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首页> 外文学位 >Exploration of the Function of a Non-Prolyl Cis-Peptide Bond in Human Plasma Platelet-Activating Factor Acetylhydrolase
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Exploration of the Function of a Non-Prolyl Cis-Peptide Bond in Human Plasma Platelet-Activating Factor Acetylhydrolase

机译：人血浆血小板活化因子乙酰水解酶中非脯氨酸顺肽键功能的探索

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摘要

Plasma platelet-activating factor acetylhydrolase (PAF-AH), also known as lipoprotein-associated phospholipase A2, is an extracellular 45 KDa monomer that circulated in plasma and associates with lipoproteins. As a group-VIIA PLA2 enzyme, it catalyzes the hydrolysis of the acetyl group at the sn-2 position of platelet-activating factor (PAF) to form the products lyso-PAF and acetate. Additionally, PAF-AH can hydrolase a wide range of substrates with a short acetyl chain at the sn-2 position like oxidized phospholipids from LDL oxidation. In order to keep PAF-AH constitutively active without undermining cellular integrity, the enzyme only targets oxidatively fragmented phospholipids. A majority of PAF-AH is bound to low-density lipoprotein (LDL), which might be correlated with inflammatory atherogenesis. Studies show that an increase of PAF-AH concentration or activity could possibly lead to an increase in risk of atherogenesis, which plays a significant role in heart diseases. Many reversible PAF-AH inhibitors were developed in the pharmaceutical industry for the use as an extra treatment for patients with heart diseases.;According to the solved structure via x-ray crystallization, there is a rare non-prolyl cis-peptide bond located between Phe-72 and Asp-73, which is far away from the active site catalytic triad formed by Ser-273, Asp-296 and His-351. Due to the high energy barrier of cis-trans isomerization, this conformational change is thought to be the rate-limit step in the protein folding process. Therefore, the existence of a cis-peptide bond usually is thought to play a great role in protein function or enhance structural stability in some way. The purpose of the existence of this non-prolyl cis-peptide in PAF-AH is still unsolved. Based on the previous work of plasma PAF-AH's cis-peptide bond, a hypothesis was formed that this non-prolyl cis-peptide bond plays a role in maintaining the binding between LDL and PAF-AH.;Work presented in this thesis aims to test this hypothesis. An E. coli expression construct of a truncated form of PAF-AH (DeltaPAF-AH) was obtained from former lab member from the Bahnson lab. In an effort to explore the function role of a cis-peptide bond at this position, two site-directed mutants (D73G and D73P) were created at the position of this cis-peptide bond. D73G mutant aims to mimic the mouse PAF-AH that is reported only bound to high density lipoprotein (HDL) and D73P mutant aims to mimic the common prolyl cis-peptide bond. Expression and purification of DeltaPAF-AH, as well as cis-peptide bond mutants, were performed using E. coli expression construct. Enzyme activity assays using a general substrate were carried out and proved that there were no functional differences between wild type and cis-peptide bond mutants, indicating the cis-peptide bond does not play a role in enzyme activity. Moreover, there are no significant differences of circular dichroism (CD) secondary structure test between wild type and mutants, eliminating the possibility that this cis-peptide bond may have a influence on protein folding. Through CD thermodynamics test, there was a significant decrease of stability of D73G mutant while D73P mutant only show a little decline of stability. Possible explanation is that D73G might have changed this position to a trans configuration, which leads to the instability of protein. In order to test this explanation, high-purity mutant protein samples were produced for further crystallization trials.
机译：血浆血小板活化因子乙酰水解酶（PAF-AH），也称为脂蛋白相关磷脂酶A2，是一种在血浆中循环并与脂蛋白缔合的细胞外45 KDa单体。作为VIIA组PLA2酶，它催化血小板活化因子（PAF）的sn-2位置处的乙酰基水解，形成溶血PAF和乙酸盐。另外，PAF-AH可以水解在sn-2位置具有短乙酰基链的多种底物，如LDL氧化产生的氧化磷脂。为了在不损害细胞完整性的情况下保持PAF-AH的组成型活性，该酶仅靶向氧化片段化的磷脂。大多数PAF-AH与低密度脂蛋白（LDL）结合，这可能与炎症性动脉粥样硬化有关。研究表明，PAF-AH浓度或活性的增加可能导致动脉粥样硬化风险的增加，这在心脏病中起重要作用。制药行业开发了许多可逆的PAF-AH抑制剂，可作为心脏病患者的额外治疗方法；根据通过X射线结晶确定的结构，在两者之间存在罕见的非脯氨酰顺肽键Phe-72和Asp-73远离由Ser-273，Asp-296和His-351形成的活性中心催化三联体。由于顺反异构化的高能垒，这种构象变化被认为是蛋白质折叠过程中的限速步骤。因此，通常认为存在顺肽键在蛋白质功能中起重要作用或以某种方式增强结构稳定性。在PAF-AH中存在这种非脯氨酰基顺式肽的目的仍未解决。基于血浆PAF-AH的顺肽键的先前工作，形成了一个假设，即该非脯氨酰的顺肽键在维持LDL和PAF-AH之间的结合中起作用。检验这个假设。 PAF-AH的截短形式的大肠杆菌表达构建体（DeltaPAF-AH）从Bahnson实验室的前实验室成员获得。为了探索顺式肽键在此位置的功能作用，在该顺式肽键的位置产生了两个定点突变体（D73G和D73P）。 D73G突变体旨在模仿仅与高密度脂蛋白（HDL）结合的小鼠PAF-AH，而D73P突变体旨在模仿常见的脯氨酰顺肽键。使用大肠杆菌表达构建体进行DeltaPAF-AH以及顺式肽键突变体的表达和纯化。进行了使用通用底物的酶活性测定，结果证明野生型和顺式肽键突变体之间没有功能差异，表明顺式肽键在酶活性中不起作用。此外，野生型和突变型之间的圆二色性（CD）二级结构测试没有显着差异，消除了这种顺肽键可能影响蛋白质折叠的可能性。通过CD热力学测试，D73G突变体的稳定性显着降低，而D73P突变体仅显示出少量的稳定性下降。可能的解释是D73G可能已将此位置更改为反式构型，从而导致蛋白质不稳定。为了检验这一解释，制备了高纯度的突变蛋白样品用于进一步的结晶试验。

著录项

  • 作者

    Xie, Shilin.;

  • 作者单位

    University of Delaware.;

  • 授予单位 University of Delaware.;
  • 学科 Biochemistry.
  • 学位 M.S.
  • 年度 2017
  • 页码 81 p.
  • 总页数 81
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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