Defining mechanisms of lipoprotein turnover and their regulation by ASGR1

脂蛋白周转的定义机制及其 ASGR1 的调节

• 批准号: 10685273
• 负责人: Tabea Moll
• 金额: $ 5.02万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10685273
• 关键词: Ablation; Address; Adherence; Affinity; Age; Antibodies; Apolipoproteins B; Asialoglycoprotein Receptor; Binding; Biological Assay; Biological Process; Blood Circulation; CRISPR/Cas technology; Cardiovascular Diseases; Cause of Death; Cellular biology; Chimeric Proteins; Cholesterol; Circulation; Clinical Trials; Complex; DNA Sequence Alteration; Data; Deposition; Diet; Doctor of Philosophy; Embryo; Endocytosis; Endosomes; Environment; Fluorescence; Gene Expression; Genes; Genetic; Harvest; High Fat Diet; Homologous Gene; Human; Individual; Institution; Knowledge; LDL Cholesterol Lipoproteins; Label; Laboratories; Larva; Lipids; Lipoprotein (a); Lipoproteins; Liver; Liver Carbohydrate-Binding Protein; Low Density Lipoprotein Receptor; Low-Density Lipoproteins; Measures; Mediating; Metabolism; Methods; Modeling; Modification; Molecular; Mutation; Optical reporter; Optics; Orthologous Gene; Patients; Phenocopy; Phenotype; Plasma; Polyacrylamide Gel Electrophoresis; Production; Proteins; Protocols documentation; Publishing; Regulation; Reporter; Reporting; Research; Research Personnel; Resources; Rodent Model; Role; Specificity; Techniques; Testing; Time; Tissues; Training; Transgenic Organisms; Triglycerides; United States; Universities; Zebrafish; cardiovascular disorder risk; cohort; course development; dietary; experience; experimental study; fluorophore; genome editing; genome wide association study; glycation; in vivo; insight; lipid metabolism; mathematical model; mutant; novel; oxidation; particle; repaired; standard of care; synergism; tool; training opportunity; transcription activator-like effector nucleases; transcriptome sequencing; uptake

High levels of plasma low-density lipoprotein (LDL) are correlated with an increased risk for cardiovascular disease (CVD). LDL is the smallest apolipoprotein-B containing lipoprotein (B-lp) and it accumulates modifications over time. These B-lp modifications may increase atherogenicity by increasing B-lp adherence to the vasculature and lowering their specificity to the LDL receptor (LDLR). However, the factors that control B-lp time in circulation, their turnover, remain to be fully defined. Current methods to study B-lp turnover rely on limited patient cohorts and require lipoprotein labeling, followed by complex mathematical modeling, that may skew the obtained data. A recent genome-wide association study (GWAS) underscores the importance to study LDL turnover. The GWAS reported lower risk for CVD, but only mildly reduced levels of LDL in individuals with a mutation in the asialoglycoprotein receptor 1 (ASGR1) gene. The reduction of LDL itself was not dramatic enough to account for the magnitude of the reduction in CVD risk. This proposal explores the hypothesis that ASGR1 modulates LDL turnover, a key understudied factor that may powerfully mediate CVD risk. I will use the optically clear zebrafish larva to obtain the first insight into general B-lp turnover in an in vivo, unperturbed context by developing multiple novel optical reporters. I generated and validated a tool to measure B-lp turnover by creating a zebrafish line that expresses the photoconvertible fluorescent protein Dendra2 fused to apolipoprotein B (ApoB). After photoconversion, Dendra2 fluoresces red and the subsequent loss of red fluorescence represents a readout of ApoB and thus B-lp turnover. I hypothesize that the general availability of lipids is a determinant of B-lp turnover and I will investigate this by genetic and dietary perturbations in zebrafish. To study the role of ASGR1 on B-lp metabolism, I identified the zebrafish ortholog of ASGR1 and created a mutant using CRISPR/Cas9. I found that the loss of ASGR1 in zebrafish does not change the total B-lp number or size. However, RNAseq analysis of ASGR1 mutants indicates that ASGR1 loss increases the expression of genes required for B-lp production and uptake. Together, these data are consistent with my hypothesis that the loss of ASGR1 increases B-lp turnover; I will directly test this by using the ApoB-Dendra2 reporter. Previous research suggests that ASGR1 binds LDLR and leads to endocytosis mediated degradation. Hence, I hypothesize that in the absence of ASGR1, LDLR escapes degradation and is more readily available. I will examine the interaction between ASGR1 and LDLR in the wild-type and ASGR1 mutants. The proposed experiments will not only generate a host of powerful new tools but will increase our understanding of B-lp regulation and provide me with exceptional training opportunities. While working on these studies, I will acquire hands-on experience with numerous ground-breaking techniques, while I expand my knowledge of lipid metabolism and CVD. Altogether, the synergy of world-renowned researchers and resources afforded by Johns Hopkins University and the Carnegie Institution create an outstanding environment to support the proposed studies and my Ph.D. training.

高水平的血浆低密度脂蛋白（LDL）与心血管疾病（CVD）的风险增加相关。 LDL是含脂蛋白（B-LP）的最小的载脂蛋白-B，随着时间的推移会累积修饰。这些B-LP的修饰可能通过增加B-LP遵守脉管系统并降低其对LDL受体（LDLR）的特异性来提高动脉粥样硬化。但是，控制循环中B-LP时间的因素，其营业额仍待完全定义。当前研究B-LP营业额的方法取决于有限的患者队列，需要脂蛋白标记，然后进行复杂的数学建模，这可能会使所获得的数据偏斜。一项最新的全基因组协会研究（GWAS）强调了研究LDL更新的重要性。 GWAS报告的CVD风险较低，但仅在Asialogolototion受体1（ASGR1）基因中突变的个体中仅轻度降低LDL水平。 LDL本身的减少不足以说明CVD风险降低的幅度。该提案探讨了ASGR1调节LDL营业额的假设，LDL营业额是一个可能有力介导CVD风险的关键因素。我将使用光学清晰的斑马鱼幼虫来通过开发多个新颖的光学记者，在体内，不受干扰的上下文中获得对一般B-LP周转的第一个见解。我通过创建斑马鱼线来生成并验证了一种测量B-LP营业额的工具，该系列斑马鱼线表达融合到载脂蛋白B（APOB）的可光转换荧光蛋白Dendra2。光转换后，Dendra2荧光红色，随后的红色荧光损失代表APOB的读数，因此代表B-LP周转率。我假设脂质的一般可用性是B-LP营业额的决定因素，我将通过斑马鱼中的遗传和饮食扰动进行调查。为了研究ASGR1在B-LP代谢中的作用，我确定了ASGR1的斑马鱼直系同源物，并使用CRISPR/CAS9创建了一个突变体。我发现斑马鱼中ASGR1的损失不会改变总B-LP数量或大小。但是，ASGR1突变体的RNASEQ分析表明，ASGR1损失会增加B-LP产生和摄取所需的基因表达。总之，这些数据与我的假设一致，即ASGR1的丢失会增加B-LP的周转率。我将通过使用APOB-DENDRA2记者直接测试。先前的研究表明，ASGR1结合LDLR并导致内吞作用介导的降解。因此，我假设在没有ASGR1的情况下，LDLR逃脱了降解，并且更容易获得。我将检查野生型和ASGR1突变体中ASGR1和LDLR之间的相互作用。拟议的实验不仅将产生许多强大的新工具，而且会增加我们对B-LP监管的理解，并为我提供出色的培训机会。在研究这些研究时，我将通过大量的开创性技术获得动手经验，而我扩大了对脂质代谢和CVD的了解。总的来说，约翰·霍普金斯大学和卡内基机构提供的世界知名研究人员和资源的协同作用创造了一个杰出的环境，以支持拟议的研究和我的博士学位。训练。