NAD and the Regulation of Sirtuin Targets

描述（由申请人提供）：沉默调节蛋白是保守的NAD+依赖性蛋白脱乙酰酶，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（芽殖酵母）Sir 2蛋白是第一个被描述的沉默调节蛋白，最初被表征为沉默交配型基因座和端粒转录沉默所需的因子，后来被表征为组蛋白脱乙酰酶。rDNA基因座和亚端粒重复序列的组蛋白脱乙酰化与酵母母细胞的复制寿命（RLS）的调节有关，RLS定义为母细胞在衰老前进行的分裂次数。SIR 2的缺失缩短了RLS，过表达延长了寿命，这可能是通过其在rDNA和端粒上的功能实现的。酵母sirtuins的ChIP测序分析显示，Sir 2及其partial Hst 1令人惊讶地共同富集在参与糖酵解、发酵、翻译和细胞壁生物合成的多个基因的开放阅读框上，所有过程在指数生长的葡萄糖培养物中高度表达，但当细胞进入二次生长转变时被抑制，酵母细胞从糖酵解/发酵向TCA循环/呼吸转变的生长时期。在初步的数据中，Sir 2和Hst 1都是有效地，特异性地抑制糖酵解基因与Sir 2和Hst 1结合到ORF所需的。这是非常有趣的，因为在哺乳动物中，Sir 2同源物SIRT 1和SIRT 6都参与控制葡萄糖代谢以维持体内平衡。我建议，酵母细胞中的糖酵解基因的调控有助于RLS的控制。第一个具体的目标是集中在确定Sir 2和Hst 1如何抑制糖酵解基因的转录时，他们被绑定到ORF。第二个具体目标旨在测试Sir 2和Hst 1是否以类似于哺乳动物sirtuins的方式控制代谢稳态，第三个具体目标集中在复制老化期间Sir 2蛋白快速周转对糖酵解基因表达的后果，并弄清楚Sir 2蛋白的年龄相关下降如何影响Sir 2在各种竞争染色质基因座中的分布。我们的目标是建立糖酵解基因调控（阻遏）作为Sir 2及其密切parastin，Hst 1的一个关键的衰老相关功能。确认这种类型的调节将提供酵母和人类之间的直接进化联系，使研究酵母中的sirtuin代谢调节成为可能，并将研究结果转化为哺乳动物。

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.