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: 10613446
• 负责人: STEVEN A FARBER
• 金额: $ 61.06万
• 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/10613446
• 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; Ligand Binding; Ligands; Lipid Binding; Lipid Mobilization; Lipids; Lipoproteins; Liver; Mammalian Cell; Maps; Mediator; Metabolic Diseases; Metabolic syndrome; Missense Mutation; Molecular; Molecular Conformation; Morbidity - disease rate; Mus; Mutate; Mutation; Mutation Analysis; N-terminal; Nature; Obesity; Phospholipids; Physiological; Plasma; Prevalence; Production; Protein Deficiency; Protein Disulfide Isomerase; Protein Inhibition; Protein Region; Protein Subunits; Proteins; Risk Factors; Risk Reduction; 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; pharmacologic; 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.

载脂蛋白B(ApoB)是许多代谢性疾病的重要标志，包括胰岛素抵抗、脂肪肝、动脉粥样硬化、肥胖和代谢综合征。抑制微粒体甘油三酯转移蛋白(MTP)是MTP和蛋白质二硫键异构酶(PDI)亚单位的异二聚体，可显著降低含有载脂蛋白B的致动脉粥样硬化脂蛋白50%，但它会导致肝脏骨质疏松症和肠脂溢症。MTP复合体传递不同的脂类，并协助产生含载脂蛋白B的脂蛋白。我们最近的工作首次证明了MTP的甘油三酯(TG)和磷脂(PL)的传递功能是可以解偶联的，并且抑制斑马鱼的TG传递活性不会导致脂肪变性，这些鱼像野生型鱼一样正常生长。我们推测，这两个脂转移结构域的原子水平细节可能为选择性药物抑制TG转移以降低血脂而不引起细胞脂质滞留的不良影响铺平道路。我们要问的基本问题是：“MTP如何区分不同的脂质配体，抑制TG转移活性的后果是什么？”目的1：表征MTP中不同的脂质结合部位：我们将用不同的脂质配体来解析MTP的结构，以获得原子水平的细节。突变分析将阐明对特定脂类结合至关重要的氨基酸残基。目的2：确定MTP和PDI亚基在脂质转移过程中的构象变化，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类。我们将在MTP的柔性环区和PDI的a‘结构域进行定点突变，以剖析其机制。尽管PDI对MTP活性是必需的，但不同PDI类似物的特异性尚不清楚。我们将检验这一假设，即其他PDI家族成员与MTP亚单位相互作用，这些相互作用具有生理后果。目的3：评估取消MTP的TG转移活性的生物学后果：在确定进一步取消TG转移的突变后，我们将确定这些突变是否支持细胞中apoB的分泌，降低小鼠的血脂，并维持鱼类的正常生长。这些研究将提供以下方面的新信息：1)MTP转移不同脂质过程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，开发新的和甘油三酯转移MTP的特异性抑制剂。