METHYLATION OF ATYPICAL PROTEIN ASPARTYL RESIDUES

哺乳动物蛋白质异戊酰甲基转移酶（PIMT）特异性地 修饰异常蛋白质 老化蛋白质中自发产生的乙酰基残基。的 该项目的实验验证了哺乳动物PIMT的假设， 在修复或降解受损的细胞蛋白质中起作用， 这一功能对于终末分化的 细胞和接近衰老的细胞。 原位杂交将用于鉴定PIMT的细胞模式 脑和睾丸中的基因表达， 有丝分裂后细胞和高水平的PIMT活性。实验将 鉴定以PIMT增加为特征的精子分化阶段 表情对脑组织的研究将识别出 PIMT水平升高的神经元。类似的研究将在 从老龄动物的脑组织，以测试是否PIMT的变化， 表达伴随着衰老过程中神经元功能的丧失。 PIMT水平将在培养的细胞模型中进行实验操作 为了确定PIMT缺失和过度表达的后果， 细胞生理学过表达或不表达的永久性细胞系 PIMT缺陷的细胞将通过高水平转染来构建 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剂量对细胞蛋白质的影响 代谢，营养应激生存能力，进入 衰老和分化成神经元表型。 一个生物化学模型将被用来确定PIMT的作用， 蛋白质修复或降解。放射性标记的钙调素将 在人工老化方案中转化为异构化形式。这些 将底物注射到非洲爪蟾卵母细胞中， 将在甲基化不存在和存在的情况下测量蛋白质 抑制剂，以确定羧基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症和神经退行性疾病 与衰老有关的疾病例如，L-异戊酰基残基 在β-淀粉样肽中的几个位置已经被确定， 老年痴呆症患者的大脑，提高了缺乏的可能性， 甲基化导致异常加工反应， 疾病甲基化介导的途径的阐明和 确定提高其效率的效应物可能 在治疗涉及缺陷的年龄相关疾病中的潜在价值 在蛋白质代谢中。

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.