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: 10314442
• 负责人: McKenna Rae Feltes
• 金额: $ 6.6万
• 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 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10314442
• 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; Structure; 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.

项目总结-代谢综合征影响多达1/3的美国人口，并与 血脂异常在肝和肠中，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。脂蛋白生物合成在调节细胞内的 消化器官中的脂质通量;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。这些知识差距一直难以调查 由于用于研究多器官现象的细胞培养模型的局限性和相对不可接近性 的哺乳动物整体动物模型，以可视化脂蛋白动力学在亚细胞水平。斑马鱼 提出了一个独特的解决方案，这些挑战-在胚胎和幼虫阶段，斑马鱼健壮 使用遗传保守的途径产生B-LPS，光学透明，能够在亚细胞上成像， 整个生物体规模，并适合遗传操作。在发育中的幼虫，母体沉积 卵黄脂在卵黄合胞体层（YSL）中被微粒体甘油三酯转运蛋白（MTP）包装成B-lps 然后被分泌以分布到其它组织。MTP缺乏的Larvae不能有效地动员脂质， 一种不正常的脂质储存，减少了光的传输，使卵黄囊的外观变暗， 通过低倍光学显微镜鉴定。我们的实验室是第一个描述“黑暗”的B-lp生物学特征的实验室。 已经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mia 2/ctage 5突变体表现出暗卵黄表型。mia 2/ctage 5的蛋白产物， Tango样（TALI）可能有助于形成大直径COPII囊泡，以转运大的B-lp货物 从急诊室到高尔基体我观察到mia 2/ctage 5缺陷鱼中的B-lps几乎完全是小的， 直径表明mia 2/ctage 5在调节B-lp大小中起关键作用。在目标1中，我将检验以下假设： 使用转基因斑马鱼系和邻近标记方法，TALI优先与大B-lps相互作用。 此外，为了检验mia 2/ctage 5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和脂质相关的靶向分析，COPII依赖性 转录因子在目标2中，我将利用暗卵黄表型来发现B-lp的新调节子 通过进行正向遗传诱变筛选来确定代谢。黑蛋黄家族将被描述为 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 将绘制等位基因并进一步研究脂质代谢缺陷。通过定义mia 2/ctage 5的作用， 在调节B-lp的大小和数量，并通过识别脂蛋白生产的新调节剂，我将扩大 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 发现，提供培训机会，以及法伯实验室和卡内基严格的学术环境 将支持我成为独立调查员的长期目标。