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

描述(申请人提供)：PCM，L-异天冬氨酸蛋白羧甲基转移酶，是一种蛋白质修复酶，用于修复由天冬氨酸或天冬酰胺自发形成异天冬氨酸(IsoAsp)造成的损伤。PCM有助于多种生物的长寿，而isAsp修复缺陷与重要的病理疾病有关，包括自身免疫、阿尔茨海默病、癫痫、脊柱裂和癌症。尽管PCM在体外修复蛋白质和恢复活性是显而易见的，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、在哪里以及何时采取行动来保护固定相大肠杆菌(衰老的模型系统)免受isAsp损害的有害影响。将解决两个具体问题：目标1：PCM如何提高老化大肠杆菌的存活率？IsAsp损伤与氧化或其他应激协同作用破坏蛋白质构象的假设将通过以下几个方面得到验证：(I)确定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学方法；(Ii)体内蛋白质聚集和氧化的测量；以及(Iii)用于探测潜在PCM底物构象的体内蛋白质折叠报告。目标2：PCM在何时何地发挥作用？PCM主要在营养恢复后具有优势的假设允许新陈代谢活动的恢复，并将通过(I)测量老化和恢复细胞、活的和不活的亚群和不同的细胞隔室中的isAsp损伤、PCM活性和相关参数来检验瞬时活跃的亚群中的PCM；以及(Ii)使用毒素-抗毒素系统来分离衰老和恢复，以在不耗尽营养的情况下诱导停滞。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：异天冬氨酸(IsoAsp)异常引起的蛋白质损伤可通过L-异天冬氨酸蛋白羧甲基转移酶(PCM)修复。最近的研究表明，未修复的isAsp与寿命降低、自身免疫力(如狼疮和糖尿病)、癫痫发作和脑肿瘤的进展有关。这项拟议的研究将有助于理解PCM保护细胞免受isAsp损伤的机制，为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提供所需的基础知识。

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.