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.

摘要 由于细胞的正常衰老和加速衰老，会发生各种特殊的化学损伤。 在某些病理背景下的损害。一种这样的化学途径是天冬氨酸降解成 异天冬氨酸残留物通过氧化剂破坏。作为一种修复机制，PIMT1是一种酶途径， 甲基化异天冬氨酸残基，生成一种异天冬氨酸甲酯，能够自发地 还原为天冬氨酸，从而逆转异天冬氨酸的损伤。PIMT1活动不足与 小鼠氧化应激增加，细胞和组织寿命缩短；然而，详细的代谢和 PIMT1的生物化学分析尚未阐明其作用。在此应用程序中，我们建议研究 PIMT1在细胞衰老中的作用。虽然将对多个组织进行分析以测试PIMT1的一般效应，但这 建议主要集中在一个关于红细胞(RBC)效应的特定中心假说上。RBC是 红细胞对健康至关重要，而红细胞功能障碍在多种疾病中起核心作用。此外，输注 红细胞压积是住院患者中最常见的一种侵入性治疗，每70人中约有1人接受这种治疗。 美国人，每年。输注的红细胞储存(出于后勤需要)长达42天 在这段时间里，它们会受到特定的细胞和生化损伤。众所周知，红细胞会失去一种必需的 通过一个关键基因产物(AE1)在正常细胞衰老和红细胞储存中发挥调节作用。然而， AE1功能障碍发生的分子机制尚不清楚。在此应用程序中，我们提供了新的 数据表明，人和小鼠红细胞的AE1结构域中都存在异天冬氨酸损伤 这需要天冬氨酸发挥作用。我们同样提供的数据表明，PIMT1通路的故障 加速了这种损害-然而，需要删除PIMT1的整个动物模型来测试 机械角色。在这种背景下，我们提供了以下具体目标，旨在批判性地检验假设 围绕PIMT1介导的氧化损伤修复作用。具体目标1：从机制上阐明 PIMT1蛋白甲基化在红细胞功能和衰老中的作用。具体目标2：互动 PIMT1氧化应激增加及其对红细胞在体内和体外老化的影响(血液储存)。PIMT1 空小鼠将与其他菌株相结合，以分离与功能相关的代谢途径 (例如G6PD缺乏)。将对这些动物应用先进的实验方法，以便分离出 特定年龄和生理条件的细胞。最后，由这些生物产生的受控生物 这些方法将通过尖端的代谢组学和蛋白质组学方法进行分析。总的来说，这些 研究将促进我们对特定的生化细胞衰老途径的作用的理解，以及 保守修复途径的机械作用(PIMT1)，以及在高级生化分析的背景下 和建模，以产生对聚焦假设的新的机械性理解和关键测试。