Mapping MTP lipid transfer activities for better therapeutics

载脂蛋白b (apoB)-含脂蛋白既是一种生物标志物，也是代谢性疾病的许多中心标志的因果介质，包括胰岛素抵抗，脂肪性肝病，动脉粥样硬化，肥胖和代谢综合征。微粒体甘油三酯转移蛋白（MTP）是一种由MTP和蛋白二硫异构酶（PDI）亚基组成的异二聚体复合物，抑制MTP可显著减少50%的特异性致动脉粥样硬化性载脂蛋白，但会导致肝纤维化和肠脂肪溢。MTP复合物转移不同的脂质并协助含载脂蛋白的脂蛋白的产生。我们最近的工作提供了第一个证据，证明MTP的甘油三酯（TG）和磷脂（PL）传递功能可以解耦，并且抑制TG传递活性不会导致斑马鱼脂肪变性，这些鱼像野生型鱼一样正常生长。我们假设，关于这两个脂质转移域的原子水平细节可能为选择性抑制TG转移到较低的血浆脂质的药理作用铺平道路，而不会引起细胞脂质保留的不利影响。我们要问的基本问题是：“MTP如何区分不同的脂质配体以及抑制TG转移活性的后果是什么？”目的1：表征MTP中不同的脂质结合位点：我们将用不同的脂质配体求解MTP结构，以获得原子水平的细节。突变分析将阐明对特定脂质结合至关重要的氨基酸残基。目的2：确定脂质转移过程中MTP和PDI亚基的构象变化，以及与MTP亚基相互作用的不同PDI家族成员：我们假设MTP和PDI亚基的构象变化是为了适应不同的脂质而发生的。我们将在MTP的柔性环区和PDI的一个域进行定点诱变，以剖析其机制。尽管PDI对MTP活性是必需的，但不同PDI的特异性尚不清楚。我们将检验其他PDI家族成员与MTP亚基相互作用以及这些相互作用具有生理后果的假设。目的3：评估消除MTP的TG转移活性的生物学后果：在进一步确定消除TG转移的突变后，我们将确定这些突变是否支持细胞中载脂蛋白ob的分泌，降低小鼠的血浆脂质，并维持正常的鱼类生长。所提出的研究将提供新的信息：1)MTP在不同脂质转移中的结构域和氨基酸；2)不同脂质转移过程中的构象变化；3)突变这些关键残留物的生化、生理和有机体后果。这一新知识将是无价的，在未来，开发新的和TG转移特异性抑制剂的MTP。

Apolipoprotein-B (apoB)-containing lipoproteins are both a biomarker and a causal mediator of many central hallmarks of metabolic disease, including insulin resistance, fatty liver disease, atherosclerosis, obesity and metabolic syndrome. Inhibition of microsomal triglyceride transfer protein (MTP), a heterodimeric complex of MTP and protein disulfide isomerase (PDI) subunits, profoundly reduces specifically atherogenic apoB- containing lipoproteins by 50%, but it causes hepatosteatosis and steatorrhea of the intestine. MTP complex transfers different lipids and assists in the production of apoB-containing lipoproteins. Our recent work provides the first evidence that the triglyceride (TG) and phospholipid (PL) transfer functions of MTP can be decoupled and that inhibition of TG transfer activity in zebrafish does not result in steatosis and these fish grow normally like wild-type fish. We hypothesize that atomic level details about these two lipid transfer domains may pave the way for selective pharmacological inhibition of TG transfer to lower plasma lipids without causing the adverse effects of cellular lipid retention. The fundamental question we are asking is: “how MTP distinguishes different lipid ligands and what are the consequences of inhibiting TG transfer activity?” Aim 1: Characterize the different lipid-binding sites in MTP: We will solve MTP structures with different lipid ligands to obtain atomic level details. Mutational analysis will elucidate amino acid residues critical for binding of specific lipids. Aim 2: Identify conformational changes in MTP and PDI subunits during lipid transfer, and different PDI family members that interact with MTP subunit: We hypothesize that conformational changes in both the MTP and PDI subunits occur to accommodate different lipids. We will perform site-directed mutagenesis in the flexible loop region of MTP and a’ domain of PDI to dissect out the mechanisms for this. Although PDI is obligatory for MTP activity, the specificity of different PDI paralogs is unknown. We will test the hypothesis that other PDI family members interact with the MTP subunit and these interactions have physiological consequences. Aim 3: Assess the biological consequences of abolishing TG transfer activity of MTP: After identifying further mutations that abolish TG transfer, we will determine whether these mutants support apoB secretion in cells, lower plasma lipids in mice, and sustain normal fish growth. The proposed studies will provide novel information about 1) the domains and amino acids in the transfer of different lipids by MTP; 2) conformational changes that occur during transfer of different lipids; and 3) biochemical, physiological and organismal consequences of mutating these critical residues. This new knowledge will be invaluable, in the future, to develop novel and TG transfer specific inhibitors of MTP.