脂质代谢中活性蛋白的结构与功能研究

【作者】 孙国涛；

【导师】 陈保生；

【作者基本信息】 中国协和医科大学 ， 生物化学与分子生物学， 2006， 博士

【副题名】1. 人载脂蛋白A-V（apoA-V）及其缺失突变体的结构与功能研究 2. 小鼠脂质营养不良相关蛋白lipin1的功能研究

【摘要】 第一部分人载脂蛋白A-Ⅴ（apoA-Ⅴ）及其缺失突变体的结构与功能研究实验背景：高甘油三酯血症是冠心病发生的独立危险因素，而血浆甘油三酯（triglyceride，TG）水平是由非遗传因素（如肥胖、饮食、酗酒、吸烟等）和遗传因素共同决定的，其中遗传因素约占21～40％。载脂蛋白A-Ⅴ（apolipoprotein A-Ⅴ，apoA-Ⅴ）基因位于载脂蛋白A-Ⅰ／C-Ⅲ／A-Ⅳ基因簇内，含有4个外显子和编码366个氨基酸。ApoA-Ⅴ主要由肝脏合成，切除信号肽后，分泌到血浆，主要分布在高密度脂蛋白（high-density lipoprotein，HDL），少量分布在极低密度脂蛋白（very low-density lipoprotein，VLDL）和乳糜微粒（chylomicron，CM）。研究显示，apoA-Ⅴ基因敲除小鼠血浆TG水平是对照小鼠的4倍；而apoA-Ⅴ转基因小鼠血浆TG水平比对照小鼠降低约1／3。另外，基因敲除小鼠血浆VLDL水平升高，而在转基因小鼠降低。一些独立的研究已经表明，apoA-Ⅴ特定单核苷酸多态（single nucleotide polymorphism，SNP）位点与人甘油三酯水平密切相关。这些研究显示apoA-Ⅴ是一个重要的血浆甘油三酯水平决定因子和一个主要的冠心病危险因素。ApoA-Ⅴ通过激活LPL发挥降甘油三酯作用的结论，还存在争议。ApoA-Ⅴ可以结合脂质（如DMPC），形成直径大约15-20nm的圆盘状复合物。已有研究表明，ApoA-Ⅴ在大肠杆菌内表达为包涵体，变性条件下纯化后，普通PH条件下，溶解度较低（＜0.1mg／ml）；极端PH条件（50mM枸橼酸钠，PH3.0）下，溶解度提高；在此条件下，ApoA-Ⅴ二级结构中α螺旋占32％。实验目的：本研究对人apoA-Ⅴ和及其不同部位发生大片段缺失的突变体的二级结构和功能进行了研究和比较，以观察不同部位的序列对apoA-Ⅴ蛋白的结构和功能的影响；我们希望这些问题的解答能够对我们更深入、更全面的认识apoA-Ⅴ降甘油三酯的机制有所帮助。实验方法：根据预测的二级结构和疏水性特征，通过DNA重组，将apoA-Ⅴ按6部分依次进行缺失，产生一系列apoA-Ⅴ缺失突变体：A-Ⅴ（△（1-51））、A-Ⅴ（△（51-128））、A-Ⅴ（△（132-188））、A-Ⅴ（△（192-238））、A-Ⅴ（△（246-299））、A-Ⅴ（△（301-343）），利用pET原核表达系统表达成熟型及突变的apoA-Ⅴ蛋白，复性后进行Ni²⁺亲和柱纯化，获得多肽链C-末端带有6个组氨酸标签（6×His tag）的高纯度蛋白（纯度＞90％）用于下面的结构和功能实验。我们利用圆二色实验分析apoA-Ⅴ的二级结构和盐酸胍变性过程；浊度澄清实验分析野生型apoA-Ⅴ和缺失突变体与脂质结合的动力学特点，寻找能与脂质特异结合的片段；脂蛋白脂酶激活实验用于明确apoA-Ⅴ是否对脂蛋白脂酶有激活作用，进一步确定发挥激活作用的片段。实验结果：圆二色（CD）实验：ApoA-Ⅴα螺旋含量为46.26±5.08％。盐酸胍诱导的化学变性圆二色（CD）实验显示，apoA-Ⅴ的变性标准自由能（△G_D⁰）为1.94 kcal／mol apoA-Ⅴ，变性中点盐酸胍浓度(D_1/2)为2.02±0.19M。浊度澄清实验：分别在第192至238位间和第301至343位间片段缺失的突变体，即A-Ⅴ（△（192-238））和A-Ⅴ（△（301-343）），的浊度澄清速率常数k_1/2值都明显小于apoA-Ⅴ（P＜0.05），尤其是A-Ⅴ（△（192-238））突变体降低更为显著，提示这些缺失突变影响了apoA-Ⅴ多肽链与磷脂的相互作用，从而破坏蛋白与脂质的结合。A-Ⅴ（△（56-227））突变体浊度澄清速率常数k_1/2值接近A-Ⅴ（△（192-238）），进一步印证前述实验结果。而A-Ⅴ（△（51-128））和A-Ⅴ（△（132-188））突变体的k_1/2值显著大于apoA-Ⅴ（P＜0.05），提示缺失这两部分促进了apoA-Ⅴ与脂质的结合。其它各突变体的脂质结合动力学与apoA-Ⅴ无明显差异（p＞0.05）。脂蛋白脂酶激活实验：apoA-Ⅴ缺失突变体对LPL的激活作用都不同程度地降低。其中，A-Ⅴ（△（1-51））、A-Ⅴ（△（51-128））、A-Ⅴ（△（246-299））和A-Ⅴ（△（301-343））突变体在40pmol时LPL激活作用约为apoA-Ⅴ的2／3，A-Ⅴ（△（132-188））突变体激活作用约为apoA-Ⅴ的1／3，而突变体A-Ⅴ（△（192-238））和A-Ⅴ（△（56-227））几乎没有LPL激活作用。初步推断载脂蛋白A-Ⅴ激活LPL的活性结构域介于第192至第227这一较短的片段。结论：1．ApoA-Ⅴ属于α螺旋含量较高的蛋白，而且不同溶液环境下，二级结构会有一定程度的变化。相对于apoA-Ⅰ，apoA-Ⅴ维持着更为松散的结构。2．ApoA-Ⅴ蛋白不同部位对其脂质结合能力的影响差别很大；多肽链靠近氨基端区域主要维持apoA-Ⅴ的低可塑性；多肽链中央区域和羧基末端在介导apoA-Ⅴ与脂质结合过程中发挥重要作用，与apoA-Ⅰ相似。3．ApoA-Ⅴ不同片段对LPL激活作用有明显不同。多肽链中央区域对LPL激活作用至关重要，第192—227位氨基酸之间的片段，可能是激活LPL的关键结构域。第二部分小鼠脂质营养不良相关蛋白lipin1的功能研究脂质营养不良和肥胖正好是脂肪代谢的两个极端，而且可以归因于一些功能分类不同的基因表达的改变。研究已经表明，lipin1突变导致脂肪细胞分化障碍和小鼠脂质营养不良。通过组织特异性的脂肪细胞和骨骼肌细胞过表达动物模型，证明其可以促进肥胖。但机制不同，在脂肪细胞，影响脂肪贮存能力；而在骨骼肌细胞则决定整体能量消耗和脂肪利用。因此，单是lipin1水平的变化就可以引起脂肪代谢的极端状态，也反应了脂肪组织和骨骼肌调控脂肪多少和能量平衡的一种机制。我们的初步实验结果如下：（一）、完整小鼠lipin1蛋白在大肠杆菌中不表达，可能跟含有串联稀有密码有关；氨基端肽段原核表达为包涵体形式，制备出的兔抗小鼠多克隆抗体，与重组蛋白可以反应。（二）、荧光定位实验证实小鼠lipin1蛋白主要定位于细胞核内，而Gly84Arg突变确实导致定位改变，主要分布于胞浆，而Cys30Arg可以抑制这一突变体的作用。（三）、伴随绿色荧光蛋白的RNAi实验表明，像酵母细胞同源蛋白SMP2一样，lipin1可能与哺乳动物细胞的核被膜、内质网结构有关系，因为干扰lipin1表达后，绿色荧光蛋白在胞浆内呈斑点状分布。但需要进一步实验验证。（四）、建立的3T3-L1细胞分化模型为进一步研究lipin1的功能奠定基础。综上所述，我们克隆了小鼠lipin1全长编码区，并制备了多克隆抗体；对lipin1的核定位问题进行了初步研究；并对lipin1表达被干扰后细胞结构变化变化进行了简单探讨。更多还原

【Abstract】 PartⅠStructural and Functional Properties of Apolipoprotein A-Ⅴand Its Deletion MutantsBackground:Hypertriglyceridemia is known to be an independent risk factor for coronaryartery disease （CAD）. In addition to non-genetic factors such as overweight, diet,heavy smoking, excessive alcohol consumption, genetic factors are important indetermining serum triglyceride levels, accounting for 21-40％.APOA-Ⅴis located proximal to the well-characterized APOA-Ⅰ/C-Ⅲ/A-Ⅳgenecluster on human 11q23. Mice expressing a human APOA-Ⅴtransgene showed adecrease in plasma triglyceride concentrations to one-third of those in control mice;conversely, knockout mice lacking apoa-ⅴhad four times as much plasmatriglycerides as controls. The levels of very low-density lipoprotein （VLDL） particleswere increased in the homozygous knockout mice and decreased in the transgenicmice compared with controls. VLDL levels in a heterozygous knockout mouse wereintermediate between homozygous knockout and control mice. In humans, singlenucleotide polymorphisms （SNPs） across the APOA-Ⅴlocus were found to besignificantly associated with plasma triglyceride levels in several independent studies.These findings indicate that APOA-Ⅴis an important determinant of plasmatriglyceride levels, a major risk factor for coronary artery disease.The APOA-Ⅴgene consists of 4 exons and encodes a 366-amino acid protein.Far-UV circular dichroism analysis reveals that apoA-Ⅴpossesses 32％α-helixcontent. Protein structure analyses predicted several amphipathic helical domains andan N-terminal signal peptide, characteristic features of lipid-binding apolipoproteins, in both human and mouse apoA-Ⅴ. The apoA-Ⅴprotein is secreted by the liver and istransport primarily on large high-density lipoprotein （HDL）.Objectives:In this study, we compared the structural and functional properties of wild-typeapoA-Ⅴ（wtapoA-Ⅴ） and its 6 deletion mutants, each occurring on a separate segment.Our aim is to explore how each segment influences the structure and function ofapoA-Ⅴ, and whether there is a relationship between the segments and functions ofapoA-Ⅴ. We hope that our findings could shed some lights on the potentialmechanisms of the prominent triglyceride-lowering effects of apoA-Ⅴ.Methods:Six deletion mutants of apoA-Ⅴwere designed, according to structure predictionand hydrophobility analysis, and generated by DNA recombition. These mutants,named as A-Ⅴ（△（1-51））, A-Ⅴ（△（51-128））, A-Ⅴ（△（132-188））, A-Ⅴ（△,（192-238））,A-Ⅴ（△（246-299））, A-Ⅴ（△（301-343））, delete corresponding fragments between thetwo number from amino-terminal to carboxyl-terminal in turn. Both of wtapoA-Ⅴandthe deletion mutants were expressed with pET30b （+） as the expression vector andBL21 （DE3） as the host bacterial, respectively. After purified by Ni²⁺ affinitychromatography, all of the recombinant proteins were examined about the propertiesof their structures and functions. Circular dichrosim （CD） was employed to determinethe secondary structure and conformation stability of apoA-Ⅴ; Turbility clearanceassay was used to assess their abilities to bind DMPC liposome; LPL activation assaywas used to observe their capacities to promote TG hydrolysis by LPL.Results:CD Assays:Theα-helix content of wild-type apoA-Ⅴ（wtapoA-Ⅴ） in detergent-binding statewas 46.26±5.08％. Guanidine was used as a chemical denaturant to assess theconformation stability of apoA-Ⅴ. CD results showed that free energy of denaturation（△G_D⁰） of wtapoA-Ⅴwas 1.94±0.14 kcal/mol apoA-Ⅴand concentration ofguanidine at the midpoint of denaturation was 2.02±0.19. Turbility Clearance Assay:The mutations with deletion from 192 to 238 and 301 to 343 showedsubstantially reduced activities of binding lipids, for their binding rate constants (K_1/2)were low enough, compared with wtapoA-Ⅴ（p＜0.05）. Specially, the former mutantfell more obviously. And mutant A-Ⅴ（△（56-227）） showed a similar decrease in lipidaffinity. While on the contrary mutants A-Ⅴ（△（51-128）） and A-Ⅴ（△（132-188））displayed increase in lipid binding rates. And other mutants exhibited the samecapacities of disrupting DMPC liposomes as wtapoA-Ⅴ（p＞0.05）.LPL Activation Assay:All mutants showed reduced activation ability to LPL. The extent of activation ofmutants A-Ⅴ（△（1-51））,A-Ⅴ（△（51-128））,A-Ⅴ（△（246-299）） and A-Ⅴ（△（301-343））were about 2/3 of wtapoA-Ⅴ. And mutant A-Ⅴ（△（132-188）） reserved approximately1/3 activation ability. Although most mutants retained some activating ability, deletionof fragment between 192 and 238 caused almost complete loss of activity.Conclusion:1. ApoA-Ⅴexhibits a highα-helix content of 46％and a low free energy of stabilityof its alpha-helical segments （△G_D⁰） of 1.94 kcal/mol. ApoA-Ⅴadopts a looselyfolded conformation in solution, compared to apoA-Ⅰ.2. ApoA-Ⅴinteracts with bilayer vesicles of dimyristoylphosphatidylcoline to formdiscoidal complexes. Each fragment of apoA-Ⅴhas very different influence onthe lipid association property of this protein. The central and C-terminal region ofpolypeptide chain seems to play important roles in mediating the interactionbetween apoA-Ⅴand lipid. And the domain proximal to N-terminus of apoA-Ⅴmay give rise to low structure plasticity.3. ApoA-Ⅴcould activate lipoprotein lipase （LPL） effectively. Our data demonstratethat the central region of polypeptide chain is of special importance for the LPLactivation function of apoA-Ⅴ. And the structure integrality of apoA-Ⅴalso playsan essential role. PartⅡLipodystrophy and obesity represent extreme and opposite ends of the adiposityspectrum and have typically been attributed to alterations in the expression or functionof distinct sets of genes. Previous sdudies have demonstrated that lipin1 deficiencyimpairs adipocyte differentiation and causes lipodystrophy in the mouse. Using twodifferent tissue-specific lipin transgenic mouse strains, it has been demonstrated thatenhanced lipin1 expression in either adipose tissue or skeletal muscle promotesobesity. Thus, variations in lipin1 levels alone are sufficient to induce extreme statesof adiposity and may represent a mechanism by which adipose tissue and skeletalmuscle modulate fat mass and energy balance.Our primary experimental results show: （A）. The mouse lipin1 protein was notsuccessfully produced in E.coli as a whole, probably dueing to the existence of severalrare codons for E.coli. And the N-terminal fragment was overproduced as inclusionbodies in E.coli BL21（DE3）. We successfully produced rabbit polyclonal antibodiesagainst this fragment. （B）. Fluorescence localiztion analysis revealed that mouselipin1 protein is predominantly located in the nucleus of 3T3-L1 cell. Point mutationanalysis revealed that Cys（30） and Gly（84） in the N-terminal region are essential tolocalization of lipin1.（C）.We eatablished a primary relation between lipin1 and thestructure of nuclear envelope and endoplasmic reticulum by RNAi test withfluorescence.（D）. The construction of adipocyte differentiation model would offer achance to study the function of lipin1 deeply.In conclusion, we got the complete coding sequence of mouse lipin1 gene andproduced rabbit polyclonal antibodies. We studied the localization of mouse lipin1protein. And we also analyzed the relation between lipin1 and cell structure.更多还原