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

在高热量培养基中生长过程中酵母寿命的调节。

• 批准号: 8718928
• 负责人: David F McCleary
• 金额: $ 3.71万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8718928
• 关键词: Affect; Aging; Aging-Related Process; Angiosperms; Animal Model; Autophagocytosis; Biological; Biological Assay; Biotinylation; Caloric Restriction; Cells; Collection; Confusion; Culture Media; Cyclic AMP-Dependent Protein Kinases; DNA; Data; Deacetylation; Diet; Disease; Exhibits; Fruit; Genes; Genetic; Gluconeogenesis; Glucose; Goals; Growth; Health; Histone Deacetylase; Human; Insulin; Insulin-Like Growth Factor I; Interphase Cell; Intervention Studies; Knock-out; Laboratories; Life; Link; Longevity; Longevity Pathway; Longitudinal Studies; Lysine; Macaca mulatta; Mass Spectrum Analysis; Measures; Mediating; Methods; Minor; Mitotic; Molecular; Partner in relationship; Pathway interactions; Peptides; Phase; Play; Population; Precipitation; Process; Property; Recombinant DNA; Recording of previous events; Regulation; Relative (related person); Research; Role; Saccharomyces cerevisiae; Signal Pathway; Sir2-like Deacetylases; Sirtuins; System; Time; Trees; Work; Yeasts; age related; base; detection of nutrient; dietary restriction; gene discovery; member; mutant; public health relevance; research study; response; sugar; telomere; tool

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.

描述（由申请人提供）：热量限制是已知可延长酵母和人类等多种物种寿命和/或健康寿命的最广泛研究的干预措施之一。酵母已被证明是发现影响人类衰老和与年龄相关疾病的基因的绝佳模式生物，因为它易于研究，并且保留了许多衰老过程中的营养感应和信号通路。最初在酵母中的研究指出 Sir2（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质疑。混淆似乎与菌株背景和培养基组成以及菌株背景和培养基组成的细微差异有关。更重要的问题在于酵母中热量限制的定义方式。在我开发的一种新的酵母热量限制范式下，实验指出了重要的 Sir2 依赖性和 Sir2 独立途径可调节时间寿命（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，介导酵母寿命的依赖于沉默调节蛋白的途径和不依赖于沉默调节蛋白的途径将继续更容易阐明，参与传统热量限制介导的长寿的基因在更广泛的热量条件下发挥作用，并且Sir2 可能通过几种已知的 Sirtuin 相关长寿途径中的一种或多种来响应糖浓度的变化，从而影响按时间顺序排列的寿命。在目标 1 中，候选遗传方法和无偏遗传方法将用于识别参与 Sir2 独立的葡萄糖调节长寿途径的基因。先前涉及热量限制介导的按时间顺序长寿的基因将在高和低葡萄糖条件下进行按时间顺序寿命的测定，并且将对酵母敲除集合的混合培养物进行按时间顺序寿命的测定，其中通过测序鉴定出短寿命和长寿命突变体与每个突变体相关的独特 DNA 条形码的计数。在热量限制条件下未能表现出寿命延长的突变体将被进一步研究。在目标 2 中，将使用候选且无偏见的方法来识别参与 Sir2 依赖性途径的基因。已知或怀疑 Sir2 相互作用途径有缺陷的突变体将在两种热量条件下进行按时间顺序的寿命分析，寻找 用于与 Sir2 的上位相互作用。此外，Sir2 的脱乙酰化目标将通过生物素化由 Sir2 特异性脱乙酰化的赖氨酸残基的过程来识别，从而使它们能够 沉淀和串联质谱鉴定。将进一步研究在高热量和低热量条件下进行差异脱乙酰化的肽在时间寿命调节中的作用。这项工作将鉴定与酵母时间老化和热量限制介导的寿命相关的新基因，并最终解决 Sir 在响应生长培养基热量条件变化而改变时间寿命方面的作用。