Molecular Mechanisms of Mammalian SIRT6 Function

描述(由申请人提供)：SIR2(沉默信息调节因子-2)蛋白质在多种模式生物的衰老和寿命调节中发挥作用。我们的长期目标是了解人类衰老和与年龄相关的病理的分子机制，以及人类Sir2因子在减弱这些过程中的作用。初步工作表明，哺乳动物中的一种Sir2蛋白SIRT6可以抑制基因组的不稳定性，并减轻几种与年龄相关的病理疾病的发生。然而，SIRT6功能的分子机制尚不清楚。因此，系统地描述SIRT6在人类细胞中发挥作用的基本分子机制，对于阐明在衰老过程中影响人类健康的基本生物学过程应该是有用的。这里，要研究的总体假设是，SIRT6通过染色质的新功能对衰老相关的细胞过程和基因组维持发挥作用。将进行一系列生化、细胞和全球蛋白质组和基因组分析，以确定SIRT6在人类细胞中的分子功能。提出了三个具体目标：1.阐明SIRT6在染色质调节中的分子机制。染色质上组蛋白的翻译后修饰在基因表达程序和DNA损伤反应中发挥着重要作用。初步结果表明，SIRT6与染色质上的组蛋白密切相关，并可能催化组蛋白的修饰。我们将确定SIRT6在染色质的酶活性，以及SIRT6水平变化对染色质结构和功能状态的影响。2.阐明人类SIRT6蛋白在基因组稳定和DNA修复中的作用。SIRT6缺陷细胞表现出更高的基因组不稳定性和对氧化和烷化DNA损伤的敏感性，这些损伤被认为是导致衰老的原因。一系列的功能和生化实验被用来阐明SIRT6在基因组维持中发挥作用的分子机制。3.阐明SIRT6调控的新的分子途径。将采用蛋白质组学和生化方法来分离和表征SIRT6底物、蛋白质结合伙伴和大分子复合体。此外，全基因组的方法将被用来确定SIRT6调节的基因和染色质上的SIRT6结合位点。

DESCRIPTION (provided by applicant): Sir2 (Silent Information Regulator-2) proteins function in aging and lifespan regulation in multiple model organisms. Our long-term goal is to understand the molecular mechanisms that underlie human aging and age-associated pathologies, and the role of human Sir2 factors in attenuating these processes. Preliminary work indicates that one mammalian Sir2 protein, SIRT6, suppresses genomic instability and attenuates the onset of several age-associated pathologies. However, the molecular mechanisms of SIRT6 function are not known. Thus, a systematic characterization of the basic molecular mechanisms through which SIRT6 functions in human cells should be instrumental for elucidating fundamental biological processes that impact on human health in aging. Here, the overall hypothesis to be investigated is that SIRT6 exerts its effects on aging-associated cellular processes and genome maintenance via novel functions at chromatin. A series of biochemical, cellular, and global proteomic and genomic analyses will be carried out to define the molecular functions of SIRT6 in human cells. Three Specific Aims are proposed: 1. To elucidate the molecular mechanisms by which SIRT6 functions in chromatin regulation. Post-translational modifications of histones at chromatin play important roles in gene expression programs and DNA damage responses. Preliminary results indicate that SIRT6 is tightly associated with, and may catalyze modifications of, histones at chromatin. We will define the enzymatic activity of SIRT6 at chromatin and the effects of altered SIRT6 levels on chromatin structure and functional states. 2. To elucidate the role of the human SIRT6 protein in genome stabilization and DNA repair. SIRT6-deficient cells show increased genomic instability and sensitivity to oxidative and alkylating DNA lesions that are proposed to contribute to aging. A series of functional and biochemical experiments are proposed to elucidate the molecular mechanisms by which SIRT6 exerts its effects on genome maintenance. 3. To elucidate novel SIRT6 regulated molecular pathways. Proteomic and biochemical approaches will be taken to isolate and characterize SIRT6 substrates, protein binding partners, and macromolecular complexes. In addition, genome wide approaches will be employed to identify SIRT6-regulated genes and SIRT6 binding sites at chromatin.