PCSK9, Lipoprotein receptors, and Atherosclerosis

PCSK9、脂蛋白受体和动脉粥样硬化

基本信息

批准号：
8248701
负责人：
SERGIO FAZIO
金额：
$ 50.22万
依托单位：
VANDERBILT UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2015-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8248701
关键词：
Active Sites Address Adipose tissue Affect Amino Acids Animals Arterial Fatty Streak Atherosclerosis Binding Biochemical Biology C-terminal Catalytic Domain Cells Cholesterol Cleaved cell Consensus Coronary Arteriosclerosis Coronary heart disease Data Development Disease Dose EGF gene Epidermal Growth Factor Excision Family Hepatic Hepatic Tissue Hepatocyte Human Hyperlipidemia In Vitro Individual Kidney Kupffer Cells Length Lipoprotein Receptor Liver Low Density Lipoprotein Receptor Low-Density Lipoproteins Lung Masks Mediating Methodology Molecular Mus N-terminal Pathway interactions Peptide Signal Sequences Plasma Predisposition Proprotein Convertases Proteins Proteolysis Publishing Regulation Reporting Rest Role Serine Serine Protease Site Solutions Structure Subtilisins Testing Therapeutic Agents Transgenic Organisms Work apolipoprotein E receptor 2 atherogenesis base cardiovascular risk factor early onset gain of function mutation heart disease prevention in vivo inhibitor/antagonist interdisciplinary approach kexin loss of function mutation macrophage mutant novel novel therapeutics protein function public health relevance receptor binding

项目摘要

DESCRIPTION (provided by applicant): Proprotein convertase subtilisin kexin type 9 (PCSK9) regulates plasma cholesterol levels by enhancing the intracellular degradation of the LDL receptor (LDLR), the primary driver of LDL clearance from plasma. Human PCSK9 is composed of 692 amino-acids. After removal of the signal peptide (residues 1-30), the secreted PCSK9 presents three domains: 1. The N-terminal pro-domain (PD, residues 31-152), which is cleaved but remains associated with the rest of the protein, and displays a highly disordered N-terminus (residues 31-60); 2. The catalytic domain (CA, residues 153-449), which contains a proteolytic site masked and neutralized by the associated PD; 3. The C-terminal domain (CD, residues 453-692), which consists of three tightly packed modules, CM1 (residues 457-527), CM2 (residues 534-601) and CM3 (residues 608-679). The consensus view is that PCSK9 binds to the epidermal growth factor type A (EGF-A) repeat of the LDLR via its CA, but does not cause direct proteolysis of the LDLR. Understanding the function of PSCK9 is central to the development of novel therapeutic agents to reduce cholesterol levels and cardiovascular risk. Although PCSK9 primarily targets the hepatic LDLR, it may also function on other lipoprotein receptors, such as apoER2, LRP1, and VLDLR, as well as on LDLR in extra-hepatic tissues. Our published work and preliminary studies have addressed the role of different PCSK9 domains in the secretion and function of the protein. We have found that: 1. Deletion of CM2 produces a protein that is secreted and functional; 2. Deletion of either CM1 or CM3 produces a protein that is neither secreted nor functional; 3. Deletion of the entire CD produces a protein that is secreted but not functional; 4. The PD of a PCSK9 fragment lacking CD facilitates the secretion of a PCSK9 fragment lacking PD; 5. The discrete CD of PCSK9 binds the ectodomain of the LDLR in solution and dose- dependently inhibits the LDLR-degrading effect of full-length PCSK9 cells; 6. PCSK9 undergoes pH-dependent self-association, which is mediated by the CA and increases the LDLR-reducing functionality; 7. Over- expression of PCSK9 reduces LDLR, apoER2, and LRP1 in HEK293T cells, and reduces LDLR levels in macrophages; 8. Transgenic expression of human PCSK9 in macrophages reduces liver LDLR and raises plasma cholesterol levels in mice. The central hypothesis of this proposal is that PCSK9 uses multiple domain interactions for secretion and function, and contributes to atherogenesis by targeting multiple lipoprotein receptors. We plan to: 1. Examine the role of the C-terminal domain in PCSK9 secretion and LDLR binding; 2. Test the hypothesis that domain interaction and self-association modulate PCSK9 secretion and function; 3. Test the hypothesis that PCSK9 secreted by macrophages modifies the biology of the atheroma through multiple effects on lipoprotein receptors. Investigating these novel roles of PCSK9's C-terminal domain and pro-domain may provide new leads in the development of inhibitors and advance our understanding of plasma cholesterol regulation. PUBLIC HEALTH RELEVANCE: PCSK9 is a newly identified protein that regulates plasma cholesterol levels in humans, thereby determining individual predisposition to coronary artery disease. By producing mutant forms of this protein, we have obtained preliminary data suggesting that there are specific structural requirements for the secretion and function of PCSK9. We are proposing to investigate these correlations in detail and to evaluate the overall impact of PCSK9 expression on lipoprotein receptors and atherosclerosis.

描述（由申请人提供）：普罗蛋白转化酶枯草蛋白Kexin型（PCSK9）通过增强LDL受体（LDLR）的细胞内降解，从而调节血浆胆固醇水平，这是血浆中LDL清除率的主要驱动力。人PCSK9由692个氨基酸组成。去除信号肽（残基1-30）后，分泌的PCSK9呈现三个结构域：1。N端亲域（PD，残基31-152），该域被切割，但仍与其他蛋白质相关，并显示高度无序的N-末端（残基31-60）； 2。催化结构域（CA，残基153-449），其中包含由相关PD掩盖并中和的蛋白水解位点； 3。C末端结构域（CD，残基453-692），由三个紧密堆积模块组成，CM1（残基457-527），CM2（残基534-601）和CM3（残基608-679）。共识的观点是，PCSK9与LDLR通过其CA的表皮生长因子A型（EGF-A）重复结合，但不会引起LDLR的直接蛋白水解。了解PSCK9的功能对于降低胆固醇水平和心血管风险的新型治疗剂的发展至关重要。尽管PCSK9主要靶向肝LDLR，但它也可能在其他脂蛋白受体（例如ApoER2，LRP1和VLDLR）以及肝外组织中的LDLR上起作用。我们发表的工作和初步研究已经解决了不同PCSK9领域在蛋白质的分泌和功能中的作用。我们发现：1。缺失CM2会产生一种分泌和功能的蛋白质； 2。删除CM1或CM3会产生既不分泌也不功能性的蛋白质。 3。整个CD的缺失会产生一种分泌但不是功能性的蛋白质。 4。缺少CD的PCSK9片段的PD有助于缺乏PD的PCSK9片段的分泌； 5。PCSK9的离散CD结合LDLR在溶液中的外生域，并依赖于剂量抑制全长PCSK9细胞的LDLR降解作用； 6。PCSK9经历了pH依赖性的自我关联，该自我关联是由CA介导的，并增加了LDLR还原功能。 7。PCSK9的过度表达降低了HEK293T细胞中的LDLR，ApoER2和LRP1，并降低了巨噬细胞中的LDLR水平； 8。巨噬细胞中人PCSK9的转基因表达减少了肝脏LDLR并提高小鼠的血浆胆固醇水平。该提议的中心假设是PCSK9使用多个域相互作用进行分泌和功能，并通过靶向多种脂蛋白受体来促进动脉粥样硬化。我们计划：1。检查C末端结构域在PCSK9分泌和LDLR结合中的作用； 2。检验域相互作用和自我关联调节PCSK9分泌和功能的假设； 3。检验以下假设：巨噬细胞分泌的PCSK9通过对脂蛋白受体的多种影响修饰了动脉粥样硬化瘤的生物学。研究PCSK9的C末端结构域和亲域的这些新作用可能会为抑制剂的发展提供新的潜在客户，并促进我们对血浆胆固醇调节的理解。公共卫生相关性：PCSK9是一种新鉴定的蛋白质，可调节人类血浆胆固醇水平，从而确定个体对冠状动脉疾病的易感性。通过产生这种蛋白质的突变形式，我们获得了初步数据，表明PCSK9的分泌和功能有特定的结构要求。我们提议详细研究这些相关性，并评估PCSK9表达对脂蛋白受体和动脉粥样硬化的总体影响。