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

鉴定 ApoB 脂蛋白生物发生和细胞内运输的新型调节因子

• 批准号: 10671051
• 负责人: McKenna Rae Feltes
• 金额: $ 7.18万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-08-01 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10671051
• 关键词: ARNT gene; Affect; Alleles; Animal Model; Apolipoproteins B; Appearance; Atherosclerosis; Biogenesis; Biological Process; Biology; Cell Culture Techniques; Clustered Regularly Interspaced Short Palindromic Repeats; Collagen; Complex; Darkness; Defect; Deposition; Diameter; Drosophila genus; Dyslipidemias; Electron Microscopy; Embryo; Embryonic Development; Environment; Ethylnitrosourea; Exhibits; Family; Fishes; Generations; Genes; Genetic Complementation Test; Genetic Screening; Genetic Transcription; Goals; Golgi Apparatus; Image; Intestines; Investigation; Knowledge; Label; Larva; Lipid Mobilization; Lipids; Lipoproteins; Liver; Location; Maps; Metabolic syndrome; Metabolism; Monitor; Mutagenesis; Mutation; Optics; Organ; Particle Size; Pathology; Pathway interactions; Phenotype; Play; Population; Production; Proteins; Regulation; Reporter; Research Personnel; Role; Study models; Testing; Tissues; Transgenic Organisms; Vesicle; Visualization; Whole Organism; Yeasts; Yolk Sac; Zebrafish; experimental study; forward genetics; genetic manipulation; light microscopy; light transmission; lipid metabolism; lipid transport; microsomal triglyceride transfer protein; mutant; novel; particle; screening; trafficking; training opportunity; transcription factor; transcriptome; transcriptome sequencing

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.

项目摘要 - 代谢综合征最多影响美国人口的1/3，并且与 血脂血症。在肝脏和肠中，脂质被分解代谢，掺入复杂脂质中，存储在脂质液滴中， 或分泌为含APOB的脂蛋白（B-LPS）。脂蛋白生物发生在控制细胞中起关键作用 消化器官中的脂质通量；但是，我们对控制B-LP容量的因素的了解很差 （尺寸），分泌和亚细胞位置（S）。这些知识差距很难调查 由于细胞培养模型的局限 哺乳动物的全动物模型可视化亚细胞水平的脂蛋白动力学。斑马鱼 为这些挑战提供了一个独特的解决方案 - 在胚胎和幼虫阶段，斑马鱼稳健 使用一般配置的途径产生B-LPS，是在光学上启用成像的光学上的成像 全体生物量表，并且适合基因操纵。在发展中的幼虫中，主要存入 通过微粒体甘油三酸酯转移蛋白（MTP）将蛋黄脂质包装到B-LPS中 然后分泌以分配到其他组织。幼虫缺乏MTP效率低下，动员脂质，导致脂质 异常的脂质存储，可降低光传输，使蛋黄囊具有简单的黑暗外观 通过低磁化光显微镜鉴定。我们的实验室是第一个表征“黑暗的B-LP生物学” 蛋黄”表型，并鉴定了几种在脂蛋白生物发生中具有已知和未知作用的基因。 最近发现MIA2/CTAGE5突变体表现出深色纱表型。 MIA2/CTAGE5的蛋白质​​产物， 探戈（Tano）（tali），可能有助于形成大直径Copii蔬菜以运输大型B-LP cargos 从急诊室到高尔基。我观察到MIA2/CTAGE5中缺乏鱼类的B-LPS几乎完全很小 直径表明MIA2/CTAGE5在调节B-LP大小中起关键作用。在AIM 1中，我将检验以下假设 Tali使用转基因斑马鱼线和接近标记方法优先与大B-LPS相互作用。 此外，为了测试MIA2/CTAGE5间接调节B-LP大小的假设 途径，我将通过RNASEQ监视转录组，并通过针对脂质相关的COPII依赖性分析 转录因子。在AIM 2中，我将利用深色Yolk表型来发现B-LP的新型调节剂 通过进行正向遗传诱变筛选来代谢。深色雅克家庭将被描述为 使用转基因APOB报告基因斑马鱼和电子显微镜的B-LP代谢缺陷。优先级 等位基因将进行映射，并进一步研究脂质代谢缺陷。通过定义MIA2/ctage5的作用 在调节B-LP的大小和数量以及通过识别脂蛋白产生的新调节剂时，我将扩大 我们对脂蛋白生物发生和相关病理的理解。提出的实验，突变体 发现的，提供的培训机会以及Farber Lab和Carnegie的严格学术环境 将支持我成为独立调查员的长期目标。