Regulation of yeast chronological lifespan during growth in high calorie media.

描述（由申请人提供）：热量限制是已知的最广泛研究的干预措施之一，可延长酵母和人类等多种物种的寿命和/或健康寿命。酵母已被证明是发现影响人类衰老和年龄相关疾病的基因的一种极好的模式生物，这是由于易于研究和保护许多衰老过程背后的营养传感和信号通路。最初的酵母研究指出，Sir 2是一种组蛋白脱乙酰酶，负责介导热量限制的延长寿命特性，而后来的研究则对这项研究提出了质疑，混淆似乎与菌株背景和培养基组成的微小差异以及酵母中定义热量限制的方式有关。在我开发的一种新的酵母热量限制范例下，实验指出了重要的Sir 2依赖性和Sir 2独立性途径，这些途径调节时间寿命（非分裂细胞在稳定期培养物中保持活力的时间），以响应葡萄糖浓度的变化。该提案的主要目标是调查和定义这些途径。我的中心假设是，在这种实验范式下，介导酵母寿命的sirtuin依赖性和sirtuin非依赖性途径将继续更容易阐明，参与传统热量限制介导的寿命的基因在更广泛的热量条件下运作，Sir 2可能通过几种已知的Sirtuin中的一种或多种影响按时间顺序的寿命，将长寿途径与糖浓度的变化联系起来。在目标1中，候选和无偏遗传方法将用于鉴定参与Sir 2非依赖性葡萄糖调节的长寿途径的基因。将在高和低葡萄糖条件下测定先前与卡路里限制介导的时间寿命有关的基因的时间寿命，并将测定酵母敲除收集物的合并培养物的时间寿命，其中通过与每个突变体相关的独特DNA条形码的测序计数鉴定短寿命和长寿命突变体。将进一步研究在卡路里限制条件下未能表现出寿命延长的突变体。在目标2中，将使用候选和无偏见的方法来识别参与Sir 2依赖性途径的基因。在已知或怀疑的Sir 2相互作用途径中有缺陷的突变体将在两种热量条件下测定时间寿命， 用于与sir 2的上位相互作用。此外，将使用生物素化被Sir 2特异性脱乙酰化的赖氨酸残基的方法来鉴定Sir 2的脱乙酰化靶标，从而允许它们的脱乙酰化。 沉淀并通过串联质谱法鉴定。肽在高热量和低热量条件下的差异脱乙酰化作用将进一步研究其在时间寿命调节中的作用。这项工作将确定与酵母时序老化和热量限制介导的寿命相关的新基因，并最终解决爵士在改变时序寿命以应对生长介质的热量条件变化中的作用。

DESCRIPTION (provided by applicant): Calorie restriction is one of the most widely studied interventions known to extend lifespan and/or healthspan in species as diverse as yeast and humans. Yeast has proven to be a fantastic model organism for the discovery of genes that influence human aging and age-related disease, due to the ease of study and the conservation of nutrient sensing and signaling pathways that underlie many aging processes. Initial studies in yeast pointed to Sir2, a histone deacetylase, as being responsible for mediating the life-extending properties of calorie restriction, while later studies called this research into question Confusion seems to relate to minor differences in strain background and media composition as well as more significant problems with the way calorie restriction is defined in yeast. Under a new yeast calorie restriction paradigm I developed, experiments point to significant Sir2-dependent and Sir2-independent pathways regulating chronological lifespan (the time a non-dividing cell remains viable in a stationary phase culture) in response to changing glucose concentrations. The major goal of this proposal is to investigate and define these pathways. My central hypotheses are that the Sirtuin-dependent and Sirtuin-independent pathways mediating yeast longevity will continue to be easier to elucidate under this experimental paradigm, that genes involved in traditional caloric restriction-mediated longevity operate across a much broader range of caloric conditions, and that Sir2 is likely affecting chronological lifespan through one or more of several known Sirtuin-linked longevity pathways in response to changes in sugar concentration. In Aim 1, both candidate and unbiased genetic approaches will be used to identify genes involved in the Sir2-independent glucose-regulated longevity pathway. Genes implicated previously in calorie restriction-mediated chronological longevity will be assayed for chronological lifespan in both high and low glucose conditions, and a pooled culture of the yeast knockout collection will be assayed for chronological longevity with short- and long-lived mutants identified by sequencing counts of unique DNA barcodes associated with each mutant. Mutants that fail to exhibit lifespan extension under calorie restriction conditions will be investigated further. In Aim 2, candidate and unbiased approaches will be used to identify genes involved in the Sir2-dependent pathway. Mutants that are defective in known or suspected Sir2- interacting pathways will be assayed for chronological lifespan in both caloric conditions, looking for epistatic interactions with sir2 . Additionally, deacetylation targets of Sir2 will be identifid using a process that biotinylates lysine residues specifically deacetylated by Sir2, allowing their precipitation and identification by tandem mass spectroscopy. Peptides subject to differential deacetylation under high and low calorie conditions will be investigated further for a role in chronological lifespan regulation. This work will identify new genes relevant to yeast chronological aging and calorie restriction-mediated longevity, and finally resolve the role of Sir in altering chronological lifespan in response to changing caloric conditions of growth media.