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: 7386267
• 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 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7386267
• 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; 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）：衰老是一种复杂而多因素的现象。涉及培养细胞中复制衰老的诱导的模型系统在阐明这种复杂过程的某些机制方面是必不可少的。因此，了解细胞衰老如何以及为什么发生对衰老研究领域至关重要。尽管有明显的相关证据表明多（ADP-核糖）和哺乳动物寿命之间存在联系，但尚未进行研究以探索PARP-1的可能作用 - 该酶在衰老中造成了90％的细胞聚（ADP-核糖）合成90％的酶。 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通过表征对模型系统中氧化损伤的反应，我们将通过质谱法和常规方法的组合来测试PARP-1的氧化损伤。在培养物中生长的正常人细胞之间建立特定的直接联系，只能在无法进一步扩散之前划分它们。这种现象被称为复制衰老，是衰老研究中广泛使用的模型。拟议的项目将分析核酶PARP-1在细胞衰老机制中的作用，以便更好地了解人类的年龄和原因。