characterized by increased PIMT expression. The studies with brain tissue will identify groups of neurons with elevated PIMT levels. Similar studies will be done with brain tissue from aging animals to test whether changes in PIMT expression accompany the loss of neuronal function during aging. PIMT levels will be experimentally manipulated in cultured cell models to determine the consequences of PIMT depletion and overexpression on cellular physiology. Permanent cell lines that either overexpress or are deficient in PIMT will be constructed by transfection with high level expression plasmids containing the PIMT gene sequence in either sense or antisense orientations. Experiments will measure the effects of PIMT dosage on cellular protein metabolism, ability to survive nutritional stress, tendency to enter senescence and differentiation to a neuronal phenotype. A biochemical model will be used to identify roles for the PIMT in protein repair or degradation. Radiolabeled calmodulins will be converted to isomerized forms in an artificial aging protocol. These substrates will be injected into Xenopus oocytes and the stability of the protein will be measured in the absence and presence of methylation inhibitors to determine if carboxyl methylation decreases or increases protein stability. Peptide mapping will be used to monitor any repair reactions. By allowing dysfunctional proteins to accumulate in cells, PIMT malfunction could contribute to infertility and neurodegenerative disorders associated with aging. For example, L-isoaspartyl residues have been identified at several locations in beta-amyloid peptides from Alzheimer-diseased brains, raising the possibility that deficiencies in methylation contribute to the aberrant processing reactions in that disease. The elucidation of the methylation-mediated pathway and the identification of effectors that increase its efficiency could have potential value in treating age-related disorders which involve defects in protein metabolism.

哺乳动物蛋白异天冬氨酰甲基转移酶 (PIMT) 修饰异常蛋白质老化蛋白质中自发产生的天冬氨酰残基。这该项目的实验检验了哺乳动物 PIMT 的假设在修复或降解受损的细胞蛋白质方面发挥作用该功能对于终末分化尤为重要细胞和接近衰老的细胞。原位杂交将用于识别 PIMT 的细胞模式大脑和睾丸等含有大量基因的组织中的基因表达有丝分裂后细胞和高水平的 PIMT 活性。实验将识别以 PIMT 增加为特征的精子分化阶段表达。对脑组织的研究将确定以下群体： PIMT 水平升高的神经元。类似的研究将与衰老动物的脑组织测试 PIMT 是否发生变化衰老过程中，表达伴随着神经元功能的丧失。 PIMT 水平将在培养细胞模型中进行实验操作确定 PIMT 耗尽和过度表达的后果细胞生理学。过度表达或过度表达的永久细胞系 PIMT缺陷将通过高水平转染构建表达质粒含有有义或反义方向的 PIMT 基因序列。实验将测量 PIMT 剂量对细胞蛋白的影响新陈代谢、营养应激生存能力、进入的倾向衰老和分化为神经元表型。将使用生化模型来确定 PIMT 在蛋白质修复或降解。放射性标记的钙调蛋白将在人工老化方案中转化为异构化形式。这些底物将被注射到非洲爪蟾卵母细胞中，并且稳定性将在甲基化不存在和存在的情况下测量蛋白质抑制剂以确定羧甲基化是否减少或增加蛋白质稳定性。肽图谱将用于监测任何修复反应。通过允许功能失调的蛋白质在细胞中积累，PIMT 功能障碍可能导致不孕和神经退行性疾病与衰老相关的疾病。例如，L-异天冬氨酰残基已在 β-淀粉样肽的多个位置被鉴定出患有阿尔茨海默病的大脑，增加了大脑缺陷的可能性甲基化导致异常加工反应疾病。甲基化介导途径的阐明和识别提高其效率的效应器可能治疗涉及缺陷的年龄相关疾病的潜在价值在蛋白质代谢中。