Date

2018

Author

Olgun, Çağla Ece

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17ß-estradiol (E2), the main circulating estrogen hormone, has an important role in the physiological and pathophysiological regulation of many tissues and organs including breast tissue. Regulation of cell proliferation, differentiation and death in target tissues is mediated by E2. The estrogen receptor (ER), a transcription factor, provides the lasting effect of E2 on cells via regulation of targeting gene expression. Previous microarray and gene expression studies in our laboratory reveal that YPEL2, which is one of the members of YPEL gene family, is an estrogen responsive gene. For understanding function of YPEL2, over-expression systems in COS7 and MCF7 were used. However, over-expression of any YPEL family gene leads to a nuclear membrane disassembly, DNA leakage and then rapid cell death. This prevented us to examine the functional features of YPEL2 in mammalian cells. In addition, the members of the YPEL gene family proteins share a remarkably high amino acid sequence homology. This high sequence homology also prevented us to assess the function of YPEL proteins by decreasing the amount of any YPEL protein using siRNA approaches. Therefore, to understand function and mechanistic aspects of YPEL2, new approaches were needed. In cells, protein functions within the context of a dynamically changing network of interacting protein partners. Thus, the identification of protein partners of protein of interest gives important information about the function of protein. In this study, to understand function of YPEL2, we used BioID system through which putative protein partners of protein of interest are defined. Putative interacting protein partners of YPEL2 were then analyzed with gene annotation tool, DAVID. According to gene clusters generated through GOTERM Biological process analysis, possible protein partners are clustered in different biological processes such as RNA processing and ribosome biogenesis. Also, our results suggest that a group of protein partners of YPEL2 is involved in the formation of stress granules. That the YPEL gene family is conserved through yeast to human with high amino acid sequence homology suggests conserved functions for Ypel proteins. In yeast there is one homolog of YPEL gene family: MOH1, on which there is a few information on structure and function. In addition to BioID studies, to assess a function to YPEL2, we wanted to generate a cell model for YPEL proteins using yeast. To establish this model, we initially wanted to characterize the functional feature of MOH1; we reasoned that a better understanding functional futures of the yeast YPEL homolog of MOH1 could provide an important experimental system to characterize YPEL2 functions. Using yeast strains, we found that the deletion of MOH1 causes a decrease in cell survival when cells are grown under nutritional depletion stress. Also, when survival of wild type and moh1∆ cells were compared under different stress conditions, survival patterns of cells differed according to stress conditions. Through these results, we conclude that MOH1 is an important factor in stress responses of cells to different stress conditions and that MOH1 affects cellular survival differently dependent upon stress conditions.

Subject Keywords

Homology (Biology)., Protein binding., Yeast.

URI

http://etd.lib.metu.edu.tr/upload/12622698/index.pdf
https://hdl.handle.net/11511/27665

Collections

Graduate School of Natural and Applied Sciences, Thesis