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

描述(申请人提供)：衰老是一个复杂的、多因素的现象。涉及在培养细胞中诱导复制衰老的模型系统在阐明这一复杂过程的一些机制方面是不可或缺的。因此，了解细胞衰老的方式和原因对衰老研究领域至关重要。尽管有重要的相关证据表明多聚ADP-核糖与哺乳动物寿命有关，但还没有研究探索PARP-1在衰老中的可能作用。PARP-1负责合成90%的细胞多(ADP-核糖)。此外，目前用于蛋白质聚(ADP-核糖基化)分析的大多数方法不能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的质谱学方法来解决这些缺点，以明确地分析体外聚(ADP-核糖基化)。然后，我们将在模型系统中通过表征聚(ADP-核糖化)对氧化损伤的反应来应用和进一步改进这些技术，以便在体内使用。最后，我们将使用质谱仪和传统方法相结合的方法来分析PARP-1在培养的衰老人成纤维细胞中的翻译后修饰、蛋白质水平和酶活性，从而检验PARP-1是细胞衰老机制中不可或缺的参与者的假设。这些研究将首次在PARP-1和人类衰老之间建立特定和直接的联系。在培养中生长的正常人类细胞在不能进一步增殖之前只能分裂固定的次数。这种现象被称为复制性衰老，是衰老研究中广泛使用的模型。这项拟议的项目将分析核酶PARP-1在细胞衰老机制中的作用，以更好地了解人类衰老的方式和原因。

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.