Molecular Mechanisms of Mammalian SIRT6 Function

描述（由申请人提供）：Sir2（沉默信息调节因子-2）蛋白在多种模式生物的衰老和寿命调节中起作用。我们的长期目标是了解人类衰老和年龄相关病理的分子机制，以及人类Sir2因子在减缓这些过程中的作用。初步研究表明，哺乳动物Sir2蛋白SIRT6抑制基因组不稳定性，并减轻几种年龄相关疾病的发病。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，将有助于阐明影响衰老过程中人类健康的基本生物学过程。在这里，要研究的总体假设是SIRT6通过染色质上的新功能对衰老相关的细胞过程和基因组维持施加影响。将进行一系列的生化、细胞和整体蛋白质组学和基因组分析，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。目的：阐明SIRT6在染色质调控中的分子机制。组蛋白在染色质上的翻译后修饰在基因表达程序和DNA损伤反应中起着重要作用。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。我们将定义SIRT6在染色质上的酶活性以及SIRT6水平改变对染色质结构和功能状态的影响。2. 目的：阐明SIRT6蛋白在基因组稳定和DNA修复中的作用。sirt6缺陷细胞表现出增加的基因组不稳定性和对氧化和烷基化DNA损伤的敏感性，这些损伤被认为是导致衰老的原因。为了阐明SIRT6在基因组维持中发挥作用的分子机制，我们提出了一系列的功能和生化实验。3. 阐明新的SIRT6调控分子通路。将采用蛋白质组学和生化方法分离和表征SIRT6底物、蛋白质结合伙伴和大分子复合物。此外，将采用全基因组方法鉴定SIRT6调控基因和染色质上的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.