Structure and Function of the LPLA2/LCAT Acyltransferase Family

描述（由申请方提供）：溶酶体磷脂酶A2（LPLA 2）在脂质降解中起主要作用，并被认为是药物诱导的磷脂质病的基础，这通常发生在服用阳离子亲脂性药物（如抗心律失常胺碘酮）的患者中。异常的LPLA 2活性也可能参与自身免疫性疾病和动脉粥样硬化的发展。LPLA 2在序列上与卵磷脂-胆固醇酰基转移酶（LCAT）50%相同，LCAT是通过高密度脂蛋白（HDL）从动脉斑块巨噬细胞逆向转运胆固醇的关键酶。LCAT基因突变导致家族性LCAT缺乏症（FLD），这是一种以低血清胆固醇酯水平和肾衰竭为特征的毁灭性疾病。没有报道LPLA 2或LCAT的原子模型，其与已知结构的其他蛋白质没有显著的同源性。因此，其底物选择性，调节和疾病表型的分子基础仍然知之甚少。在这项提案中，我们通过对LPLA 2新的1.8“晶体结构的功能分析，LCAT原子结构的测定，通过电子显微镜对与HDL颗粒结合的LCAT进行成像，绘制已知引起遗传疾病的体细胞突变，以及研究酰基受体选择性差异的结构基础，来解决这一关键的知识空白。为了支持我们的目标，我们提供了LPLA 2在各种配体状态下的多个高分辨率结构，LCAT-HDL复合物的负染色图像，以及完全糖基化的LCAT的低分辨率晶体结构。这些研究的预期结果是更好地理解在脂质代谢中起关键作用的结构上未表征的真核酶家族的机制。我们的结构和功能研究将有助于解释遗传疾病的分子基础，并最终帮助设计改进的生物治疗药物和小分子LCAT激活剂，以治疗脂质相关疾病，如动脉粥样硬化和LCAT缺乏症。

DESCRIPTION (provided by applicant): Lysosomal phospholipase A2 (LPLA2) plays a major role in lipid degradation and is believed to underlie drug-induced phospholipidosis, which commonly occurs in patients taking cationic lipophilic drugs such as the antiarrhythmic amiodarone. Aberrant LPLA2 activity may also be involved in development of autoimmune disease and atherosclerosis. LPLA2 is 50% identical in sequence to lecithin-cholesterol acyltransferase (LCAT), a key enzyme in reverse cholesterol transport from arterial plaque macrophages via high density lipoproteins (HDL). Genetic mutations in LCAT are responsible for Familial LCAT Deficiency (FLD), a devastating disease characterized by low serum cholesterol ester levels and renal failure. There are no reported atomic models for either LPLA2 or LCAT, which do not have significant homology to other proteins of known structure. Thus, the molecular bases for their substrate selectivity, regulation, and disease phenotypes remain poorly understood. In this proposal, we address this critical gap in knowledge via functional analysis of our new 1.8 � crystal structure of LPLA2, determination of the atomic structure of LCAT, imaging LCAT bound to HDL particles by electron microscopy, mapping somatic mutations known to cause genetic disease, and investigating the structural basis for differences in acyl acceptor selectivity. In support of our aims, we provide multiple high resolution structure of LPLA2 in various ligand states, negative stained images of LCAT-HDL complexes, and a low resolution crystal structure of fully glycosylated LCAT. The expected outcome of these studies is a better mechanistic understanding of a structurally uncharacterized family of eukaryotic enzymes that play key roles in lipid metabolism. Our structural and functional studies will help explain the molecular basis for genetic disease and ultimately assist in the design of improved biotherapeutics and small molecule LCAT activators to treat lipid-related disorders such as atherosclerosis and LCAT deficiency.