Human RIP1 in yeast: A high-throughput model for necrosis

Necrosis has long been regarded a passive form of cell death, seemingly not requiring genetic regulators that are characteristic of programmed cell death (PCD). However, extensive evidence has emerged recently that strongly argues for the existence of genes controlling the onset and progression of necrosis. Among the important discoveries, characterization of Receptor-Interacting Protein (RIP) 1, a kinase that drives necrosis in response to different lethal stimuli (ie. cytokines, DNA damage, etc.) has presaged a paradigm shift in the PCD field and demonstrated that necrosis is a programmed molecular mechanism with profound implications to human health and disease. The yeast Saccharomyces cerevisiae employs orthologs of canonical pro-apoptotic molecules, such as caspases, endonuclease G and apoptosis-inducing factor, among others, to execute PCD that is highly reminiscent of mammalian apoptosis. In addition, yeast has been used to examine roles for various subcellular processes in PCD, including mitochondrial and vacuolar function, and more recently, lipid metabolism. Technically, the use of yeast in PCD research offers many benefits: simple plating assays can precisely determine the survival rate of a yeast culture upon induction of death. Moreover, sophisticated staining techniques are used to distinguish between specific modes of cell death (ie. apoptosis or necrosis).Additionally, since yeast, unlike higher eukaryotes, retain viability when compromised for respiration, they present a convenient context in which to investigate the mechanistic details of mitochondrial functions in PCD. Thus, many of the same features of the yeast model that have benefited apoptosis research make it a promising tool for studying necrosis. Despite the absence of apparent orthologs of RIP1, our preliminary data indicate a clear pro-necrotic role for RIP1 in yeast. This suggests that putative substrates of RIP1 kinase activity are likely conserved in yeast and that the characterization of the effects of heterologously expressing RIP1 can lead to insights into it function in human cells. This reasoning parallels that regarding heterologously expressed Bax protein, which induces apoptosis in yeast, and the study of which has resulted in numerous important findings that bear relevance to mammalian apoptotic mechanisms. Herein, we propose a yeast model for the investigation of necrosis. Specifically, we propose a high-throughput genetic and metabolomic approach to identify novel necrotic regulators and regulatory mechanisms. This will be undertaken by studying the lethal effects of heterologously expressed human RIP1, including the identification of yeast genes that both promote and impede RIP1-induced necrosis in a high-throughput and exhaustive manner. Additionally, using co-immunoprecipitation experiments, I will identify yeast proteins that interact with heterologously expressed RIP1. In parallel, I plan to perform a lipidomic analysis of RIP1-expressing yeast cells, as a means to identify potential lipid biomarkers and/or mediators of the necrotic response. This would represent the first examination of alterations in lipid metabolite levels in cells undergoing necrosis. Since we expect that the results obtained to pertain to the necrotic process across species, the final stage of my proposed research will involve using our findings in yeast to predict novel necrotic regulatory mechanisms in mammalian cells. In conclusion, it is worthy to note the renewed interest in necrosis research of late, with a focus on its recently acknowledged regulated nature. For this reason, I am particularly enthusiastic to use RIP1-expressing yeast as a high-throughput, genetically-tractable and evolutionarily conserved model for mammalian cell necrosis. I am confident that this will uncover novel mechanisms by which necrosis is governed and executed