PIMT1 in Red Blood Cell aging in vivo and in vitro

摘要 各种特定的化学损伤发生作为正常细胞衰老的结果，以及加速细胞衰老。 在某些病理情况下的损害。一种这样的化学途径是将酒石酸盐降解为 通过氧化剂损伤的异丙基残留物。作为一种修复机制，PIMT 1是一种酶途径， 甲基化异丙基残基，产生异丙基甲基酯，然后能够自发 转化为天冬氨酸，从而逆转异戊酰损伤。PIMT 1活性不足与 小鼠中氧化应激增加和细胞和生物体寿命缩短;然而，详细的代谢和 PIMT 1作用的生化分析尚未阐明。在本申请中，我们建议研究 PIMT 1在细胞衰老中的作用虽然将分析多个组织以测试PIMT 1的一般效应，但这 该提案主要关注关于红细胞（RBC）效应的特定中心假设。rbc是 红细胞对健康至关重要，红细胞功能障碍在多种疾病中起着核心作用。此外，输血 红细胞是最常见的单一住院侵入性治疗，大约每70人中就有1人接受 美国人每年输注的红细胞储存（作为后勤必需品）长达42天， 此时它们会遭受特定的细胞和生化损伤。已知红细胞会失去一种重要的 在正常细胞老化和红细胞储存中，通过关键基因产物（AE 1）发挥调节功能。但 AE 1功能障碍发生的分子机制尚不清楚。在本申请中，我们提供了新颖的 数据表明，在人和鼠RBC的AE 1中，在AE 1的结构域中， 这需要一个系统来运作。我们同样提出的数据表明，PIMT 1通路的失败， 加速了这种损伤-然而，需要缺失PIMT 1的整个动物模型来测试这种损伤。 机械的作用。在这种情况下，我们提出了以下具体目标，旨在严格检验假设 PIMT 1介导的氧化损伤修复的作用。具体目标1： PIMT 1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 PIMT 1上增加的氧化应激及其对体内和离体RBC老化（血液储存）的影响。PIMT 1 空小鼠将与设计用于分离功能相关的代谢途径的其他品系组合 (e.g. G6 PD缺陷）。先进的实验方法将应用于这些动物，以分离 特定年龄和生理条件的细胞。最后，由这些产生的受控生物 将通过最先进的代谢组学和蛋白质组学方法对这些方法进行分析。总的来说，这些 研究将促进我们对生化细胞衰老的特定途径的作用的理解， 保守修复途径（PIMT 1）的机制作用以及先进生化分析的背景 和建模，以产生新的机械理解和重点假设的关键测试。

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.