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），并随着时间的推移积累修饰。这些B-lp修饰可能通过增加B-lp对血管的粘附性和降低它们对LDL受体（LDLR）的特异性而增加动脉粥样硬化性。然而，控制B-lp流通时间的因素，即它们的周转率，仍有待充分确定。目前研究B-lp转换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（GWAS）强调了研究LDL转换的重要性。GWAS报告了较低的CVD风险，但在asialal糖蛋白受体1 （ASGR1）基因突变的个体中，LDL水平仅轻度降低。低密度脂蛋白本身的降低不足以解释心血管疾病风险降低的幅度。本研究探讨了ASGR1调节LDL转换的假设，这是一个尚未得到充分研究的关键因素，可能有力地介导心血管疾病的风险。我将使用光学透明的斑马鱼幼虫，通过开发多种新型光学报告器，首次深入了解在体内不受干扰的情况下的一般B-lp周转。我生成并验证了一种测量B-lp转换的工具，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。在光转化后，Dendra2发出红色荧光，随后红色荧光的丢失代表ApoB的读数，从而代表B-lp的转换。我假设脂质的普遍可用性是B-lp转换的决定因素，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在B-lp代谢中的作用，我鉴定了ASGR1在斑马鱼中的同源基因，并利用CRISPR/Cas9构建了一个突变体。我发现斑马鱼中ASGR1的缺失并不会改变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。总之，这些数据与我的假设一致，即ASGR1的缺失会增加B-lp的周转；我将直接使用ApoB-Dendra2报告程序进行测试。先前的研究表明，ASGR1结合LDLR并导致内吞介导的降解。因此，我假设在没有ASGR1的情况下，LDLR可以避免降解，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。提出的实验不仅会产生一系列强大的新工具，而且会增加我们对B-lp调控的理解，并为我提供特殊的培训机会。在这些研究中，我将获得许多突破性技术的实践经验，同时扩大我在脂质代谢和心血管疾病方面的知识。总之，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为支持拟议的研究和我的博士学位培养创造了一个良好的环境。

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.