Structure-function studies of a lipid-binding class I MHC-like protein may lead to a possible treatment for type 2 diabetes

世界卫生组织预测，到2030年，糖尿病将成为第七大死因。锌a2糖蛋白（ZAG）是一种脂肪因子，可以分解人体脂肪细胞（脂解）。生化研究越来越多的证据表明ZAG与糖尿病有关，但其涉及脂质与mhc样槽结合的生化机制尚不清楚。本项目将采用基于ZAG脂质结合研究的最先进的结构、生物物理和计算方法来确定ZAG诱导的脂质分解的分子机制。学生将受益于独特的跨学科培训机会，在一个设备齐全的免疫学实验室。项目背景：ZAG具有一类mhc样蛋白折叠，其a1和a2结构域螺旋之间有一个开放的顶端凹槽。然而，与MHC不同的是，ZAG是可溶的，不固定在质膜上，并且与催乳素诱导蛋白而不是b2微球蛋白结合。人类ZAG的原始晶体结构显示其主槽（PDB 1T7Z）的电子密度无法识别。我们发现这个密度是聚乙二醇，一种结晶助剂（参考文献4）。我们还发现ZAG含有一个紧密结合的锌离子[参考文献1]，预计位于a1和a2结构域螺旋附近。今年，通过结合荧光滴定和荧光检测的分析性超离心，我们意外地发现在ZAG凹槽中至少有两个不同的脂质结合位点（参考文献2）。目的：鉴定ZAG的脂质结合特性，揭示其作用的分子机制。研究计划：我们将使用重组大肠杆菌和人血浆纯化ZAG。通过荧光标记重组ZAG，我们可以从血浆中识别出含有ZAG的部分，并从中分离出ZAG。由此，我们可以建立抗体或脂质包被亲和柱，从人血浆中分离天然ZAG。首先，我们将使用LC-MS质谱法鉴定ZAG的内在脂质，使用血浆纯化的ZAG，加入离心分离的血浆富脂/贫蛋白部分。化学脂质文库将筛选紧密结合的脂质。其次，我们将ZAG重新结晶，并将晶体浸泡在富含脂质的血浆中，以促进具有最强亲和力的脂质的结合。晶体结构将识别结合的脂质及其构象。晶体也将暴露在锌中，以确定ZAG中强锌结合位点。第三，在确定了结合最紧密的脂质后，用荧光标记的脂质进行竞争实验，将确定观察到的与ZAG凹槽结合的结合脂质的亲和力。

Strategic Research Priority: Bioscience for HealthAbstract The WHO predicts that diabetes will be the seventh leading cause of death in 2030. Zinc a2 glycoprotein (ZAG) is an adipokine that breaks down human fat cells (lipolysis). There is growing evidence from biochemical studies to suggest that ZAG is relevant to diabetes, however its biochemical mechanism involving lipid binding to its MHC-like groove remains unknown. This project will use state-of-the-art structural, biophysical and computational methods based on lipid-binding studies to ZAG to identify the molecular mechanism of ZAG-induced lipolysis. The student will benefit from unique cross-disciplinary training opportunities in a well-equipped immunology laboratory. Project Background: ZAG possesses a class I MHC-like protein fold with an open apical groove between its a1 and a2 domain helices. However ZAG is distinct from MHC by being soluble and not anchored to plasma membranes, and associates with prolactin-inducible protein rather than B2-microglobulin. The original crystal structure of human ZAG revealed unidentifiable electron density in its major groove (PDB 1T7Z). We found that this density is polyethylene glycol, a crystallization adjuvant (Ref.4). We also showed that ZAG contains one tightly-bound zinc ion [Ref.1], predicted to lie close to the a1 and a2 domain helices. This year, by combining fluorescent titrations and fluorescent-detected analytical ultracentrifugation, we have unexpectedly shown that there are at least two distinct lipid binding sites in the ZAG groove (Ref.2). Aim: The lipid-binding properties of ZAG will be identified to uncover its molecular mechanism of action. Plan of investigation: We will use E. coli recombinant and human plasma-purified ZAG. By fluorescent-tagging recombinant ZAG, we can identify the ZAG-containing fraction from plasma, and isolate ZAG from this. From this, we can create an antibody or lipid-coated affinity column to isolate native ZAG from human plasma. First we will use LC-MS mass spectrometry to identify ZAG's intrinsic lipid using plasma-purified ZAG, adding a plasma lipid-rich/protein-poor fraction separated by centrifugation to this. Chemical-lipid libraries will screen for tightly-bound lipids. Second, we will re-crystallise ZAG, and soak the crystals in a lipid-rich plasma fraction in order to facilitate the binding of the lipid with the strongest affinity. The crystal structure will identify the bound lipid and its conformation. Crystals will also be exposed to zinc to identify the strong zinc binding site in ZAG. Third, having identified the tightest bound lipids, competition experiments with fluorescent-labelled lipids will identify the affinities of the bound lipids observed to bind to the ZAG groove.