Structural Biology of Pattern Recognition Receptors

模式识别受体的结构生物学

基本信息

批准号：
7593991
负责人：
Jeffrey C Boyington
金额：
$ 57.12万
依托单位：
NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
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/7593991
关键词：
Adhesions Advanced Glycosylation End Products Affinity Air Area Arteries Atherosclerosis Attention Binding Blood Vessels Cardiac Myocytes Cessation of life Chimeric Proteins Cholesterol Chronic Computer software Crystallization Crystallography Cultured Cells DNA Data Deposition Diabetes Mellitus Disease Dissociation Double-Stranded RNA Dust Escherichia coli Exposure to Extracellular Space Family Fatty Acids Fibrosis Foam Cells Functional disorder Future Gel Chromatography Generations Genetic Glucose HMGB1 Protein Human Immune response Immune system Inflammation Inflammatory Bowel Diseases Intake Integrins Lead Learning Length Ligand Binding Ligands Lipids Low Density Lipoprotein oxidation Lung Microbe Modeling Molecular Mutagenesis N-terminal Natural regeneration Necrosis Nucleotides Numbers Oxidants Patients Pattern recognition receptor Play RNA Rage Rattus Reactive Oxygen Species Research Resolution Role Rupture Serum Signal Transduction Site Societies Sorting - Cell Movement Specificity Surface Plasmon Resonance Thrombosis Time Tissues Vascular Endothelium Vascular Permeabilities Widespread Disease base cigarette smoking design drug development exhaust in vivo insight interest macrophage maltose-binding protein mathematical model medical implant monocyte mouse Smc1l1 protein mouse Smc1l2 protein oxidized low density lipoprotein particle pathogen prevent receptor research study scavenger receptor sensor structural biology

项目摘要

Research Accomplishments: Progress was made primarily in the area of RAGE (Receptor for advanced glycation end products). RAGE is expressed on macrophages, SMC, vascular endothelium and cardiac myocytes. Diabetes-associated vascular dysfunction can be prevented by in vivo blockade of RAGE. Upon interaction with glycated proteins, RAGE elicites a proinflammatory cascade that includes hyperpermiability of blood vessels, monocyte adhesion and generation of reactive oxygen species. RAGE is also a signal transduction receptor for proinflammatory S100/calgranulins, HMGB1/amphoterin and the integrin Mac-I. In an attempt to optimize the crystallization of RAGE for higher resolution diffraction, several more soluble fragments of human RAGE including fusion proteins were designed and successfully expressed in E coli. These included further shortening the linker with the N-terminal maltose-binding protein (MBP) fusion, altering the length of the C-terminus in the second domain, removing the second domain altogether and including Rat Rage in addition to human RAGE. Preliminary data from our lab has shown for the first time that RAGE recognizes both DNA and RNA as ligands with submicromolar affinity. We pursued this further using gel filtration and surface plasmon resonance (SPR, Biacore) experiments. Only dsDNA and dsRNA bound to RAGE; ssDNA did not. The curved scatchard plot and subsequent mathematical modeling (using Origin software) revealed that the binding is most likely follows a one-dimensional lattice model in which RAGE binds with no sequence specificity to dsDNA. This type of binding results in an initial high binding affinity of RAGE to DNA that is proportional to the length of DNA. However, as more ligand (e.g. RAGE) molecules bind to the DNA, and available sites are reduced, the affinity also decreases in a manner that resembles negative cooperativity. An analysis of several Biacore experiments and subsequent modeling revealed a binding footprint of approximately 8 nucleotides and an intrinsic dissociation constant (for one binding footprint) of 10-20 nanomolar with no positive cooperativity. This suggests that RAGE may be a sensor of some sort for naked DNA or RNA in various extracellular spaces where RAGE is expressed. Such a function may play a role in detecting necrotic or damaged cells, signaling the potential presence of a pathogen. Potential future experiments include crystallizing RAGE with dsDNA or dsRNA fragments and functional studies with RAGE in cell cultures to more fully explore the potential functional roles of RAGE in pathogen recognition and signaling.

研究成果：主要在 RAGE（高级糖基化终产物受体）领域取得了进展。 RAGE 在巨噬细胞、SMC、血管内皮和心肌细胞上表达。体内阻断 RAGE 可以预防糖尿病相关的血管功能障碍。在与糖化蛋白相互作用后，RAGE 引发促炎级联反应，包括血管渗透性过高、单核细胞粘附和活性氧的产生。 RAGE 也是促炎性 S100/钙颗粒蛋白、HMGB1/两性蛋白和整合素 Mac-I 的信号转导受体。为了优化 RAGE 结晶以获得更高分辨率的衍射，设计了几种更可溶的人 RAGE 片段（包括融合蛋白）并在大肠杆菌中成功表达。其中包括进一步缩短与 N 端麦芽糖结合蛋白 (MBP) 融合体的接头、改变第二个结构域中 C 端的长度、完全删除第二个结构域以及除了人 RAGE 之外还包括 Rat Rage。我们实验室的初步数据首次表明，RAGE 将 DNA 和 RNA 识别为具有亚微摩尔亲和力的配体。我们使用凝胶过滤和表面等离子共振（SPR，Biacore）实验进一步研究了这一点。仅 dsDNA 和 dsRNA 与 RAGE 结合；单链DNA没有。斯卡卡曲线图和随后的数学模型（使用 Origin 软件）表明，这种结合很可能遵循一维晶格模型，其中 RAGE 与 dsDNA 的结合没有序列特异性。这种类型的结合导致 RAGE 与 DNA 的初始高结合亲和力与 DNA 的长度成正比。然而，随着更多配体（例如 RAGE）分子与 DNA 结合，可用位点减少，亲和力也会以类似于负协同性的方式降低。对多个 Biacore 实验和后续建模的分析揭示了大约 8 个核苷酸的结合足迹和 10-20 纳摩尔的内在解离常数（对于一个结合足迹），没有正协同性。这表明 RAGE 可能是表达 RAGE 的各种细胞外空间中裸露 DNA 或 RNA 的某种传感器。这种功能可能在检测坏死或受损细胞中发挥作用，从而发出病原体潜在存在的信号。未来可能的实验包括用 dsDNA 或 dsRNA 片段结晶 RAGE 以及在细胞培养物中进行 RAGE 功能研究，以更全面地探索 RAGE 在病原体识别和信号转导中的潜在功能作用。