PCSK9, Lipoprotein receptors, and Atherosclerosis

描述(申请人提供)：枯草杆菌蛋白原转换酶9(PCSK9)通过促进低密度脂蛋白受体(LDLR)的细胞内降解来调节血浆胆固醇水平，低密度脂蛋白受体是从血浆中清除低密度脂蛋白的主要驱动力。人PCSK9由692个氨基酸组成。在去除信号肽(残基1-30)后，分泌的PCSK9呈现三个结构域：1.N-末端前结构域(PD，残基31-152)，它被切割，但仍与蛋白质的其余部分结合，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)，包含一个被相关PD掩蔽和中和的蛋白水解区；3.C-末端结构域(CD，残基453-692)，由三个紧密结合的模块组成，CM1(残基457-527)，CM2(残基534-601)和CM3(残基608-679)。普遍认为，PCSK9通过其CA与LDLR的A型表皮生长因子(EGF-A)重复序列结合，但不直接引起LDLR的蛋白分解。了解PSCK9的功能对于开发降低胆固醇水平和心血管风险的新型治疗药物至关重要。虽然PCSK9主要针对肝脏低密度脂蛋白受体，但它也可能作用于其他脂蛋白受体，如ApoER2、LRP1和VLDLR，以及肝外组织中的低密度脂蛋白受体。我们已发表的工作和初步研究已经解决了不同的PCSK9结构域在蛋白质的分泌和功能中的作用。我们发现：1.CM2的缺失产生一种分泌的和有功能的蛋白质；2.CM1或CM3的缺失产生一种既不分泌也不具有功能的蛋白质；3.整个CD的缺失产生一种分泌但不具有功能的蛋白质；4.缺乏CD的PCSK9片段的PD促进缺乏PD的PCSK9片段的分泌；5.PCSK9的离散CD结合溶液中LDLR的胞外结构域，并剂量依赖地抑制全长PCSK9细胞的LDLR降解作用；6.PCSK9经历由CA介导的pH依赖的自结合，并增加LDLR还原功能；7.过表达PCSK9可降低HEK293T细胞的低密度脂蛋白受体、ApoER2和LRP1，并降低巨噬细胞的低密度脂蛋白受体水平；8.人PCSK9基因在巨噬细胞中的转基因表达可降低小鼠肝脏低密度脂蛋白受体的表达，并提高小鼠的血浆胆固醇水平。这一建议的中心假设是PCSK9利用多个结构域的相互作用进行分泌和功能，并通过靶向多个脂蛋白受体而促进动脉粥样硬化的形成。我们计划：1.检测C-末端结构域在PCSK9分泌和LDLR结合中的作用；2.检验结构域相互作用和自结合调节PCSK9分泌和功能的假说；3.检验巨噬细胞分泌的PCSK9通过对脂蛋白受体的多方面影响而改变动脉粥样硬化生物学的假说。研究PCSK9的S C端区和前C端区的这些新的作用，可能会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.