Grant: Discovery Grants Program - Individual

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.