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Influence Of Protein/lipid Interactions On Signal Transduction

蛋白质/脂质相互作用对信号转导的影响

基本信息

批准号：
7732092
负责人：
Drake Curtis Mitchell
金额：
$ 103.82万
依托单位：
NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/7732092
关键词：
1-Butanol 7-dehydrocholesterol AY9944 Acute Adipocytes Adipose tissue Affect Alcohols Animal Model Antigen Presentation Antigen-Presenting Cells Antigens Biochemical Biological Models Biological Preservation Bos taurus Butanols Calorimetry Cattle Cell membrane Cells Cholesterol Chronic Complex Mixtures Condition Cytolysis Cytotoxic T-Lymphocytes Defect Detergents Diet Differential Scanning Calorimetry Docosahexaenoic Acids Equilibrium Ethanol Exhibits F-Actin Fatty Acids Fatty acid glycerol esters Fluorescence Anisotropy Fluorescence Spectroscopy G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors GTP-Binding Proteins Goals Guidelines Histocompatibility Antigens Class I Immune response Immunity Individual Inherited Isomerism Kinetics Lecithin Ligands Link Lipids MHC Class I Genes Mediterranean Diet Membrane Membrane Fluidity Membrane Lipids Membrane Proteins Metabolic Diseases Micelles Modeling Molecular Molecular Conformation Molecular Weight Motion Neurotransmitters Obesity Oleic Acid Oleic Acids Palmitic Acids Partition Coefficient Pathway interactions Pharmaceutical Preparations Pharmacologic Substance Phospholipids Photoreceptors Phototransduction Process Property Protein Binding Protein Conformation Proteins Range Rate Rattus Receptor Mediated Signal Transduction Relative (related person)Retinal Rhodopsin Rod Outer Segments Role Saturated Fatty Acids Scanning Series Signal Pathway Signal Transduction Signal Transduction Pathway Smell Perception Smith-Lemli-Opitz Syndrome Specificity Staging Stereoisomer Surface System T-Lymphocyte Taste Perception Techniques Testing Time Tissues Titrations Transition Temperature Triglycerides Variant Vesicle Vision Visual system structure Water alcohol exposure cholesterol biosynthesis design feeding member pathogen physical property prevent protein function protein protein interaction receptor reconstitution relating to nervous system retinal rods rod outer segment disc tert-Butyl Alcohol visual phototransduction

项目摘要

This project is designed to assess the role of membrane lipid composition, especially polyunsaturated phospholipids, in modulating G protein-coupled receptor (GPCR) signal transduction and to elucidate the mechanism of action of ethanol in these systems. GPCRs are ubiquitous components of signal transduction pathways, including taste, smell, vision, and many neurotransmitter systems. GPCRs are also targets of a great many pharmaceutical drugs. The visual transduction pathway of the retinal rod photoreceptor is the best characterized member of this receptor superfamily and is being used as a model system in these studies. Six projects were completed this year; 1) Determination of the interplay between polyunsaturated lipids and cholesterol in determining protein stability, 2) Analysis of the biophysical properties of retinal tissue in Smith-Lemli-Opitz Syndrome, 3) Established a link between the early stages of lipid overload and antigen presentation that is exacerbated by saturated fatty acids in non-adipose cells, 4) Determined the effects of membrane phospholipid composition on alcohol partition coefficients, 5) Investigated structural specificity in the interaction of rhodopsin and alcohols, 6) Investigated a wide variety of detergents to determine the requirements for optimal preservation of membrane protein structural stability and function. Purified bovine rhodopsin was reconstituted into vesicles consisting of 1-stearoyl-2-oleoyl phosphatidylcholine or 1-stearoyl-2-docosahexaenoyl phosphatidylcholine with and without 30 mol% cholesterol. Rhodopsin stability was examined using differential scanning calorimetry (DSC). The thermal unfolding transition temperature (Tm) of rhodopsin was scan rate dependent, demonstrating the presence of a rate-limited component of denaturation. Both Tm and Ea varied with bilayer composition. Cholesterol increased Tm both in the presence and absence of DHA acyl chains. In contrast, cholesterol lowered Ea in the absence DHA acyl chains, but raised Ea in the presence of 20 mol% DHA-containing phospholipid. Maximal kinetic stability was found within the range of acyl chain order found in native bovine rod outer segment disk membranes. The results demonstrate that membrane composition has distinct effects on the thermal versus kinetic stabilities of membrane proteins, and suggests that a balance between membrane constituents with opposite effects on acyl chain packing may be required for maximum protein stability. Smith-Lemli-Opitz syndrome (SLOS) is caused by an inherited defect in the last step in cholesterol biosynthesis, leading to abnormal accumulation of 7-dehydrocholesterol (7DHC) and decreased cholesterol (Chol) levels. We evaluated alterations in the biochemical and physical properties of retinal rod outer segment (ROS) membranes in an animal model, using fluorescence spectroscopy and DPH. At one month of AY9944 treatment, there were modest alterations in fatty acid composition, but no significant differences in spectroscopic parameters. However, at three months, membrane fatty acid unsaturation was dramatically reduced and fluorescence anisotropy values were decreased, relative to controls. The DPH probe exhibited decreased rotational motion and increased orientational order in ROS membranes from three-month AY9944-treated rats, relative to controls. These findings indicate reduced ROS membrane fluidity in the SLOS rat model, relatively to controls, likely due primarily to dramatic reduction membrane DHA levels. Lipid overload, associated with metabolic disorders, occurs when saturated fatty acids (SFAs) accumulate in non-adipose tissues. Cells of these tissues use MHC class I molecules to present antigen to T cells in order to eliminate pathogens. Since obesity is associated with impaired immune responses, we tested the hypothesis that the early stages of lipid overload with SFAs alters MHC class I antigen presentation. Antigen presenting cells (APCs) were fed either the saturated palmitic acid (PA), abundant in the high fat Western diet, or the monounsaturated oleic acid (OA), a component of the Mediterranean diet. PA-treatment lowered APC lysis by activated cytotoxic T lymphocytes and inhibited APC ability to stimulate nave T cells. Inhibition of immune responses with PA was due to a significant reduction in MHC class I surface expression, inhibition in the rate of APC-T cell conjugation, and lowering of plasma membrane F-actin levels. OA-treatment had no effect on antigen presentation and upon feeding with PA, prevented the phenotypic effects of PA. OA-treatment conferred protection against changes in antigen presentation by accumulating fatty acids into triglyceride-rich lipid droplets of APCs. Our findings establish for the first time a link between the early stages of lipid overload and antigen presentation and suggest that dietary SFAs in non-adipose cells of obese individuals could impair immunity. Using isothermal titration calorimetry we have established that the membrane-water partition coefficient for alcohol varies with phospholipid composition. Partitioning into the membrane is particularly sensitive to phospholipid headgroup composition. Alcohol partitioning is 4 to 5 times higher in a PC/PS membrane than in a PC/PE membrane and 2 to 3 times than in a pure PC membrane. We have begun to investigate the effects of phospholipid acyl chain composition and find that the presence of DHA acyl chains increases the partition coefficient by a factor of 2 in a PC membrane. We will extend this study to investigate alcohol partitioning in more complex mixtures, with particular emphasis on the changes in membrane composition associated with chronic ethanol exposure, such as increased cholesterol content and reduced levels of polyunsaturated acyl chains. We have used 5 structural isomers of butanol to examine possible structural specificity in the interaction of alcohols with rhodopsin. R and S stereoisomers of 1-butanol have identical effects on both the activating conformation change of rhodopsin and rhodopsins thermal stability. The most structurally compact isomer, tert-butanol, had the smallest effect on these processes, while the most extended isomer, 1-butanol, had the greatest effect on both processes. Selected variation of membrane and alcohol concentrations strongly implies that all effects on protein conformation change and stability are due to alcohol molecules acting at the lipid-protein interface. Due to the continuing challenge of reconstituting purified membrane proteins in quantities sufficient for many for many biophysical techniques, there is increasing use of membrane proteins solubilized in detergents. We analyzed the effects of anionic, cationic, zwitterionic and non-ionic detergents on the conformation changes involved in rhodopsin activation and deactivation, which occur over time scales from microseconds to hundreds of seconds. The fastest conformation changes, associated with formation of the G protein-binding conformation, were significantly distorted by all detergents. The slowest conformation changes, associated with release of ligand, proceeded with native-like kinetics in detergents with a high micelle molecular weight, regardless of detergent ionic character. The results will provide membrane biophysicists with useful guidelines for determining when it is appropriate to study solubilized membrane proteins and which detergents may provide reasonably native conditions.

本项目旨在评估膜脂组成，特别是多不饱和磷脂，在调节G蛋白偶联受体（GPCR）信号转导中的作用，并阐明乙醇在这些系统中的作用机制。gpcr是信号转导途径的普遍组成部分，包括味觉、嗅觉、视觉和许多神经递质系统。gpcr也是许多药物的靶标。视网膜杆状光感受器的视觉转导通路是该受体超家族中最具特征的成员，并且在这些研究中被用作模型系统。今年完成了六个项目；1)确定多不饱和脂质和胆固醇在决定蛋白质稳定性中的相互作用，2)分析Smith-Lemli-Opitz综合征视网膜组织的生物物理特性，3)建立非脂肪细胞中饱和脂肪酸加剧的脂质过载与抗原递呈之间的联系，4)确定膜磷脂组成对酒精分配系数的影响。5)研究了视紫红质与醇相互作用的结构特异性；6)研究了多种洗涤剂，以确定最佳保存膜蛋白结构稳定性和功能的要求。