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: 10526282
• 负责人: McKenna Rae Feltes
• 金额: $ 6.76万
• 依托单位: CARNEGIE INSTITUTION OF WASHINGTON, D.C.
• 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 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10526282
• 关键词: ARNT gene; Affect; Alleles; Animal Model; Apolipoproteins B; Appearance; Atherosclerosis; Biogenesis; Biological Process; Biology; Caliber; Cell Culture Techniques; Clustered Regularly Interspaced Short Palindromic Repeats; Collagen; Complement; Complex; Defect; Deposition; Drosophila genus; Dyslipidemias; Electron Microscopy; Embryo; Embryonic Development; Environment; Ethylnitrosourea; Exhibits; Family; Fishes; Generations; Genes; 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; 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 - 代谢综合征影响着多达 1/3 的美国人口，并且与 血脂异常。在肝脏和肠道中，脂质被分解代谢，结合成复合脂质，储存在脂滴中， 或作为含 ApoB 的脂蛋白 (B-lps) 分泌。脂蛋白生物合成在细胞调控中发挥着关键作用 消化器官中的脂质流动；然而，我们对控制 B-lp 容量的因素了解甚少 （大小）、分泌和脂化的亚细胞位置。这些知识差距很难调查 由于研究多器官现象的细胞培养模型的局限性和相对难以接近 哺乳动物整体动物模型，以在亚细胞水平上可视化脂蛋白动态。斑马鱼 为这些挑战提供了独特的解决方案——在胚胎和幼虫阶段，斑马鱼稳健地 使用遗传保守途径产生 B-lps，光学透明，能够在亚细胞上成像 整个生物体规模，并且适合基因操作。在发育中的幼虫中，母体沉积 卵黄脂质通过卵黄合胞层 (YSL) 中的微粒体甘油三酯转移蛋白 (MTP) 包装成 B-lps 然后分泌并分布到其他组织。缺乏 MTP 的幼虫无法有效地动员脂质，导致 异常的脂质储存会减少光透射，使卵黄囊呈现黑色外观，很容易 通过低倍光学显微镜鉴定。我们的实验室是第一个描述“暗生物”B-lp 生物学特征的实验室。 蛋黄”表型，并鉴定了几个在脂蛋白生物合成中具有已知和未知作用的基因。 最近发现 mia2/ctage5 突变体表现出深色卵黄表型。 mia2/ctage5的蛋白质​​产物， Tango-like (TALI)，可能促进大直径 COPII 囊泡的形成，以运输大型 B-lp 货物 从急诊室到高尔基体。我观察到 mia2/ctage5 缺陷鱼中的 B-lps 几乎完全是小 直径表明 mia2/ctage5 在调节 B-lp 大小中起着关键作用。在目标 1 中，我将检验以下假设： TALI 使用转基因斑马鱼系和邻近标记方法优先与大型 B-lps 相互作用。 此外，为了检验 mia2/ctage5 通过调节转录间接调节 B-lp 大小的假设 途径，我将通过 RNAseq 和脂质相关、COPII 依赖性的靶向分析来监测转录组 转录因子。在目标 2 中，我将利用暗蛋黄表型来发现 B-lp 的新调节因子 通过进行正向遗传诱变筛选来进行代谢。黑蛋黄家族的特征是 使用转基因 ApoB 报告基因斑马鱼和电子显微镜观察 B-lp 代谢缺陷。高优先级 等位基因将被绘制并进一步研究脂质代谢的缺陷。通过定义mia2/ctage5的作用 在调节 B-lp 大小和数量以及通过识别脂蛋白产生的新型调节剂方面，我将拓宽 我们对脂蛋白生物发生和相关病理学的理解。实验提出，突变体 法伯实验室和卡内基的发现、提供的培训机会以及严格的学术环境 将支持我成为一名独立调查员的长期目标。