Repair of Isoaspartyl Damage to Proteins: in vivo Roles in Aging E. coli

描述（由申请人提供）：PCM（L-异天冬酰基蛋白羧甲基转移酶）是一种蛋白质修复酶，可修复由天冬氨酸或天冬酰胺自发形成异天冬氨酸（isoAsp）引起的损伤。PCM有助于多种生物体的长寿，并且isoAsp修复中的缺陷与重要的病理状况有关，包括自身免疫、阿尔茨海默病、癫痫、脊柱裂和癌症。虽然PCM在体外修复蛋白质并恢复活性是很清楚的，但关于该酶在体内的作用的重要问题仍有待回答。这项拟议的研究将由首席研究员和12-15名本科生在未来三年内进行，将解决PCM如何，在何处以及何时起作用，以保护静止期大肠杆菌细菌（老化模型系统）免受isoAsp损伤的有害影响。两个具体的问题将得到解决：目标1：PCM如何提高老龄化E的生存。大肠杆菌？isoAsp损伤与氧化或其他应激协同作用以使蛋白质构象不稳定的假设将通过以下方法进行测试：（i）鉴定PCM介导的蛋白质修复和其他维持系统之间相互作用的遗传方法;（ii）蛋白质聚集和氧化的体内测量;和（iii）用于探测潜在PCM底物构象的体内蛋白质折叠报告基因。目标2：PCM在何时何地发挥作用？PCM主要在营养物恢复后是有利的这一假设允许代谢活性的恢复，并且在瞬时活性亚群中将通过以下方式进行测试：（i）测量老化和恢复细胞、存活和不存活亚群以及不同细胞区室中的isoAsp损伤、PCM活性和相关参数;以及（ii）使用毒素-抗毒素系统将老化与恢复分离以诱导停滞而不消耗营养物。从这项工作中获得的见解将有助于理解并最终改善衰老和各种退行性疾病的影响。公共卫生相关性：由异常的异丙基氨基酸（isoAsp）引起的蛋白质损伤可以通过L-异丙基蛋白羧基甲基转移酶（PCM）修复。最近的研究将未修复的isoAsp与寿命缩短、自身免疫（例如，狼疮和糖尿病）、癫痫发作和脑肿瘤进展。拟议的研究将有助于了解PCM保护细胞免受isoAsp损伤影响的机制，提供更好地理解并最终更好地治疗衰老和退行性疾病的原因和后果所需的基础知识。

DESCRIPTION (provided by applicant): PCM, the L-isoaspartyl protein carboxyl methyltransferase, is a protein-repair enzyme that repairs damage caused by spontaneous formation of isoaspartate (isoAsp) from aspartate or asparagine. PCM contributes to longevity in a variety of organisms, and defects in isoAsp repair have been linked to important pathological conditions, including autoimmunity, Alzheimer'sdisease, epilepsy, spina bifida and cancer. Although it is clear that PCM repairs proteins and restores activity in vitro, important questions about the enzyme's roles in vivo remain to be answered. The proposed research, to be conducted by the principal investigator and 12-15 undergraduate students over the next three years, will address how, where and when PCM acts to protect stationary-phase Escherichia coli bacteria (a model system for aging) from the deleterious effects of isoAsp damage. Two specific questions will be addressed: Aim 1: How does PCM enhance survival of aging E. coli? The hypothesis that isoAsp damage acts synergistically with oxidative or other stresses to destabilize protein conformation will be tested by: (i) a genetic approach to identify interactions between PCM-mediated protein repair and other maintenance systems; (ii) in vivo measurement of protein aggregation and oxidation; and (iii) an in vivo protein folding reporter used to probe conformation of potential PCM substrates. Aim 2: When and where does PCM exert its effect? The hypothesis that PCM is advantageous primarily after restoration of nutrients allows resumption of metabolic activity and in transiently active subpopulations will be tested by (i) measuring isoAsp damage, PCM activity and related parameters in aging and recovering cells, viable and inviable subpopulations and different cellular compartments; and (ii) separating aging from recovery using a toxin-antitoxin system to induce stasis without depleting nutrients. Insights gained from this work will contribute to understanding and eventually ameliorating the effects of aging and a variety of degenerative diseases. PUBLIC HEALTH RELEVANCE: Protein damage resulting from abnormal isoaspartyl amino acids (isoAsp) can be repaired by the L-isoaspartyl protein carboxyl methyltransferase (PCM). Recent research links unrepaired isoAsp with reduced longevity, autoimmunity (e.g., lupus and diabetes), epileptic seizures and progression of brain tumors. The proposed research will contribute to understanding the mechanisms by which PCM protects cells from the effects of isoAsp damage, providing the foundational knowledge needed to better understand and eventually better treat the causes and consequences of aging and degenerative disease.