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.

研究成就：进展主要是在愤怒区域（晚期糖基化终产物的受体）领域取得的进展。愤怒以巨噬细胞，SMC，血管内皮和心肌细胞表示。通过体内阻断愤怒，可以预防与糖尿病相关的血管功能障碍。与糖化蛋白相互作用后，激怒了一种促炎级联反应，其中包括血管过高的性能，单核细胞粘附和活性氧的产生。 RAGE也是促炎S100/calgranulins，HMGB1/Amphoterin和整合素MAC-I的信号转导受体。为了优化更高分辨率衍射的愤怒的结晶，设计并成功地表达了包括融合蛋白在内的人类愤怒的几个可溶性片段。其中包括进一步缩短连接器，使用N末端麦芽糖结合蛋白（MBP）融合，改变了第二个域中的C端长度，完全消除了第二个结构域，除了人类的愤怒外，还包括rage愤怒。我们实验室的初步数据首次表明，RAGE将DNA和RNA视为具有亚粒度亲和力的配体。我们使用凝胶过滤和表面等离子体共振（SPR，BIACORE）实验进一步追求这一点。只有dsdna和dsRNA绑定到愤怒； ssDNA没有。弯曲的SCATCHARD图和随后的数学建模（使用原始软件）表明，结合很可能是遵循的一维晶格模型，在该模型中，RAGE结合而没有对DSDNA的序列特异性。这种类型的结合导致愤怒与DNA的初始高结合亲和力与DNA长度成正比。但是，随着更多的配体（例如愤怒）分子与DNA结合，可用位点减少，亲和力也以类似于负合作的方式降低。对几个BIACORE实验和随后的建模的分析表明，约有8个核苷酸的结合足迹和10-20纳摩尔的固有离解常数（一个结合足迹），而没有正合作。这表明在表达愤怒的各种细胞外空间中，愤怒可能是某种裸体DNA或RNA的传感器。这样的功能可能在检测坏死或受损细胞中发挥作用，表明病原体的潜在存在。潜在的未来实验包括用dsDNA或dsRNA片段结晶的愤怒以及在细胞培养物中rage的功能研究，以更充分地探索愤怒在病原体识别和信号传导中的潜在功能作用。