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

战略研究重点：健康生物科学摘要世界卫生组织预测，糖尿病将成为2030年的第七大死因。锌α 2糖蛋白（ZAG）是一种脂肪因子，可分解人体脂肪细胞（脂解）。越来越多的生化研究证据表明ZAG与糖尿病相关，但其涉及脂质与MHC样沟结合的生化机制仍不清楚。该项目将使用最先进的结构，生物物理和计算方法的基础上的脂质结合研究ZAG，以确定ZAG诱导脂解的分子机制。学生将在设备齐全的免疫学实验室中受益于独特的跨学科培训机会。项目背景：ZAG具有I类MHC样蛋白折叠，在其a1和a2结构域螺旋之间具有开放的顶沟。然而，ZAG与MHC的不同之处在于它是可溶的，不锚定在质膜上，并且与催乳素诱导蛋白而不是B2-微球蛋白相关。人ZAG的原始晶体结构在其大沟（PDB 1 T7 Z）中显示出无法识别的电子密度。我们发现该密度是聚乙二醇，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]，预计靠近a1和a2结构域螺旋。今年，通过结合荧光滴定和荧光检测的分析超离心，我们意外地发现ZAG沟中至少有两个不同的脂质结合位点（参考文献2）。目的：研究ZAG的脂质结合特性，揭示其作用的分子机制。 研究方案：采用E.大肠杆菌重组和人血浆纯化的ZAG。通过荧光标记重组ZAG，我们可以从血浆中鉴定含有ZAG的组分，并从中分离ZAG。由此，我们可以创建抗体或脂质包被的亲和柱，以从人血浆中分离天然ZAG。首先，我们将使用LC-MS质谱法来鉴定ZAG的内在脂质，使用血浆纯化的ZAG，向其中加入通过离心分离的血浆脂质丰富/蛋白质贫乏部分。化学脂质文库将筛选紧密结合的脂质。其次，我们将使ZAG重结晶，并将晶体浸泡在富含脂质的血浆部分中，以促进具有最强亲和力的脂质结合。晶体结构将识别结合的脂质及其构象。晶体也将暴露于锌以鉴定ZAG中的强锌结合位点。第三，已经鉴定了最紧密结合的脂质，用荧光标记的脂质的竞争实验将鉴定观察到的结合到ZAG沟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.