Characterization of novel Programmed Cell death (PCD) pathways in yeast

酵母中新型程序性细胞死亡（PCD）途径的表征

• 批准号: RGPIN-2016-04812
• 负责人: Greenwood, Michael
• 金额: $ 2.26万
• 依托单位: Royal Military College of Canada
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2018
• 资助国家: 加拿大
• 起止时间: 2018-01-01 至 2019-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=652435
• 关键词: Characterization; novel; Programmed; Cell; death

Understanding Programmed Cell Death (PCD) is complicated by the existence of many different sub-forms that exists within and between the different types of PCD. The yeast S. cerevisiae undergoes a form of intrinsic apoptosis comparable to that seen in metazoans. By screening a mammalian expression cDNA library in yeast undergoing PCD, we identified a number of pro-survival sequences and we have now characterized 10 of these. We have made some interesting observations including the fact that human 14-3-3β/α prevents apoptotic like PCD as well as preventing PCD in response to prolonged autophagy. Presently, we are characterizing the anti-PCD function of human H-ferritin as well as a putative yeast ferritin encoded by the orphan gene RGI1. Within this context we propose the following 3 aims:***1- Characterization of copper, leucine deprivation and iron PCD- We found differences in the way our pro-survival sequences behave with respect to PCD inducing stresses. All clones can prevent copper mediated PCD, a few can prevent the effects of prolonged leucine deprivation while only the putative yeast ferritin RGI1p can prevent both copper and iron PCD. We will examine the different characteristics of the PCD that we see with copper, leucine deprivation and iron. We will use biochemical and cytological markers that differentiate between necrosis and apoptosis and examine mutants defective in autophagy, apoptosis and necrosis. We will also screen our cDNA library in yeast using the minimum concentration of iron that causes complete growth inhibition. ***2- Characterization of ferritin and intracellular iron mediated cell death- Yeast is a commonly used model for the study of iron metabolism. We are currently carrying out an analysis of the human and of our putative yeast ferritin (RGI1) with respect to their ability to store, scavenge and protect from iron in yeast. Ferritins are also general pro-survival proteins that can prevent PCD in response to ROS generating stresses. These studies support the model that levels of free iron are increased and serve to induce stress mediated PCD. A role for iron as stress induced intracellular 2nd messenger mediator of PCD in yeast has received little attention. Here we will examine the total as well as the labile iron pools notably when cells are treated with all 3 PCD inducing agents. Other experiments will include examining characterizing the effects of cell permeable iron chelator on PCD. ***3- Characterizing the pro-survival potential of novel Bax suppressors- From our original screen of a cardiac cDNA library, numerous uncharacterized sequences of interest remain. We propose to characterize the mechanism by which two orphan genes as well as 3 of the ones involved in the electron transport chain prevent PCD. These are methodologically well-established and are used as more facile projects to be initiated by undergrad student researchers and serve as second projects for grad students.**

程序性细胞死亡(PCD)的理解是复杂的，因为存在于不同类型的PCD内部和之间的许多不同的亚型。酿酒酵母经历了一种与后生动物相似的内在凋亡形式。通过在酵母中筛选PCD过程中的哺乳动物表达cDNA文库，我们鉴定了一些促进生存的序列，现在我们已经鉴定了其中的10个。我们已经做了一些有趣的观察，包括人类14-3-3β/α可以防止像PCD一样的细胞凋亡，也可以防止PCD对长时间的自噬的反应。目前，我们正在鉴定人类H-铁蛋白的抗PCD功能，以及由孤儿基因RGI1编码的一种可能的酵母铁蛋白。在此背景下，我们提出了以下3个目标：*1-铜、亮氨酸剥夺和铁PCD的特征-我们发现我们的促生存序列在PCD诱导压力下的表现方式不同。所有克隆都可以阻止铜介导的PCD，少数克隆可以防止长时间亮氨酸剥夺的影响，而只有可能的酵母铁蛋白RGI1p可以防止铜和铁的PCD。我们将研究铜、亮氨酸缺乏和铁的PCD的不同特征。我们将使用区分坏死和凋亡的生化和细胞学标记，并检测在自噬、凋亡和坏死方面存在缺陷的突变株。我们还将使用导致完全抑制生长的铁的最低浓度在酵母中筛选我们的cDNA库。*2-铁蛋白和细胞内铁介导的细胞死亡酵母是研究铁代谢的常用模型。我们目前正在对人类和我们推定的酵母铁蛋白(RGI1)进行分析，以了解它们在酵母中存储、清除和保护铁的能力。铁蛋白也是一般的促进生存的蛋白质，可以防止PCD在ROS产生压力时做出反应。这些研究支持这一模型，即游离铁水平增加，并有助于诱发应激介导的PCD。在酵母中，铁作为应激诱导的PCD细胞内第二信使中间体的作用还很少受到关注。在这里，我们将检查总的以及不稳定的铁库，特别是当细胞用所有3种PCD诱导剂处理时。其他实验将包括研究细胞可渗透铁络合剂对PCD的影响。*3-表征新的Bax抑制子的促生存潜力-从我们最初对心脏cDNA文库的筛选中，仍有大量感兴趣的未表征序列。我们建议描述两个孤立基因以及参与电子传递链的3个基因预防PCD的机制。这些都是方法成熟的项目，被用作本科生研究人员发起的更简单的项目，并作为研究生的第二项目。