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

基本信息

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
项目类别：
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

项目摘要

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) 的脂蛋白既是生物标志物，也是许多代谢疾病核心特征的因果介质，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。 MTP 和蛋白质二硫键异构酶 (PDI) 亚基的异二聚体复合物，可显着减少特异性致动脉粥样硬化的含 apoB 的脂蛋白50%，但它会导致肝脂肪变性和肠道脂肪泻，MTP 复合物会转移不同的脂质，并协助产生含 apoB 的脂蛋白。 MTP 的转移功能可以解耦，并且抑制斑马鱼的 TG 转移活性不会导致脂肪变性，并且这些鱼像野生型一样正常生长我们追求关于这两个脂质转移结构域的原子水平细节可能为选择性药理抑制 TG 转移以降低脂质血浆铺平道路，而不会引起细胞脂质滞留的不利影响。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 中的不同脂质结合位点：我们将用不同的脂质配体解析 MTP 结构，以获得原子水平的详细信息。阐明对特定脂质结合至关重要的氨基酸残基：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：我们寻求 MTP 和 PDI 亚基都发生构象变化。为了适应不同的脂质，我们将在 MTP 的柔性环区域和 PDI 的 a' 结构域中进行定点诱变，以剖析其机制，尽管 PDI 是必需的。对于 MTP 活性，不同 PDI 旁系同源物的特异性尚不清楚，我们将测试其他 PDI 家族成员与 MTP 亚基相互作用的假设，并且这些相互作用具有生理后果：评估消除 MTP TG 转移活性的生物学后果。在确定了废除 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、降低小鼠血浆脂质并维持鱼类的正常生长。拟议的研究将提供有关 1) 结构域和功能的新信息。通过 MTP 转移不同脂质时的氨基酸；2) 不同脂质转移过程中发生的构象变化；以及 3) 这些关键残基突变的生化、生理和生物学后果对于未来来说将是非常宝贵的。开发新型的 TG 转移特异性 MTP 抑制剂。