Identifying novel regulators of the biogenesis and intracellular trafficking of ApoB lipoproteins

点击翻译按钮获取中文摘要

PROJECT SUMMARY - Metabolic syndrome affects up to 1/3 of the US population and is associated with dyslipidemia. In liver and intestine, lipid is catabolized, incorporated into complex lipids, stored in lipid droplets, or secreted as ApoB-containing lipoproteins (B-lps). Lipoprotein biogenesis plays a key role in governing cellular lipid flux in digestive organs; however, we have a poor understanding of the factors controlling B-lp capacity (size), secretion, and subcellular location(s) of lipidation. These knowledge gaps have been difficult to investigate due to the limitations of cell culture models for studying multi-organ phenomena and the relative inaccessibility of mammalian whole-animal models to visualize lipoprotein dynamics at the subcellular level. The zebrafish presents a unique solution to these challenges – in the embryonic and larval stages, the zebrafish robustly produces B-lps using genetically conserved pathways, is optically clear enabling imaging on the subcellular to whole-organism scale, and is amenable to genetic manipulation. In the developing larva, maternally deposited yolk lipid is packaged into B-lps by microsomal triglyceride transfer protein (MTP) in the yolk syncytial layer (YSL) and then secreted for distribution to other tissues. Larvae deficient in MTP inefficiently mobilize lipid resulting in abnormal lipid storage that reduces light transmission, giving the yolk sac a dark appearance that is easily identified by low-magnification light microscopy. Our lab was the first to characterize the B-lp biology of the “dark yolk” phenotype and has identified several genes with known and unknown roles in lipoprotein biogenesis. mia2/ctage5 mutants were recently found to exhibit the dark-yolk phenotype. The protein product of mia2/ctage5, Tango-like (TALI), may facilitate the formation of large diameter COPII vesicles to transport large B-lp cargos from the ER to the Golgi. I observe that B-lps in mia2/ctage5 deficient fish are almost exclusively small in diameter suggesting mia2/ctage5 plays a key role in regulating B-lp size. In aim 1, I will test the hypothesis that TALI preferentially interacts with large B-lps using transgenic zebrafish lines and a proximity labeling approach. Further, to test the hypothesis that mia2/ctage5 indirectly regulates B-lp size by modulating transcriptional pathways, I will monitor the transcriptome by RNAseq and by targeted analysis of lipid-related, COPII-dependent transcription factors. In aim 2, I will exploit the dark-yolk phenotype to uncover novel regulators of B-lp metabolism by conducting a forward genetic mutagenesis screen. Dark-yolk families will be characterized for defects in B-lp metabolism using transgenic ApoB reporter zebrafish and electron microscopy. High priority alleles will be mapped and further investigated for defects in lipid metabolism. By defining the role of mia2/ctage5 in regulating B-lp size and number and by identifying novel regulators of lipoprotein production, I will broaden our understanding of lipoprotein biogenesis and related pathologies. The experiments proposed, mutants discovered, training opportunities provided, and rigorous academic environment of the Farber Lab and Carnegie will support my long-term goal of becoming an independent investigator.