Dietary restriction, aging, and the proteasome

描述（由申请人提供）：蛋白酶体是一种细胞内蛋白酶，负责大量的蛋白质水解，包括氧化蛋白和大量缺乏氧化修饰的已建立的短寿命蛋白的降解。越来越多的证据表明，蛋白酶体-蛋白水解途径的抑制是正常衰老的一部分，蛋白酶体功能的抑制可能导致蛋白质组中许多与年龄相关的改变，包括与年龄相关的氧化蛋白的增加。然而，由于缺乏严格的生化和蛋白质组学分析，使得这些估计在很大程度上是假设和理论的。饮食限制（DR）是唯一已知的能够持续可靠地延长哺乳动物平均寿命和最长寿命的方法。大量研究表明，DR可以改善与年龄相关的氧化蛋白增加，并抑制多种与年龄相关的蛋白质组改变。我们实验室的数据表明，DR改善了与年龄相关的蛋白酶体功能损伤，蛋白酶体功能的保存可能有助于DR对蛋白质组的有益作用。本提案的重点是利用严格的蛋白质组学和生化分析来验证DR改善与年龄相关的蛋白酶体功能损伤的假设，因为它直接影响蛋白酶体复合物，在正常衰老过程中，蛋白酶体活性的保存有助于DR诱导的蛋白质组（氧化和非氧化蛋白质）的影响。验证这一假说的具体目的如下：(1)确定DR对蛋白酶体亚基表达、蛋白酶体生物发生、蛋白酶体组成和蛋白酶体氧化的影响；(2)确定DR对蛋白酶体个体肽酶活性和蛋白质降解能力的影响；(3)在体外培养的原代中枢神经系统细胞中，确定蛋白酶体功能受损对蛋白质组的细胞类型特异性影响，包括蛋白质氧化；(4)确定在体外蛋白酶体抑制后表现出表达改变或氧化增加的蛋白质，在体内衰老过程中是否表现出类似的变化；(5)确定DR是否改善了蛋白质组的改变，包括体内衰老和体外蛋白酶体抑制过程中观察到的氧化蛋白的增加。

DESCRIPTION (provided by applicant): The proteasome is an intracellular protease that is responsible for a significant amount of proteolysis, including the degradation of oxidized proteins and a large number of established short-lived proteins lacking oxidative modifications. Increasing evidence suggests that inhibition of the proteasome-proteolytic pathway occurs as a part of normal aging, with inhibition of proteasome function likely contributing to numerous age-related alterations in the proteome, including age-related increases in oxidized protein. However, the lack of a rigorous biochemical and proteomic analysis has made such estimations largely hypothetical and theoretical. Dietary restriction (DR) is the only known manipulation to consistently and reliably increase average and maximal lifespan in mammals. Numerous studies have demonstrated that DR can ameliorate age-related increases in oxidized protein, and suppress a variety of age-related alterations to the proteome. Data from our laboratory demonstrates that DR ameliorates age-related impairments in proteasome function, with the preservation of proteasome function possibly contributing to the beneficial effects of DR on the proteome. The focus of this proposal is to utilize rigorous proteomic and biochemical analysis to test the hypothesis that DR ameliorates age-related impairments in proteasome function as the result of its direct effects on the proteasome complex, with the preservation of proteasome activity contributing to DR-induced effects on the proteome (oxidized and non-oxidized proteins) during normal aging. The specific aims for testing this hypothesis are as follows: (1) To determine the effects of DR on proteasome subunit expression, proteasome biogenesis, proteasome composition, and proteasome oxidation; (2) To determine the effects of DR on the individual peptidase activities and protein degrading capabilities of the proteasome; (3) To determine the cell-type specific effects of impaired proteasome function on the proteome, including protein oxidation, in primary CNS cell cultures in vitro; (4) To determine if proteins which exhibit altered expression or increased oxidation following proteasome inhibition in vitro, exhibit similar changes during aging in vivo; (5) To determine if DR ameliorates alterations in the proteome, including increases in oxidized protein, observed during aging in vivo and following proteasome inhibition in vitro.