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: 10472028
• 负责人: Tabea Moll
• 金额: $ 4.68万
• 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/10472028
• 关键词: 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; Clinical Trials; Complex; DNA Sequence Alteration; Data; Deposition; Diet; Doctor of Philosophy; Embryo; Endocytosis; Environment; Fluorescence; Gene Expression; Genes; Genetic; Harvest; Hepatic; High Fat Diet; Homologous Gene; Human; Individual; Institution; Knowledge; LDL Cholesterol Lipoproteins; Label; Laboratories; Larva; Lead; Lipids; Lipoprotein (a); Lipoproteins; Liver; 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; base; 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）风险增加相关。 LDL 是包含脂蛋白 (B-lp) 的最小载脂蛋白 B，并且会随着时间的推移而积累修饰。这些 B-lp 修饰可能通过增加 B-lp 对脉管系统的粘附性并降低其对 LDL 受体 (LDLR) 的特异性来增加动脉粥样硬化性。然而，控制 B-lp 流通时间及其周转时间的因素仍有待完全确定。目前研究 B-lp 周转率的方法依赖于有限的患者群体，并且需要脂蛋白标记，然后进行复杂的数学模型，这可能会扭曲所获得的数据。最近的一项全基因组关联研究 (GWAS) 强调了研究 LDL 周转率的重要性。 GWAS 报告称，脱唾液酸糖蛋白受体 1 (ASGR1) 基因突变的个体的 CVD 风险较低，但 LDL 水平仅轻度降低。 LDL 本身的降低幅度不足以解释 CVD 风险降低的幅度。该提案探讨了 ASGR1 调节 LDL 周转率的假设，这是一个尚未得到充分研究的关键因素，可能会有力地介导 CVD 风险。我将使用光学透明的斑马鱼幼虫，通过开发多种新颖的光学报告基因，在体内、不受干扰的环境中，首次深入了解 B-lp 的一般周转情况。我通过创建表达与载脂蛋白 B (ApoB) 融合的光转换荧光蛋白 Dendra2 的斑马鱼系，生成并验证了一种测量 B-lp 周转率的工具。光转换后，Dendra2 发出红色荧光，随后红色荧光的丧失代表 ApoB 的读出，从而代表 B-lp 转换。我假设脂质的普遍可用性是 B-lp 周转的决定因素，我将通过斑马鱼的遗传和饮食扰动来研究这一点。为了研究 ASGR1 对 B-lp 代谢的作用，我鉴定了 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 的丢失不会改变 B-lp 的总数或大小。然而，ASGR1 突变体的 RNAseq 分析表明 ASGR1 缺失会增加 B-lp 产生和摄取所需基因的表达。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；我将使用 ApoB-Dendra2 报告基因直接测试这一点。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 不会降解并且更容易获得。我将研究野生型和 ASGR1 突变体中 ASGR1 和 LDLR 之间的相互作用。拟议的实验不仅会产生许多强大的新工具，而且会增加我们对 B-lp 调节的理解，并为我提供特殊的培训机会。在从事这些研究的过程中，我将获得许多突破性技术的实践经验，同时扩展我对脂质代谢和心血管疾病的知识。总而言之，约翰霍普金斯大学和卡内基研究所提供的世界知名研究人员和资源的协同作用创造了一个出色的环境来支持拟议的研究和我的博士学位。训练。