NAD and the Regulation of Sirtuin Targets

描述(由申请人提供)：sirtuins是保守的NAD+依赖的蛋白去乙酰基酶，有助于调节与衰老和年龄相关疾病相关的细胞途径和功能。酿酒酵母(芽接酵母)Sir2蛋白是第一个被描述的sirtuin蛋白，最初被认为是沉默交配型基因座和端粒转录沉默所必需的因子，后来被鉴定为组蛋白脱乙酰酶。RDNA位点和亚端粒重复序列上的组蛋白去乙酰化与酵母母细胞复制寿命(RLS)的调节有关，RLS被定义为母亲在衰老之前进行的分裂数量。Sir2的缺失缩短了RLS，过度表达延长了寿命，这可能是通过其在rDNA和端粒上的功能实现的。对酵母sirtuins的芯片测序分析表明，Sir2及其伴生体Hst1出人意料地共富集在参与糖酵解、发酵、翻译和细胞壁生物合成的多个基因的开放阅读框上，所有这些过程在指数级生长的葡萄糖培养物中都高度表达，但当细胞进入二次生长转换时受到抑制，这是酵母细胞将代谢从糖酵解/发酵转移到TCA循环/呼吸的时期。在初步数据中，Sir2和Hst1都是有效和特异地抑制糖酵解基因所必需的，Sir2和Hst1与ORF结合。这非常耐人寻味，因为在哺乳动物中，Sir2同源基因SIRT1和SIRT6都被认为与控制葡萄糖代谢以维持体内平衡有关。我认为酵母细胞中糖酵解基因的调节有助于控制RLS。第一个具体目标是确定当Sir2和Hst1与ORF结合时如何抑制糖酵解基因的转录。第二个特定目的是测试Sir2和Hst1是否以类似于哺乳动物sirtuins的方式控制代谢动态平衡；第三个特定目标是研究复制衰老过程中Sir2蛋白快速周转对糖酵解基因表达的影响，并找出与年龄相关的Sir2蛋白下降如何影响Sir2在各个相互竞争的染色质基因座上的分布。我们的目标是建立糖酵解基因调节(抑制)作为Sir2及其近邻Hst1与衰老相关的关键功能。确认这种类型的调控将在酵母和人类之间提供直接的进化联系，使研究酵母中sirtuin代谢调控成为可能，并将发现转化为哺乳动物。

DESCRIPTION (provided by applicant): The sirtuins are conserved NAD+-dependent protein deactylases that contribute to the regulation of cellular pathways and functions associated with aging and age-related diseases. The Saccharomyces cerevisiae (budding yeast) Sir2 protein was the first sirtuin to be described and was originally characterized as a factor required for transcriptional silencing at the silent mating-type loci and telomeres, and then later characterized as histone deacetylase. Histone deacetylation at the rDNA locus and sub-telomeric repeats has been linked to the regulation of replicative lifespan (RLS) of yeast mother cells, which is defined as the number of divisions a mother performs before senescing. Deletion of SIR2 shortens RLS and overexpression extends lifespan, presumably through its function at the rDNA and telomeres. ChIP-sequencing analysis of the yeast sirtuins revealed that Sir2 and its paralog, Hst1, are surprisingly co-enriched on the open reading frames of multiple genes involved in glycolysis, fermentation, translation, and cell wall biosynthesis, all processes that ae highly expressed in exponentially growing glucose cultures, but are then repressed when cells enter the diauxic shift, the period in growth when yeast cells shift their metabolism from glycolysis/fermentation toward the TCA cycle/respiration. In preliminary data, Sir2 and Hst1 are both required for efficiently, and specifically repressing glycolytic genes with Sir2 and Hst1 bound to the ORF. This is very intriguing because in mammals, the Sir2 homologs SIRT1 and SIRT6 have both been implicated in controlling glucose metabolism in order to maintain homeostasis. I propose that the regulation of glycolytic genes in yeast cells contributes to the control of RLS. The first specific aim is focused on determining how Sir2 and Hst1 repress transcription of glycolytic genes when they are bound to the ORF. The second specific aim is designed to test whether Sir2 and Hst1 control metabolic homeostasis in a manner similar to mammalian sirtuins, and the third specific aim is focused on the consequences of rapid Sir2 protein turnover during replicative aging on glycolytic gene expression, and figuring out how the age- associated decline of Sir2 protein impacts the distribution of Sir2 across the various competing chromatin loci. The goal is to establish glycolytic gene regulation (repression) as a critical aging-related function of Sir2 and its close paralog, Hst1. Confirmation of this type of regulation would provide a direct evolutionary link between yeast and humans, making it possible to study sirtuin metabolism regulation in yeast, and translate the findings to mammals.