Defining mechanisms of lipoprotein turnover and their regulation by ASGR1

血浆低密度脂蛋白(LDL)水平高与心血管疾病(CVD)风险增加相关。低密度脂蛋白是最小的载脂蛋白-B含脂蛋白(B-LP)，随着时间的推移，它会积累修饰。这些B-LP修饰可能通过增加B-LP与血管系统的粘附性并降低其对低密度脂蛋白受体(LDLR)的特异性来增加动脉粥样硬化。然而，控制B-LP在循环中的时间及其周转率的因素仍未完全确定。目前研究B-LP周转率的方法依赖于有限的患者队列，需要脂蛋白标记，然后进行复杂的数学建模，这可能会扭曲所获得的数据。最近的一项全基因组关联研究强调了研究低密度脂蛋白周转的重要性。Gwas报告了患心血管疾病的风险较低，但在ASGR1基因突变的个体中，低密度脂蛋白水平仅略有降低。低密度脂蛋白本身的降低还不足以说明心血管疾病风险降低的幅度。这项建议探索了ASGR1调节低密度脂蛋白周转的假设，低密度脂蛋白是一个未被研究的关键因素，可能有力地调节心血管疾病的风险。我将使用光学透明的斑马鱼幼虫，通过开发多个新颖的光学记者，在体内、不受干扰的情况下首次深入了解一般的B-LP周转。我创建了一个斑马鱼品系，表达融合到载脂蛋白B(ApoB)上的可光转换荧光蛋白Dendra2，从而生成并验证了一个测量B-LP营业额的工具。光转化后，Dendra2发出红色荧光，随后的红色荧光损失代表ApoB的读数，从而B-LP周转。我假设脂肪的普遍可获得性是B-LP周转的一个决定因素，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在B-LP代谢中的作用，我鉴定了ASGR1的斑马鱼同源基因，并使用CRISPR/Cas9创建了一个突变体。我发现斑马鱼中ASGR1的缺失不会改变B-LP的总数或大小。然而，对ASGR1突变体的RNAseq分析表明，ASGR1缺失增加了B-LP产生和摄取所需基因的表达。总而言之，这些数据与我的假设一致，即ASGR1的缺失会增加B-LP的成交量；我将使用ApoB-Dendra2报告直接检验这一点。以前的研究表明，ASGR1与LDLR结合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可以逃脱降解，并且更容易获得。我将研究ASGR1和LDLR在野生型和ASGR1突变体中的相互作用。拟议中的实验不仅将产生许多强大的新工具，而且将增加我们对B-LP监管的理解，并为我提供特殊的培训机会。在从事这些研究的同时，我将获得许多开创性技术的实践经验，同时我还将扩大我对脂代谢和心血管疾病的了解。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，以支持拟议的研究和我的博士培训。

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.