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Deciphering the Structural Organization of Scavenger Receptor Class B Type I and Its Impact on Cholesterol Transport.

机译：解释B类清道夫受体的结构组织及其对胆固醇转运的影响。

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摘要

Atherosclerosis is characterized as an inflammatory disease involving interactions between low- and high-density lipoproteins (LDL and HDL, respectively), components of the immune system (macrophages and T cells), and endothelial and smooth muscle cells of the arterial wall. These interactions can lead to the formation of lesions in the artery wall, which can then cause myocardial infarction or stroke upon rupture. Since the risk for cardiovascular disease is inversely correlated with plasma HDL levels, HDL is considered to be “atheroprotective”. The primary atheroprotective characteristics of HDL can be attributed to its role in reverse cholesterol transport, the body's endogenous mechanism for cholesterol excretion. The last step of reverse cholesterol transport involves the binding of HDL to its receptor, scavenger receptor class B type I (SR-BI), followed by subsequent transfer of HDL-cholesteryl ester (CE) into the liver for disposal. One approach to improving cholesterol flux out of the body is to enhance HDL-CE removal via SR-BI. We hypothesize that efficient HDLcholesterol transport is dependent upon the structural organization of SR-BI at the plasma membrane. To test this hypothesis, we designed a spectrum of experiments that tested the importance of specific regions within the extracellular domain of SR-BI and also probed the native oligomeric status of SR-BI within the plasma membrane.;First, we investigated a stretch of hydrophobic residues within the Nterminal half of the extracellular domain of SR-BI and determined that this region (specifically a span of amino acids from position 67 to 221) was critical for HDL binding, selective uptake of HDL-cholesteryl ester (HDL-CE), efflux of free cholesterol (FC) to HDL, and redistribution of FC within the plasma membrane. These results suggest that this hydrophobic region may interact with the plasma membrane.;Second, by creating single cysteine (Cys, C) and Cys-less SR-BI mutants, we determined that all Cys residues in the extracellular domain participated in intramolecular disulfide bond formation, and that the presence of these bonds was required for HDL binding and selective uptake of HDL-CE; however, all but one of these residues (C323) was dispensable for cholesterol efflux, while none were required for redistribution of cholesterol within the plasma membrane. These results suggest that a specific disulfide bonding pattern is required to maintain SR-BI in a conformation that supports these cholesterol transport functions.;We next exploited CD36, a related protein that can bind HDL but is unable to mediate selective uptake of HDL-CE. We created a panel of SR-BI/CD36 chimeric receptors to further delineate the boundaries of the functional subdomains within the extracellular domain and observed that key sub-domains within the N-terminal half of the extracellular domain, that coincidentally overlap with important hydrophobic regions identified in our earlier study, are responsible for HDL binding and selective uptake of HDL-CE. We also identified a subdomain in the C-terminal half of the extracellular domain that may overlap with putative dimerization motifs.;Finally, we utilized bimolecular fluorescence complementation coupled to fluorescence resonance energy transfer (BiFC-FRET) to investigate the oligomeric organization of SR-BI in live cells in the presence and absence of ligand (i.e. HDL). The novelty of this technique lies in its ability to detect interactions between three proteins of interest. Our BiFC-FRET studies suggested that SR-BI exists as a constitutive oligomer in the absence of HDL ligand; binding of ligand then causes a conformational change within the oligomeric complex.;Determining the mechanisms of the cholesterol transport functions of SRBI is crucial in understanding its protective role against atherosclerosis. As the SR-BI/HDL interaction is critical to lowering plasma cholesterol levels, an improved understanding of the structure-function relationships within this receptor-ligand complex will hopefully lead the development of new therapeutic strategies to combat this devastating disease.
机译：动脉粥样硬化的特征是炎性疾病，涉及低密度脂蛋白和高密度脂蛋白（分别为LDL和HDL），免疫系统的组成部分（巨噬细胞和T细胞）以及动脉壁的内皮和平滑肌细胞之间的相互作用。这些相互作用可导致在动脉壁上形成病变，然后在破裂时可引起心肌梗塞或中风。由于心血管疾病的风险与血浆HDL水平成反比，因此HDL被认为具有“抗动脉粥样硬化作用”。 HDL的主要抗动脉粥样硬化特性可归因于其在胆固醇逆向运输中的作用，胆固醇是体内胆固醇排泄的内源性机制。胆固醇逆向转运的最后一步涉及将HDL与其受体I类清道夫受体（SR-BI）结合，随后将HDL-胆固醇酯（CE）转移至肝脏中进行处置。改善胆固醇从体内流出的一种方法是通过SR-BI增强HDL-CE的去除。我们假设有效的HDL胆固醇运输取决于质膜上SR-BI的结构组织。为了验证这一假设，我们设计了一系列实验，测试了SR-BI胞外域内特定区域的重要性，并探查了SR-BI在质膜内的天然寡聚状态。 SR-BI胞外域N末端一半内的疏水残基，并确定该区域（特别是从67到221位氨基酸的跨度）对于HDL结合，选择性摄取HDL-胆固醇酯（HDL-CE）至关重要，游离胆固醇（FC）向HDL的外流以及FC在质膜内的重新分布。这些结果表明该疏水区域可能与质膜相互作用。其次，通过产生单个半胱氨酸（Cys，C）和少半胱氨酸的SR-BI突变体，我们确定了胞外域中的所有半胱氨酸残基均参与了分子内二硫键HDL结合和HDL-CE的选择性摄取需要这些键的存在；然而，这些残基（C3​​23）中只有一个残基可用于胆固醇外排，而在质膜内不需要再分配胆固醇。这些结果表明，需要一种特定的二硫键结合模式才能将SR-BI维持在支持这些胆固醇转运功能的构象中;我们接下来开发了可与HDL结合但无法介导HDL-CE选择性摄取的相关蛋白CD36。 。我们创建了一组SR-BI / CD36嵌合受体，以进一步描绘细胞外域内功能性亚域的边界，并观察到细胞外域N端一半内的关键子域与重要的疏水性区域重合在我们较早的研究中鉴定出的结果与HDL结合和HDL-CE的选择性摄取有关。我们还在细胞外结构域的C末端一半中发现了一个亚域，该亚域可能与假定的二聚化基序重叠。最后，我们利用双分子荧光互补偶联荧光共振能量转移（BiFC-FRET）来研究SR-的寡聚结构存在和不存在配体（即HDL）时，活细胞中的BI。该技术的新颖之处在于它能够检测三种目标蛋白之间的相互作用。我们的BiFC-FRET研究表明，在没有HDL配体的情况下，SR-BI以结构性低聚物的形式存在。配体的结合随后在寡聚复合物中引起构象变化。；确定SRBI的胆固醇转运功能的机制对于理解其对动脉粥样硬化的保护作用至关重要。由于SR-BI / HDL相互作用对于降低血浆胆固醇水平至关重要，因此对这种受体-配体复合物中的结构-功能关系的更好理解将有望引领新的治疗策略的发展，以对抗这种破坏性疾病。

著录项

  • 作者

    Kartz, Gabriella Alyssa.;

  • 作者单位

    The Medical College of Wisconsin.;

  • 授予单位 The Medical College of Wisconsin.;
  • 学科 Health Sciences Pharmacology.;Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 207 p.
  • 总页数 207
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 高分子化学（高聚物）;
  • 关键词

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