Naturally occurring site-directed mutagenesis in free radical theory of aging

描述(由申请人提供)：活性氧物种(ROS)在衰老过程中起着重要作用。ROS对DNA、蛋白质、脂质和小分子的氧化抑制了这些生物成分的作用。在老化过程中，ROS对蛋白质的氧化很大程度上归因于蛋白质的失活和降解。甲基化是蛋白质氧化的一种方式，在衰老过程中起着重要作用。它发生在铁催化的过氧化氢(H_2O_2)依赖的氨基酸侧链氧化反应中。蛋白质的羰化作用被认为使蛋白质的功能失活，并将受损的蛋白质标记为蛋白酶体依赖的降解。虽然人们认为碳化的蛋白质不会发生电子还原，但我的实验室发现了蛋白质去羰化的机制，在这种机理中，可以通过电子还原来消除羰基。此外，我们的初步实验证实谷氧还蛋白-1(Grx1)在蛋白质中起催化作用 去甲基化。易受铁催化氧化的主要氨基酸残基包括脯氨酸和精氨酸，这两种氨基酸都被氧化成含有羰基的谷氨酰半醛。脯氨酸残基被氧化成5-羟基脯氨酸，再被氧化成谷氨酰半醛。虽然5-羟基脯氨酸氧化为谷氨酰半醛的过程是可逆的，但是否发生了5-羟基脯氨酸的氧化还不清楚。然而，根据我们最近对蛋白质去甲基化的研究结果，我推测，通过Grx1等酶的催化，将Pro残基氧化为谷氨酰半醛的反应是完全可逆的。因此，我还假设，从精氨酸产生的谷氨酰半醛也可以转化为脯氨酸。此外，谷氨酰半醛可以被氧化成谷氨酸。这提出了一个革命性的概念，即铁催化的氧化可以在蛋白质结构中将精氨酸转化为脯氨酸，精氨酸转化为谷氨酸，或将精氨酸转化为谷氨酸，从而导致自然发生的定点突变。我推测，这些修饰会导致蛋白质功能的改变，从而导致衰老。R03项目的目的是为蛋白质结构中精氨酸-脯氨酸、精氨酸-谷氨酸和脯氨酸-谷氨酸转化的发生提供证据，这些转化是新的自然发生的定点突变过程。该申请的目标将通过追求三个具体目标来实现：1)确定蛋白质结构中5-羟基脯氨酸还原为脯氨酸的过程；2)鉴定与老化过程中精氨酸-脯氨酸转化一致的蛋白质修饰；以及3)鉴定老化过程中与精氨酸-谷氨酸或Pro-谷氨酸转化一致的蛋白质修饰。这项拟议的工作具有很高的创新性，因为它将首次解决涉及自然发生的定点突变的生物机制，并提供一种新的ROS作用机制。结果将是意义重大的，因为它们有望提供一种新的衰老机制，并帮助开发延缓人类衰老过程的策略。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) play an important role in aging. Oxidation of DNA, proteins, lipids, and small molecules by ROS inhibit the actions of these biological components. Protein oxidation by ROS during the aging process is largely attributed to the inactivation and the degradation of proteins. Carbonylation is one mode of protein oxidation that is important in the aging process. It occurs in response to iron-catalyzed, hydrogen peroxide (H2O2)-dependent oxidation of amino acid side chains. Protein carbonylation has been thought to inactivate protein functions and to mark the damaged proteins for proteasome-dependent degradation. While carbonylated proteins are believed not to undergo the electron reduction, my laboratory discovered the protein de-carbonylation mechanism, in which carbonyl groups can be eliminated through the electron reduction. Further, our preliminary experiments identified that glutaredoxin-1 (Grx1) plays a catalytic role in protein de-carbonylation. Major amino acid residues that are susceptible to iron-catalyzed oxidation include proline and arginine, both of which get oxidized to become glutamyl semialdehyde that contains a carbonyl group. Proline residues are oxidized to 5-hydroxyproline that is further oxidized to glutamyl semialdehyde. While the oxidation of 5-hydroxyproline to glutamyl semialdehyde is readily reversible, whether the oxidation of proline to 5-hydroxyproline occurs is unclear. However, based on our recent results on protein de-carbonylation, I hypothesize that the reaction for the oxidation of proline residues to glutamyl semialdehyde is fully reversible through the catalysis by reducing enzymes such as Grx1. Consequently, I also hypothesize that glutamyl semialdehyde that is produced from arginine can also be converted to proline. Further, glutamyl semialdehyde can be oxidized to glutamic acid. This suggests a revolutionizing concept that iron-catalyzed oxidation can convert arginine to proline, arginine to glutamic acid, or proline to glutamic acid within the protein structure, resulting in the occurrence of naturally occurring site-directed mutagenesis. I hypothesize that these modifications result in altered protein functions and contribute to aging. The objective of this R03 project is to provide evidence for the occurrence of arginine-proline, arginine-glutamic acid, and proline-glutamic acid conversions within the protein structure, as novel naturally occurring site-directed mutagenesis processes. The objective of the application will be accomplished by pursuing three specific aims: 1) Define the reduction of 5- hydroxyproline to proline within the protein structure 2) Identify the protein modification that is consistent with the arginine-proline conversion in aging; and 3) Identify protein modifications that are consistent with arginine-glutamic acid or proline-glutamic acid conversion in aging. The proposed work is highly innovative, as it will address for the first time a biologic mechanism that involves naturally occurring site-directed mutagenesis and provide a novel mechanism of ROS actions. Results will be significant because they are expected to provide a new mechanism of aging and help developing strategies to delay the aging process in humans.