Quantitative Analysis of PARP-1 and PolyADP-ribosylation in Cellular Senescence

细胞衰老中 PARP-1 和 PolyADP-核糖基化的定量分析

• 批准号: 7498979
• 负责人: Yvette M Edmonds
• 金额: $ 3.58万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2010-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7498979
• 关键词: ADP ribosylation; Address; Age; Aging; Biological Models; Cell Aging; Cells; Complex; Cultured Cells; Enzymes; Fibroblasts; Human; In Vitro; Length; Longevity; Mass Spectrum Analysis; Mediating; Methods; Modeling; Modification; Niacinamide; Nuclear; Numbers; Phyllanthus emblica; Poly Adenosine Diphosphate Ribose; Polymers; Post-Translational Protein Processing; Process; Proliferating; Protein Analysis; Research; Role; Techniques; Testing; Time; density; in vivo; novel; response; senescence

DESCRIPTION (provided by applicant): Aging is a complicated and multifactorial phenomenon. Model systems involving the induction of replicative senescence in cultured cells have been indispensable in elucidating some of the mechanisms underlying this complex process. An understanding of how and why cellular senescence occurs is thus critical to the field of aging research. Despite the fact that there is significant correlative evidence suggesting a connection between poly(ADP-ribose) and mammalian longevity, no studies have been done to explore a possible role for PARP-1 - the enzyme responsible for synthesis of 90% of cellular poly(ADP-ribose) - in senescence. Furthermore, most methods currently being used for analysis of protein poly(ADP-ribosylation are incapable of making convincing distinctions between covalent modification and non-covalent association, and are thus fraught with imprecision. We propose to address these weaknesses first by developing novel mass spectrometric methods for the unambiguous analysis of in vitro poly(ADP-ribosylation. We will then apply and further refine these techniques for in vivo use by characterizing the poly(ADP-ribosylation) response to oxidative damage in a model system. Finally, we will test the hypothesis that PARP-1 is an integral player in mechanisms of cellular senescence by using a combination of mass spectrometry and conventional methods to analyze PARP-1 post-translational modifications, protein levels, and enzymatic activity in aging human fibroblasts grown in culture. These studies will for the first time establish a specific and direct connection between PARP-1 and human aging. Normal human cells growing in culture can divide only a set number of times before they become incapable of further proliferation. This phenomenon, known as replicative senescence, is a widely-used model in aging studies. The proposed project will analyze the role of the nuclear enzyme PARP-1 in mechanisms of cellular senescence, in order to better understand how and why humans age.

描述（由申请人提供）： 衰老是一种复杂且多因素的现象。涉及诱导培养细胞复制衰老的模型系统对于阐明这一复杂过程背后的一些机制是不可或缺的。因此，了解细胞衰老如何以及为何发生对于衰老研究领域至关重要。尽管有重要的相关证据表明聚（ADP-核糖）与哺乳动物寿命之间存在联系，但尚未进行任何研究来探索 PARP-1（负责合成 90% 细胞聚（ADP-核糖）的酶）的可能作用。 ADP-核糖） - 衰老。此外，目前用于分析蛋白质聚（ADP-核糖基化）的大多数方法无法令人信服地区分共价修饰和非共价结合，因此充满了不精确性。我们建议首先通过开发新型质谱来解决这些弱点明确分析体外聚（ADP-核糖基化）的方法。然后，我们将通过表征聚（ADP-核糖基化）对氧化的反应来应用并进一步完善这些技术以用于体内使用最后，我们将通过结合质谱法和传统方法来分析 PARP-1 翻译后修饰、蛋白质水平和细胞衰老机制，来检验 PARP-1 是细胞衰老机制中不可或缺的参与者这一假设。这些研究首次在培养物中生长的衰老人类成纤维细胞中建立了 PARP-1 与人类衰老之间的特定且直接的联系，在培养物中生长的正常人类细胞在无法分裂之前只能分裂一定次数。更远扩散。这种现象被称为复制衰老，是衰老研究中广泛使用的模型。该项目将分析核酶 PARP-1 在细胞衰老机制中的作用，以便更好地了解人类衰老的方式和原因。