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

描述（由申请人提供）：衰老是一个复杂的多因素现象。涉及诱导培养细胞复制衰老的模型系统在阐明这一复杂过程的一些机制中是不可或缺的。因此，了解细胞衰老是如何以及为什么发生的，对衰老研究领域至关重要。尽管事实上，有显着的相关证据表明之间的连接聚（ADP-核糖）和哺乳动物的寿命，没有研究已经做了探索PARP-1的可能作用-负责合成90%的细胞聚（ADP-核糖）-在衰老。此外，目前用于分析蛋白质聚ADP-核糖基化的大多数方法不能在共价修饰和非共价缔合之间做出令人信服的区分，因此充满了不精确性。我们建议首先通过开发新的质谱方法来明确分析体外聚（ADP-核糖基化）来解决这些弱点。然后，我们将应用并进一步完善这些技术在体内使用的聚（ADP-核糖基化）的氧化损伤的模型系统中的特征。最后，我们将测试的假设，即PARP-1是一个不可或缺的球员在细胞衰老的机制，通过使用质谱和常规方法相结合，分析PARP-1的翻译后修饰，蛋白质水平和酶活性在老化的人成纤维细胞培养。这些研究将首次建立PARP-1与人类衰老之间的特定和直接联系。在培养物中生长的正常人类细胞在无法进一步增殖之前只能分裂一定次数。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。该项目将分析核酶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.