Apo-B Domains and Lipoprotein Structure and Assembly

富含甘油三酯的脂蛋白(Tag-LP)的组装涉及到小hdl大小的初始形成。 具有中性脂核的原始颗粒和将TAG和磷脂(PL)添加到 创造新生的极低密度脂蛋白。ApoB(B0.5)的N端20.5%与卵黄蛋白(LV)有同源性，LV包括 一个N-末端的β桶，一个由17个两亲性α螺旋(Aah)和2个β片域组成的区域，A和C 床单。由于某些点突变和失败，N端的适当折叠是分泌所必需的 以形成二硫键为目标进行降解。617和B20.5的有限蛋白降解表明apoB 与LV的同源性是近似的，但不是精确的。分离蛋白水解物及其表达产物的结构测定 用CD、核磁共振和X射线衍射法确定B20.5结构域将在分子水平上确定。这个 在MTP缺陷的C127细胞中研究载脂蛋白B初级序列驱动的脂类组装 用C-末端截断的形式转染。分子伴侣在折叠、脂化和蛋白质合成中的作用 在C127和肝癌来源的细胞中都进行了质量控制研究。从B20.5到B29的地区新兵 表面分子，主要是光致发光到新生颗粒。随着多肽从B20.5延长到B29， 表面分子的数量从每个粒子大约5个增加到大约70个。B32和B41之间序列招募了近200个TAG(大约1个TAG/2.3AA)，其形成具有低温EM可辨别核心的新生粒子 这在B32.5中是没有的。生物物理分析表明，B37和B41多肽必须直接与 核心。该区域包含24个两亲性β链(AP)，长度为12-15个氨基酸。共识12 AA APS和 27个氨基酸P片肽结合到油/水(O/W)界面，具有弹性，在高压下不被驱替。 载脂蛋白B还含有aah的a2和a3两个主要区域。共识AAH多肽与O/W和空气/水结合 但在临界压力下被喷射到水相中。本机apoB还绑定到 Tag/W接口，不能被高压完全移位，但apoB的部分在以下情况下脱落 压缩，并在曲面再次展开时快速恢复。因此，我们建议灵活 ApoB的部分是Aah，不可交换的部分是AbetaS。这个项目通过以下方式剖析了复杂的途径 哪种“坏胆固醇”(载脂蛋白B相关的胆固醇，极低密度脂蛋白，低密度脂蛋白)是由肝脏制造和分泌的。这个 长期目标是找到减少坏胆固醇、心脏病发作和中风的程序或药物。

The assembly of triadyglycerol-rich lipoproteins (TAG-LP) involves the initial formation of a small HDL sized primordial particle with a neutral lipid core and a later process which adds TAG and phospholipid (PL) to create nascent VLDL. The N-terminal 20.5% of apoB(B0.5) has homology to lipovitellin (LV) that consists of an N-terminal beta barrel, a region of 17 amphipathic alpha helices (AaH) and 2 beta sheet domains, the A and C sheets. Proper folding of the N-terminal is necessary for secretion since certain point mutations and failure to form disulfide bonds target it to degradation. Limited proteolysis of 617 and B20.5 indicates that apoB homology to LV is approximate but not exact. Structural determination of isolated proteolytic and expressed domains using CD, NMR and X-ray diffraction will define B20.5 domains at the molecular level. The assembly with lipids as driven by the primary sequence of apoB is studied in MTP-deficient C127 cells transfected with C-terminally truncated forms. The role of molecular chaperones in folding, lipidation and in quality control is studied both in C127 and hepatoma-derived cells. The region from B20.5 to B29 recruits surface molecules, mainly PL to the nascent particle. As the peptide lengthens from B20.5 to B29 the number of surface molecules increases from approximately 5 to about 70 per particle. The sequence between B32 and B41 recruits almost 200 TAG (about 1 TAG/2.3 aa) which form nascent particles with a cryo-EM discernable core which is not seen in B32.5. Biophysical analysis shows that B37 and B41 peptides must interact directly with the core. This region contains 24 amphipathic beta strands (ApS) 12-15 aa long. Consensus 12 aa ApS and 27 aa p sheet peptides bind to oil/water (O/W) interfaces, are elastic and are not displaced at high pressure. ApoB also contains 2 major regions a2 and a3 of AaH. Consensus AaH peptides bind to O/W and air/water interfaces, but are ejected at a critical pressure into the aqueous phase. Native apoB also binds to the TAG/W interface and cannot be fully displaced by high pressure, but parts of the apoB come off when compressed and snap back on rapidly when the surfaces are again expanded. Thus, we suggest the flexible parts of apoB are AaH and the non-exchangeable part is AbetaS. This project dissects the complex pathway by which "bad cholesterol" (apoB associated cholesterol, VLDL, LDL) is made and secreted by the liver. The long term goal is to find procedures or drugs to reduce bad cholesterol, heart attack and stroke.