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-新陈代谢综合征影响多达三分之一的美国人口，并与 血脂异常。在肝脏和肠道中，脂肪被分解代谢，结合成复杂的脂质，储存在脂滴中， 或以含载脂蛋白B的脂蛋白(B-LP)的形式分泌。脂蛋白生物发生在细胞调控中的关键作用 消化器官中的脂质流量；然而，我们对控制B-LP能力的因素了解很少 (大小)、分泌和亚细胞定位(S)。这些知识差距一直很难调查 由于用于研究多器官现象的细胞培养模型的局限性以及相对难以获得 建立哺乳动物全动物模型，在亚细胞水平上可视化脂蛋白动力学。斑马鱼 为这些挑战提供了一个独特的解决方案--在胚胎和幼体阶段，斑马鱼顽强地 使用遗传保守的途径产生B-LP，光学清晰，能够在亚细胞上成像 整个生物体的规模，并服从于基因操作。在发育中的幼虫中，母体沉积 卵黄脂通过卵黄合胞层中的微粒体甘油三酯转运蛋白(MTP)包裹到B-脂蛋白中。 然后分泌出来，分发到其他组织。缺乏MTP的幼虫不能有效地动员脂肪，导致 不正常的脂肪储存，减少透光性，使卵黄囊容易出现黑色外观 经低倍光学显微镜鉴定。我们的实验室是第一个将B-LP生物学特征描述为 并发现了几个在脂蛋白生物发生中具有已知和未知作用的基因。 Mia2/ctage5突变体最近被发现表现为暗卵黄表型。Mia2/ctage5的蛋白产物， 探戈样(TALI)，可促进大直径COPII囊泡的形成，以运输大的B-LP货物 从急诊室到高尔基。我观察到MIA2/ctage5缺陷鱼中的B-Lp几乎都是小的 直径提示Mia2/ctage5在调节B-LP大小中起关键作用。在目标1中，我将测试假设 TALI优先使用转基因斑马鱼品系和邻近标记方法与大的B-LP相互作用。 此外，为了验证Mia2/ctage5通过调节转录水平间接调节B-LP大小的假设 途径，我将通过RNAseq和通过对脂类相关的、COPII依赖的靶向分析来监控转录组 转录因子。在目标2中，我将利用暗蛋黄表型来发现B-LP的新调控因子 通过进行正向遗传突变筛查来进行代谢。深色蛋黄家族的特征将是 利用转基因ApoB报告斑马鱼和电子显微镜研究B-LP代谢缺陷。高优先级 等位基因将被定位，并进一步研究脂代谢缺陷。通过定义Mia2/ctage5的作用 在调节B-LP的大小和数量以及确定脂蛋白产生的新调节因素方面，我将扩大 我们对脂蛋白生物发生和相关病理的理解。实验提出，变种人 法伯实验室和卡内基的发现、提供的培训机会和严谨的学术环境 将支持我成为一名独立调查员的长期目标。