Mapping MTP lipid transfer activities for better therapeutics

绘制 MTP 脂质转移活性以实现更好的治疗

• 批准号: 10391443
• 负责人: STEVEN A FARBER
• 金额: $ 60.57万
• 依托单位: NYU LONG ISLAND SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10391443
• 关键词: 3-Dimensional; Abetalipoproteinemia; Address; Adverse effects; Amino Acids; Apolipoproteins B; Atherosclerosis; Bacteria; Binding; Binding Sites; Biochemical; Biochemistry; Biological; Biological Markers; Blood; Cause of Death; Cells; Cellular biology; Complex; Crystallization; Developmental Biology; Diabetes Mellitus; Drosophila genus; Drug Side Effects; Family; Family member; Fatty Liver; Fishes; Future; Genetic; Growth; Human; Individual; Insulin Resistance; Intestines; Knowledge; Ligands; Lipid Binding; Lipid Mobilization; Lipids; Lipoproteins; Liver; Mammalian Cell; Mediator of activation protein; Metabolic Diseases; Metabolic syndrome; Missense Mutation; Molecular; Molecular Conformation; Morbidity - disease rate; Mus; Mutate; Mutation; Mutation Analysis; N-terminal; Nature; Obesity; Pharmacology; Phospholipids; Physiological; Plasma; Prevalence; Production; Protein Disulfide Isomerase; Protein Inhibition; Protein Region; Protein Subunits; Proteins; Risk Factors; Role; Scientist; Site-Directed Mutagenesis; Specificity; Steatorrhea; Structure; System; Tertiary Protein Structure; Testing; Therapeutic; Triglycerides; Work; Yolk Sac; Zebrafish; apolipoprotein B-48; cardiovascular disorder risk; cardiovascular risk factor; design; fatty liver disease; flexibility; in vivo; inhibitor; lipid transfer protein; loss of function; member; microsomal triglyceride transfer protein; mortality; mutant; new therapeutic target; novel; overexpression; paralogous gene; protein complex; protein structure; side effect; skills; small molecule; structural biology

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.

含载脂蛋白B（apoB）的脂蛋白既是代谢性疾病的许多中心标志的生物标志物，也是代谢性疾病（包括胰岛素抵抗、脂肪肝疾病、动脉粥样硬化、肥胖和代谢综合征）的致病介质。抑制微粒体甘油三酯转移蛋白（MTP）（MTP和蛋白质二硫键异构酶（PDI）亚基的异二聚体复合物），可将致动脉粥样硬化的含apoB的脂蛋白显著降低50%，但会导致脂肪肝和肠脂肪变性。MTP复合物转移不同的脂质，并协助生产含apoB的脂蛋白。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）转移功能可以解耦，并且斑马鱼中TG转移活性的抑制不会导致脂肪变性，并且这些鱼像野生型鱼一样正常生长。我们假设，原子水平的细节，这两个脂质转移域可能铺平了道路，选择性药理学抑制TG转移，以降低血脂，而不会造成细胞脂质潴留的不良影响。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？”目标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解析MTP结构，以获得原子水平的细节。突变分析将阐明特定脂质结合的关键氨基酸残基。目标二：识别脂质转移过程中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。我们将在MTP的柔性环区域和PDI的a'结构域中进行定点突变，以剖析其机制。尽管PDI对于MTP活性是必需的，但是不同PDI旁系同源物的特异性是未知的。我们将测试的假设，其他PDI家族成员与MTP亚基相互作用，这些相互作用有生理后果。目标三：评估消除MTP的TG转移活性的生物学后果：在鉴定了消除TG转移的进一步突变后，我们将确定这些突变体是否支持细胞中的apoB分泌，降低小鼠的血浆脂质，并维持正常的鱼类生长。拟议的研究将提供有关以下内容的新信息：1）MTP转移不同脂质中的结构域和氨基酸; 2）不同脂质转移过程中发生的构象变化; 3）突变这些关键残基的生化、生理和生物后果。这一新的知识将是非常宝贵的，在未来，开发新的和TG转移特异性抑制剂的MTP。