PIMT1 in Red Blood Cell aging in vivo and in vitro

PIMT1在体内和体外红细胞老化中的作用

• 批准号: 10405591
• 负责人: Angelo D'Alessandro
• 金额: $ 60.58万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10405591
• 关键词: Affect; Affinity; Age; Aging; Aldehyde-Lyases; American; Animal Model; Animals; Anions; Asparagine; Aspartate; Back; Binding; Binding Sites; Biochemical; Biochemical Pathway; Biology; Blood; Blood Banks; Blood Transfusion; Blood donor; Cell Aging; Cell Separation; Cell Survival; Cell membrane; Cell physiology; Cells; Cellular biology; Charge; Chemicals; Childhood; Chlorides; Data; Dehydration; Disease; Dissection; Docking; Energy Metabolism; Enzymes; Erythrocyte Transfusion; Erythrocytes; Esters; Exposure to; Failure; Flow Cytometry; Functional disorder; G6PD gene; Glucose; Glucosephosphate Dehydrogenase Deficiency; Glyceraldehyde-3-Phosphate Dehydrogenases; Glycolysis; Goals; Health; Hemoglobin; Homeostasis; Hospitals; Human; Human body; In Vitro; Individual; Inpatients; Knockout Mice; Logistics; Longevity; Lung; Masks; Mediating; Medical; Membrane Proteins; Metabolic; Metabolic Pathway; Metabolic stress; Metabolism; Methodology; Methylation; Methyltransferase; Modeling; Molecular; Mus; N-terminal; NADP; New York; Organism; Oxidants; Oxidation-Reduction; Oxidative Stress; Oxides; Oxygen; Pathologic; Pathology; Pathway interactions; Pentosephosphate Pathway; Peripheral; Persons; Physiological; Play; Population; Procedures; Protein Biosynthesis; Protein Methylation; Proteins; Proteomics; Reaction; Recycling; Regulation; Role; Sickle Cell; Site; Succinimides; System; Testing; Time; Tissues; Toxicology; Transfusion; Translations; Vertebral column; age related; cell age; cell type; cofactor; deamidation; design; gene product; in vivo; metabolomics; mouse model; mutant; novel; novel therapeutic intervention; oxidant stress; oxidation; oxidative damage; repaired; senescence; sensor; stressor; tool

ABSTRACT A variety of specific chemical damage occurs as a result of normal cellular senescence, as well as accelerated damage in the context of certain pathologies. One such chemical pathway is the degradation of aspartates into isoaspartyl residues through oxidant damage. As a repair mechanisms, PIMT1 is an enzymatic pathway that methylates isoaspartyl residues, creating an isoaspartyl methyl ester that is capable of then spontaneously reverting into aspartate, thus reversing isoaspartyl damage. Insufficient PIMT1 activity has been associated with increased oxidant stress and shorter cellular and organism lifespan in mice; however, a detailed metabolic and biochemical analysis of the role of PIMT1 has not been elucidated. In this application, we propose to study the role of PIMT1 in cellular aging. While multiple tissues will be analyzed to test general effects of PIMT1, this proposal mainly focusses on a specific central hypothesis regarding effects in red blood cells (RBCs). RBCs are essential to health, and dysfunction of RBCs plays a central role in multiple diseases. In addition, transfusion of RBCs is the single most common inpatient invasive therapy, being given to approximately 1 out of every 70 Americans, annually. RBCs that are transfused are stored (as a logistical necessity) for up to 42 days, during which time they undergo specific cellular and biochemical damage. RBCs are known to lose an essential regulatory function through a key gene product (AE1) in normal cellular aging and in RBC storage. However, the molecular mechanism by which AE1 dysfunction occurs has been unknown. In this application we provide novel data demonstrating that isoaspartyl damage occurs in AE1 of both human and murine RBCs in a domain of AE1 that requires aspartates to function. We likewise present data suggesting that failure of PIMT1 pathways accelerates this damage – however whole animal modeling with deletion of PIMT1 is required to test a mechanistic role. In this context, we offer the following specific aims, designed to critically test hypotheses around the role of PIMT1 mediated repair of oxidant damage. Specific Aim 1: Mechanistic elucidation of the role of protein methylation by PIMT1 in the function and senescence of RBCs. Specific Aim 2: the interaction of increased oxidant stress on PIMT1 and its effects on RBCs aging in vivo and ex vivo (blood storage). PIMT1 null mice will be combined with additional strains designed to isolate metabolic pathways of functional relevance (e.g. G6PD deficient). Advanced experimental methodologies will be applied to these animals in order to isolate cells of particular age and physiological conditions. Finally, the controlled biologies generated from these approaches will be analyzed by cutting edge metabolomic and proteomic methodologies. In aggregate, these studies will advance our understanding of the role of specific pathways of biochemical cellular aging, of the mechanistic role of a conserved repair pathway (PIMT1), and in the context of advanced biochemical analysis and modeling to generate novel mechanistic understanding and critical testing of focused hypotheses.

抽象的 由于正常的细胞感染而发生了多种特异性化学损伤，并且加速了 在某些病理的背景下损害。这样的化学途径之一是天冬氨酸的降解 异ast素通过氧化物损伤保留。作为维修机制，PIMT1是一种酶促途径 甲基盐异源自然保留，形成一个能够发起人的异冬酯甲酯 恢复天冬氨酸，从而逆转异源损伤。 PIMT1活性不足与 氧化应激增加，小鼠的细胞和生物体寿命较短；但是，详细的代谢和 PIMT1作用的生化分析尚未阐明。在此应用中，我们建议研究 PIMT1在细胞衰老中的作用。虽然将多次分析以测试PIMT1的一般效应，但 提案主要集中于有关红细胞（RBC）影响的特定中心假设。 RBC是 对健康至关重要，RBC的功能障碍在多种疾病中起着核心作用。另外，输血 RBC是最常见的内科入侵疗法，每70个中约有1个 美国人，每年。在 他们在哪个时候受到特定的细胞和生化损害。众所周知，RBC失去了必不可少的 通过正常细胞衰老和RBC存储中的关键基因产物（AE1）调节功能。但是， AE1功能障碍发生的分子机制尚不清楚。在此应用程序中，我们提供小说 数据表明，异源损伤发生在AE1域中的人类和鼠RBC的AE1中 这需要天冬氨酸才能起作用。我们同样呈现数据表明PIMT1途径失败 加速了这种损害 - 但是，需要删除PIMT1的整个动物建模才能测试A 机械作用。在这种情况下，我们提供以下特定目标，旨在批判性检验假设 围绕PIMT1介导的氧化物损伤修复的作用。特定目标1：机械阐明 PIMT1蛋白甲基化在RBC的功能和感应中的作用。特定目标2：互动 氧化物对PIMT1及其对RBC衰老体内和体内（血液储存）的影响的增加。 PIMT1 NULL小鼠将与旨在隔离功能相关的代谢途径的其他菌株结合 （例如G6PD缺陷）。先进的实验方法将应用于这些动物以隔离 特定年龄和身体状况的细胞。最后，从这些生物产生的受控生物 方法将通过最先进的代谢组和蛋白质组学方法来分析。总体而言，这些 研究将促进我们对生化细胞衰老特定途径的作用的理解， 保守修复途径（PIMT1）的机械作用，并在先进的生化分析的背景下 并建模以产生新的机械理解和对集中假设的批判性测试。