STRUCTURAL BIOLOGY--EARLY EVENTS IN LIPOPROTEIN ASSEMBLY

结构生物学——脂蛋白组装的早期事件

• 批准号: 6847165
• 负责人: DONALD M SMALL
• 金额: $ 21.27万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-01-26 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6847165
• 关键词: X ray crystallography; amphiphilicity; apolipoprotein B; binding sites; blood lipoprotein biosynthesis; electron microscopy; gene mutation; lipid bilayer membrane; membrane lipids; molecular assembly /self assembly; molecular chaperones; nuclear magnetic resonance spectroscopy; phospholipids; protein binding; protein folding; protein sequence; protein structure function; recombinant proteins; structural biology; transfection; triglycerides; very low density lipoprotein

The process of assembly of triacylglycerol rich lipoproteins (TAG-LP) is a complicated process involving the initial formation of a small primordial (120-200 AD) particle with a neutral lipid (TAG and CE) core and a later process which adds TAG and phospholipid (PL) to increase the size to that of a nascent VLDL (300-600 A D). Once synthesized apoB can follow 2 pathways: if lipid is available, apoB is secreted on nascent TAG rich particles-if lipid is deficient, apoB is degraded. Mammary derived C27 cells make no apolipoproteins, secreted no lipid and have no microsomal TAG transfer protein (MTP). These cells are used to study assembly directed only by the primary sequence of apoB. When C127 cells are transfected with cDNA for C-terminal truncated apoB forms, they efficiently secrete the N-terminal 17% of apoB (B17) in a lipid poor state but secrete apoB29, B32.5, B37, and B41 with progressively increasing amounts of lipids. We show that B29 binds PL and DAG, B32 binds PL and TAG, while the sequences between B32 and B41 bind mainly TAG. Thus, specific sites for PL and DAG binding appear in the sequence between B20 and B29 while specific TAG sites occur from B32 to B41. Structural analysis of B37 and B41 particles indicated that apoB must interact directly with the core. These truncated forms are secreted more efficiently when oleate is supplied and degraded if lipids are deficient. Several intermediate folding forms have been identified in the assembly process and these forms bind a variety of chaperones. Some chaperones appear to be involved in early folding events and others in targeting unlipidated, misfolded forms toward the degradation path/ A search for potential lipid binding sequences in B41 indicates that there are amphipathic beta strands (AbetaS) located between B21 and B41 probably organized in sheets of 2 to 4 strands. A consensus 27 aa amphipathic beta sheet was synthesized and bound avidly to a hydrocarbon/water interface lowering the interfacial tension from 50 to 22 Mn/M. The sheet bound elastically and could not be displaced from the oil/water interface when compressed. An ideal property for apoB binding to the hydrophobic core of TAG-LP.

富含三酰甘油的脂蛋白（TAG-LP）的组装过程是一个复杂的过程，包括最初形成具有中性脂质（TAG和CE）核心的小原始（120-200 AD）颗粒，以及随后添加TAG和磷脂（PL）以将尺寸增加到新生VLDL（300-600 AD）的尺寸的过程。一旦合成，apoB可以遵循2个途径：如果脂质可用，apoB分泌在新生的TAG丰富的颗粒上-如果脂质缺乏，apoB被降解。乳腺来源的C27细胞不产生载脂蛋白，不分泌脂质，也没有微粒体TAG转移蛋白（MTP）。这些细胞用于研究仅由apoB的一级序列指导的组装。当C127细胞用C-末端截短的apoB形式的cDNA转染时，它们以脂质贫乏的状态有效地分泌N-末端17%的apoB（B17），但分泌apoB 29、B32.5、B37和B41，其中脂质的量逐渐增加。我们发现B29结合PL和DAG，B32结合PL和TAG，而B32和B41之间的序列主要结合TAG。因此，PL和DAG结合的特异性位点出现在B20和B29之间的序列中，而特异性TAG位点出现在B32至B41之间。B37和B41粒子的结构分析表明，apoB必须直接与核心相互作用。这些截短的形式更有效地分泌时，油酸供应和降解，如果脂质不足。 在组装过程中已经鉴定了几种中间折叠形式，并且这些形式结合多种分子伴侣。一些分子伴侣似乎参与了早期折叠事件，而另一些分子伴侣则参与了靶向降解途径的非脂化、错误折叠形式。对B41中潜在脂质结合序列的搜索表明，B21和B41之间存在两亲性β链（AbetaS），可能组织成2至4条链的片层。合成了共有27个氨基酸的两亲性β折叠，并与烃/水界面紧密结合，将界面张力从50 Mn/M降低到22 Mn/M。当被压缩时，片材弹性地结合并且不能从油/水界面移位。apoB与TAG-LP疏水核心结合的理想特性。