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: 2016
• 资助国家: 加拿大
• 起止时间: 2016-01-01 至 2017-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=615319
• 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 14-3-3ß/a 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.

理解程序性细胞死亡（PCD）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子形式。酵母S.酿酒酵母经历一种与后生动物中所见相当的内在凋亡形式。通过筛选哺乳动物表达的cDNA文库在酵母经历PCD，我们确定了一些促生存序列，我们现在已经确定了其中10个。我们已经做了一些有趣的观察，包括人14-3- 3 β/a防止凋亡样PCD以及防止PCD响应于延长的自噬的事实。目前，我们正在表征人类H-铁蛋白以及由孤儿基因RGI 1编码的假定酵母铁蛋白的抗PCD功能。在这方面，我们提出以下三个目标： 1-铜、亮氨酸剥夺和铁PCD的表征-我们发现我们的促存活序列在PCD诱导应激方面的行为方式存在差异。所有的克隆都可以防止铜介导的PCD，少数克隆可以防止长期亮氨酸剥夺的影响，而只有推定的酵母铁蛋白RGI 1 p可以防止铜和铁PCD。我们将研究我们在铜，亮氨酸剥夺和铁中看到的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，并检查自噬、凋亡和坏死缺陷的突变体。我们还将使用导致完全生长抑制的最低浓度的铁在酵母中筛选我们的cDNA文库。 2-铁蛋白和细胞内铁介导的细胞死亡的表征-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（RGI 1）进行分析，以了解它们储存、清除和保护酵母中铁的能力。铁蛋白也是一般的促存活蛋白，其可以防止响应于ROS产生的应激的PCD。这些研究支持游离铁水平增加并用于诱导应激介导的PCD的模型。铁作为应激诱导的酵母细胞内PCD的第二信使介质的作用很少受到关注。在这里，我们将检查总的以及不稳定的铁池，特别是当细胞用所有3种PCD诱导剂处理时。其他实验将包括检查表征细胞渗透性铁螯合剂对PCD的影响。 3-描述新型Bax抑制子的促生存潜力-从我们最初的心脏cDNA文库筛选中，仍然存在许多未表征的感兴趣序列。我们建议的机制，其中两个孤儿基因，以及3个参与电子传递链防止PCD的特点。这些都是方法上完善的，并被用来作为更容易的项目，由本科生研究人员发起，并作为格拉德生的第二个项目。