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

衰老自由基理论中自然发生的定点突变

• 批准号: 8730824
• 负责人: YUICHIRO Justin SUZUKI
• 金额: $ 7.78万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-15 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8730824
• 关键词: Address; Aging; Aging-Related Process; Amino Acid Sequence; Amino Acids; Animals; Arginine; Biological; Catalysis; Cells; DNA; Electrons; Enzymes; Free Radicals; Glutamic Acid; Grx1 protein; Human; Hydrogen Peroxide; Hydroxyproline; Iron; Knowledge; Laboratories; Lipids; Mediating; Metals; Modification; Peptide Sequence Determination; Play; Post-Translational Protein Processing; Process; Proline; Proteins; Reaction; Reactive Oxygen Species; Role; Side; Site-Directed Mutagenesis; Stress; Testing; Therapeutic Agents; Time; Work; base; biological systems; carbonyl group; design; free radical oxygen; innovation; multicatalytic endopeptidase complex; novel; novel therapeutics; oxidation; protein degradation; protein function; protein structure; public health relevance; research study; response; senescence; small molecule; theories

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.

描述（由申请人提供）：活性氧（ROS）在衰老中起重要作用。ROS对DNA、蛋白质、脂质和小分子的氧化抑制了这些生物组分的作用。衰老过程中ROS对蛋白质的氧化主要是由于蛋白质的失活和降解。羰基化是蛋白质氧化的一种模式，在衰老过程中很重要。它发生在响应铁催化，过氧化氢（H2 O2）依赖性氧化的氨基酸侧链。蛋白质羰基化被认为是破坏蛋白质功能和标记蛋白酶体依赖性降解的受损蛋白质。虽然羰基化的蛋白质被认为不经历电子还原，但我的实验室发现了蛋白质去羰基化机制，其中羰基基团可以通过电子还原消除。此外，我们的初步实验证实，谷氧还蛋白-1（Grx 1）在蛋白质合成中起催化作用， 脱羰基。对铁催化氧化敏感的主要氨基酸残基包括脯氨酸和精氨酸，它们都被氧化成含有羰基的谷氨酰半醛。脯氨酸残基被氧化为5-羟脯氨酸，5-羟脯氨酸被进一步氧化为谷氨酰半醛。虽然5-羟基脯氨酸氧化为谷氨酰半醛是容易可逆的，但脯氨酸氧化为5-羟基脯氨酸是否发生尚不清楚。然而，根据我们最近的研究结果，蛋白质脱羰基，我推测，脯氨酸残基氧化成谷氨酰半醛的反应是完全可逆的，通过还原酶，如Grx 1的催化。因此，我还假设由精氨酸产生的谷氨酰半醛也可以转化为脯氨酸。此外，谷氨酰半醛可被氧化成谷氨酸。这表明了一个革命性的概念，即铁催化氧化可以将精氨酸转化为脯氨酸，精氨酸转化为谷氨酸，或在蛋白质结构内将脯氨酸转化为谷氨酸，从而导致天然发生的定点突变的发生。我假设这些修饰导致蛋白质功能改变并导致衰老。该R 03项目的目的是为蛋白质结构内的精氨酸-脯氨酸、精氨酸-谷氨酸和脯氨酸-谷氨酸转化提供证据，作为新型天然定点突变过程。本申请的目的将通过追求三个具体目标来实现：1）确定蛋白质结构内5-羟基脯氨酸还原为脯氨酸; 2）鉴定与老化中的精氨酸-脯氨酸转化一致的蛋白质修饰;以及3）鉴定与老化中的精氨酸-谷氨酸或脯氨酸-谷氨酸转化一致的蛋白质修饰。拟议的工作是高度创新的，因为它将首次解决涉及自然发生的定点诱变的生物学机制，并提供一种新的ROS作用机制。结果将是重要的，因为它们有望提供一种新的衰老机制，并有助于制定延缓人类衰老过程的策略。