Obesity is a major health problem of the 21st century. Identification of novel approaches for prevention and treatment of obesity is of great importance for public health. Obesity specifically refers to an excessive amount of adipose tissue, where lipids are stored in specialized organelles called lipid droplets (LDs). Recent studies in the autophagy field suggest that LDs can be selectively delivered to and degraded by lysosomes. This discovery has opened an exciting, new opportunity to correct lipid metabolism in humans by modulating lipophagy, the selective autophagy (or the cell's "self-eating") of LDs. I have studied selective autophagy for the past 12 years and identified several proteins required for the selective degradation of another organelle, the peroxisome. Recently, we found that acyl-CoA binding proteins (ACBPs), Acb1 and another protein we identified as Atg36, are essential for selective autophagy of peroxisomes (pexophagy) and Ape1 complexes (Cvt pathway) in the Pichia pastoris yeast. Therefore, the first goal of my project is to elucidate the molecular role o ACBPs in selective autophagy in P. pastoris. We predict that the role of ACBPs in selective autophagy is conserved from yeast to mammals. Thus, the second goal is to elucidate the physiological role of ACBPs in mammalian cells. We will characterize the role of ACBD5 and DBI, the mammalian orthologs of Atg36 and Acb1, respectively, in both pexophagy and lipophagy. The third goal is to develop the obesity model yeast, Yarrowia lipolytica, as a simple lipophagy model and study the molecular role of ACBPs in lipophagy. My long- term goals are to develop an independent research program on lipophagy, uncover its molecular mechanism and the role of ACBPs in this process. Knowledge of the proteins specifically involved in the autophagic degradation of LDs might help to design new therapeutic approaches to cure obesity in humans. To complete my transition from (1) yeast to mammalian cells, (2) pexophagy to lipophagy and (3) mentored to an independent research, I will continue to receive training in working with mammalian cell lines and in monitoring mammalian pexophagy and lipophagy. I will do it through collaboration with Dr. Till and consultation with Dr. Cuervo, experts on mammalian pexophagy and lipophagy, respectively. I will expand my knowledge on the structure and function of ACBPs through consultation with an ACBP expert, Dr. Loomis. I will also receive extensive training on lipid biology in the lab of my co-mentor, Dr. Field, an expert on cellular lipids and lipid-binding proteins. This training is essential to accomplish lipid profiing of ACBP mutants and to study interactions of ACBPs with phospholipids. To increase my knowledge on selective autophagy and LDs, I will continue to attend both autophagy and lipid biology meetings. For the training in public speaking, I will practice giving talks at the Toastmasters International. In 2012, I will give a talk on Atg36 at the Gordon Research Seminar on Autophagy (Ventura, CA). To become a successful independent investigator, I will also receive training in biostatistics, scientific management, writing and grant writing by taking classes and through personal meetings with UCSD faculties. I will write the manuscript on the role of ACBPs in selective autophagy and submit it to one of the top-ranked journals by the end of the 2nd year of the K01 award. Additionally, I will write my first R01 grant on the molecular mechanism of lipophagy and submit it to NIDDK by the end of the 3rd year. I will start searching for a tenure-track faculty position during the 4th year and accept an offer by the end of the 4-year K01 award. Having developed an independent research direction on lipophagy, I will continue to uncover its molecular mechanism and the role of ACBPs in this process in both yeast and mammalian cells. My mentor, Dr. Subramani, will closely monitor my research and career development during the K01 award. My proposal consists of the three Specific Aims.
Aim 1 will address two alternative, but not mutually exclusive, roles of ACBPs in selective autophagy in P. pastoris: (1) the role in bridging cargo and the phagophore, an autophagic isolation membrane, via interaction of ACBPs with the phagophore protein, Atg8 (yeast two-hybrid and co-immunoprecipitation studies) and (2) the role in generating phagophore membrane curvature via the interaction of ACBPs with phospholipids and remodeling of the phagophore membrane (in vitro filter and liposome binding assays, lipid profiling of ACBP mutants).
Aim 2 will explore the physiological role of ACBPs in selective autophagy in mammalian cells. We will study the role of ACBD5 and DBI in pexophagy using human cervical cancer, hepatocarcinoma and teratocarcinoma cells and a new pexophagy reporter, mRFP-GFP-SKL. Our pilot studies suggest that human ACBD5 is essential for pexophagy. I will also address the role of ACBD5 and DBI in lipophagy using an established rat hepatocyte cell line and lipophagy assays. Finally, Aim 3 will develop the first simple model for lipophagy using Y. lipolytica. We will knock out the Y. lipolytica ATG36 and ACB1 genes, and elucidate their roles in lipophagy using fluorescence microscopy and a novel Tgl3-GFP processing assay. We expect studies on ACBPs in yeast and mammalian cells will extend our understanding of organelle homeostasis and provide new targets for the prevention and treatment of obesity in humans.
Over one-third of American adults are obese with obesity being one of the main risk factors for type 2 diabetes, coronary heart disease and stroke. This proposal will address the mechanism of the recently discovered natural process of fat breakdown through autophagy (literally, cell's self-eating). The genes required for the autophagy of fat will be identified and characterized. Knowledge of these genes will help us design new therapeutic strategies to cure obesity in humans.
|Nazarko, Taras Y; Ozeki, Katharine; Till, Andreas et al. (2014) Peroxisomal Atg37 binds Atg30 or palmitoyl-CoA to regulate phagophore formation during pexophagy. J Cell Biol 204:541-57|
|Nazarko, Taras Y (2014) Atg37 regulates the assembly of the pexophagic receptor protein complex. Autophagy 10:1348-9|