Defining mechanisms of lipoprotein turnover and their regulation by ASGR1

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是最小的含载脂蛋白-B的脂蛋白（B-lp），并且其随时间积累修饰。这些B-lp修饰可通过增加B-lp对血管的粘附并降低其对LDL受体（LDLR）的特异性来增加致动脉粥样硬化性。然而，控制B-LP在流通中的时间的因素，即它们的周转率，仍然有待完全确定。目前研究B-lp周转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周转的重要性。GWAS报告了CVD的风险较低，但在脱唾液酸糖蛋白受体1（ASGR 1）基因突变的个体中，LDL水平仅轻度降低。LDL本身的降低并不足以解释CVD风险降低的幅度。该提案探讨了ASGR 1调节LDL周转的假设，LDL周转是一个关键的未充分研究的因素，可能有力地介导CVD风险。我将使用光学透明的斑马鱼幼虫，以获得第一次洞察一般B-LP营业额在体内，不受干扰的情况下，通过开发多个新的光学记者。我通过创建一个表达与载脂蛋白B（Apo B）融合的光转换荧光蛋白Dendra 2的斑马鱼品系，生成并验证了一种测量B-lp周转率的工具。在光转化后，Dendra 2发红色荧光，随后红色荧光的损失代表ApoB的读数，因此代表B-lp周转。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干扰来研究这一点。为了研究ASGR 1对B-lp代谢的作用，我鉴定了ASGR 1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ASGR 1的丢失不会改变B-lp的总数或大小。然而，ASGR 1突变体的RNAseq分析表明，ASGR 1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ASGR 1的缺失增加了B-lp的周转率;我将通过使用ApoB-Dendra 2报告基因直接测试这一点。先前的研究表明，ASGR 1结合LDLR并导致内吞介导的降解。因此，我假设在缺乏ASGR 1的情况下，LDLR逃避降解，更容易获得。我将研究野生型和ASGR 1突变体中ASGR 1和LDLR之间的相互作用。拟议的实验不仅将产生一系列强大的新工具，而且将增加我们对B-lp调控的理解，并为我提供特殊的培训机会。在从事这些研究的同时，我将获得许多突破性技术的实践经验，同时扩大我对脂质代谢和CVD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创造了一个出色的环境，以支持拟议的研究和我的博士学位。训练

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.