Linked Protein Repair, Proteolysis, and Oxidation in Aging

描述（由申请人提供）：衰老过程中人体功能丧失的一个重要部分可能是由于受损蛋白质的积累。负责人体大部分催化和结构操作的蛋白质会随着时间的推移而自发分解。随着生物体年龄的增长，蛋白质会积累足够的化学损伤，使其失去活性，甚至有毒。衰老生物体的成功可能取决于它们首先识别哪些蛋白质受损，然后修复或清除这些蛋白质的能力。在这个提议中，我们想要了解生物体如何整合蛋白质修复和蛋白质水解途径来阻止受损蛋白质的积累。我们特别感兴趣的是，如何通过l-异天冬氨酸-（d -天冬氨酸）蛋白O-甲基转移酶和特定蛋白水解降解反应启动的分子修复组合，将一种主要类型的自发损伤，即蛋白质天冬氨酸和天冬酰胺残基的异构化和外消旋化最小化。我们建议使用小鼠、酵母（酿酒酵母）和线虫（秀丽隐杆线虫）作为模型系统。这些系统中的每一个都有优势，可以帮助我们破译可能也适用于人类的途径。我们将首先研究小鼠中蛋白质修复和蛋白质水解途径之间的联系。我们将重点关注缺乏蛋白质修复甲基转移酶的动物中使用的途径。我们之前已经确定，修复缺陷小鼠体内受损天冬氨酸残留的积累在大约60天后趋于稳定。与此同时，小鼠尿液中受损肽的水平增加，表明去除未修复蛋白质的蛋白水解系统被激活。我们建议表征这一后备系统，并找出其在正常老化过程中的作用。然后，我们将研究在缺乏蛋白质修复甲基转移酶的酿酒酵母中含有受损天冬氨酸残基的蛋白质的代谢。我们已经证明，含有受损天冬氨酸残留物的蛋白质不会在酵母中积累，尽管它们的形成速度似乎与其他生物体相同。因此，我们提出酵母具有特定的蛋白质水解系统来防止这些改变的蛋白质的积累，并将通过生物化学和遗传方法的结合来表征它们。最后，我们将比较小鼠和衰老蠕虫的修复/蛋白水解反应。我们实验室以前的工作表明，在线虫C.秀丽隐杆线虫中，蛋白质水解可能与蛋白质修复耦合。我们将描述缺乏l -异天冬氨酸甲基转移酶的蠕虫，重点关注蠕虫的两个幼虫阶段，这两个阶段专门用于生存，并且在缺乏修复酶的情况下似乎受到的影响最大。蠕虫和小鼠在修复、信号传导和蛋白水解系统方面的相似性表明，我们在这里学到的东西对人类健康很重要。7. 我们想要了解人类细胞如何进行有助于健康衰老的分子修复和替换过程，以及这些途径中的缺陷如何导致疾病。人体功能所必需的蛋白质分子在自发的化学过程中不断被降解。除非受损分子得到修复或替换，否则它们的积累会减缓或停止正常的生理功能。

DESCRIPTION (provided by applicant): A significant part of the loss of human function in aging may be due to the build-up of damaged proteins. Proteins, responsible for most of the catalytic and structural operations of the body, can spontaneously break down with time. As organisms age, proteins can accumulate enough chemical damage to become inactivated, or even toxic. The success of aging organisms may depend upon their ability to first recognize which proteins are damaged, and then to either repair or remove these species. In this proposal, we want to understand how organisms integrate protein repair and proteolytic pathways to stem the accumulation of damaged proteins. We are particularly interested in how a major type of spontaneous damage, the isomerization and racemization of protein aspartyl and asparaginyl residues, is minimized by a combination of molecular repair initiated by the L-isoaspartyl-(D-aspartyl) protein O- methyltransferase enzyme and specific proteolytic degradation reactions. We propose to use mice, yeast (Saccharomyces cerevisiae), and nematode worms (Caenorhabditis elegans) as model systems. Each of these systems has advantages to aid us in deciphering the pathways that may also be used in humans. We will first examine the links between protein repair and proteolysis pathways in mice. We will focus on pathways used in animals lacking the protein repair methyltransferase. We have previously established that the accumulation of damaged aspartyl residues in repair deficient mice levels off after about 60 days of age. At the same time, the levels of damaged peptides in the urine of the mice increases, suggesting that a proteolytic system to remove the unrepaired proteins is activated. We propose to characterize this back-up system and to find its role in the normal aging process. We will then examine the metabolism of proteins containing damaged aspartyl residues in the yeast S. cerevisiae that lacks the protein repair methyltransferase. We have shown that proteins containing damaged aspartyl residues do not accumulate in yeast, although they appear to be formed at the same rate as in other organisms. We thus propose that yeast have specific proteolytic systems to prevent the accumulation of these altered proteins and will characterize them by a combination of biochemical and genetic approaches. Finally, we will compare the repair/proteolysis responses of mice to those that occur in aging worms. Previous work in our laboratory has suggested that proteolysis may be coupled to protein repair in the nematode C. elegans. We will characterize worms deficient in the L-isoaspartyl methyltransferase, focusing on two larval stages of worms that are specialized for survival and that appear to be most affected in the absence of the repair enzyme. Similarities in repair, signaling, and proteolysis systems in worms and mice suggest that what we learn here will be important for human health. 7. PROJECT NARRATIVE We want to understand how human cells can perform molecular repair and replacement processes that contribute to healthy aging and how defects in these pathways lead to disease. Protein molecules essential for body functions are continuously being degraded by spontaneous chemical processes. Unless damaged molecules are repaired or replaced, their accumulation can slow or stop normal physiological functions.