PCSK9, Lipoprotein receptors, and Atherosclerosis

描述（由申请方提供）：前蛋白转化酶枯草杆菌蛋白酶kexin 9型（PCSK 9）通过增强LDL受体（LDLR）的细胞内降解来调节血浆胆固醇水平，LDLR是LDL从血浆中清除的主要驱动因素。人PCSK 9由692个氨基酸组成。在去除信号肽（残基1-30）后，分泌的PCSK 9呈现三个结构域：1. N-末端前结构域（PD，残基31-152），其被切割但保持与蛋白质的其余部分缔合，并且显示高度无序的N-末端（残基31-60）; 2.催化结构域（CA，残基153-449），其含有被相关PD掩蔽和中和的蛋白水解位点; 3. C-末端结构域（CD，残基453-692），其由三个紧密包装的模块CM 1（残基457-527）、CM 2（残基534-601）和CM 3（残基608-679）组成。共识是PCSK 9通过其CA与LDLR的A型表皮生长因子（EGF-A）重复序列结合，但不会引起LDLR的直接蛋白水解。了解PSCK 9的功能对于开发新型治疗药物以降低胆固醇水平和心血管风险至关重要。尽管PCSK 9主要靶向肝脏LDLR，但它也可能对其他脂蛋白受体（如apoER 2、LRP 1和VLDLR）以及肝外组织中的LDLR起作用。我们已发表的工作和初步研究已经解决了不同PCSK 9结构域在蛋白质分泌和功能中的作用。我们发现：1. CM 2的缺失产生分泌的和功能性的蛋白质; 2. CM 1或CM 3的缺失产生既不分泌也不起作用的蛋白质; 3.整个CD的缺失产生分泌但无功能的蛋白质; 4.缺乏CD的PCSK 9片段的PD促进缺乏PD的PCSK 9片段的分泌; 5. PCSK 9的离散CD结合溶液中LDLR的胞外域，并剂量依赖性地抑制全长PCSK 9细胞的LDLR降解作用; 6. PCSK 9经历pH依赖性自缔合，其由CA介导并增加LDL R降低功能性; 7. PCSK 9的过表达降低HEK 293 T细胞中的LDLR、apoER 2和LRP 1，并降低巨噬细胞中的LDLR水平; 8.人PCSK 9在巨噬细胞中的转基因表达降低了小鼠的肝脏LDLR并提高了血浆胆固醇水平。 该提议的中心假设是PCSK 9使用多个结构域相互作用进行分泌和功能，并通过靶向多个脂蛋白受体促进动脉粥样硬化形成。我们计划：1。检查C端结构域在PCSK 9分泌和LDLR结合中的作用; 2.检验结构域相互作用和自缔合调节PCSK 9分泌和功能的假设; 3.检验巨噬细胞分泌的PCSK 9通过对脂蛋白受体的多种作用改变动脉粥样硬化生物学的假设。研究PCSK 9的C-末端结构域和前结构域的这些新作用可能为抑制剂的开发提供新的线索，并促进我们对血浆胆固醇调节的理解。

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.