Molecular Determinants of Endotoxin Interaction with Toll-like Receptor4

内毒素与 Toll 样受体4相互作用的分子决定因素

• 批准号: 8280134
• 负责人: Theresa Lee Gioannini
• 金额: --
• 依托单位: IOWA CITY VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8280134
• 关键词: Acute Lung Injury; Address; Adjuvant; Affinity; Agonist; Antimicrobial Resistance; Atherosclerosis; Autoimmune Process; Bacterial Infections; Binding; Biochemical; CD14 gene; Cell surface; Cells; Collaborations; Complex; Dependence; Detection; Development; Disease; E protein; Elements; Endotoxins; Event; Frequencies; Glycolipids; Goals; Gram-Negative Bacteria; Hemin; Host Defense; Host resistance; Immune; Immune response; Immunologics; Infection; Inflammatory; Inflammatory Response; Injury; Invaded; Iowa; Knowledge; Lead; Ligands; Link; Lipid A; Molecular; Natural Immunity; Nature; Pathology; Pathway interactions; Pattern Recognition; Physiological; Population; Proteins; Reaction; Reagent; Receptor Activation; Regulation; Risk; Role; Sepsis; Signal Pathway; Signal Transduction; Specific qualifier value; Stimulus; Structure; Surface; Syndrome; System; Testing; Therapeutic Agents; Tissues; Toll-like receptors; Variant; Veterans; Water; Work; acute liver disease; adaptive immunity; base; design; extracellular; insight; lipopolysaccharide-binding protein; microbial; molecular recognition; novel; prophylactic; receptor; receptor coupling; response; toll-like receptor 4; tool

DESCRIPTION (provided by applicant): ABSTRACT Toll-like receptors couple molecular recognition of conserved and structurally unique microbial molecules to rapid mobilization of innate immune effector systems and later induction of adaptive immunity. Toll-like receptor 4 (TLR4) is essential for the recognition of even minute amounts of endotoxin (E), unique glycolipids abundantly present on the surface of Gram negative bacteria (GNB), that are responsible for host responses to many GNB. Potent responses to E require the sequential action of several extracellular and cell surface host proteins, including lipopolysaccharide-binding protein (LBP), CD14, MD-2 for delivery to and activation of TLR4. Endotoxin-dependent responses elicited through TLR4 lead to activation of MyD88- and TRIF-dependent signaling pathway. Variations in endotoxin structure - as occurs naturally among GNB - and variations in levels of host cell proteins involved in E binding and signal transduction make possible great diversification of host cell responses to E. The normally protective responses to E may contribute to severe host pathology by exacerbating systemic inflammatory responses when local infection is not contained, as in GNB sepsis. Inappropriate signaling, whether too extensive or too modest, may contribute to a variety of infectious and inflammatory diseases including sepsis, atherosclerosis, autoimmune syndromes, liver disease, and acute lung injury. The novel reagents and experimental approaches we have developed allowed detection and quantitative analysis of specific, high affinity (pM) E- protein and TLR4 interactions under physiologically relevant conditions and so permitted determination of the mechanism of action of several natural and synthetic regulators of TLR4 activation. The discovery and purification of stable, water-soluble monomeric complexes of E.MD-2 that, depending on the structure of bound E or MD-2, can act at pM concentrations as TLR4 agonists or antagonists, have provided unique reagents to probe the remaining mysteries of endotoxin-triggered TLR4 activation and the structural limits of pattern recognition of endotoxin by MD-2 and TLR4. Increasing evidence suggests physiologic and patho-physiologic roles of TLR4 extending beyond host responses to invading GNB. Thus, knowledge of the molecular and cellular rules regulating TLR4 function should provide new insights into what determines the nature and the strength of TLR4-dependent responses in settings including but not limited to GNB infection. The long-term goals of our work are to better understand the mechanism(s) by which TLR4-dependent cellular responses to GNB endotoxin and host-derived "danger molecules" are regulated. More specifically, the work is focused on understanding 1) the mechanism(s) by which E interactions with MD-2 specify activation or antagonism of TLR4 and the contrasting structural and molecular requirements for hemin activation of TLR4; 2) the effects of the naturally occurring polymorphic variant substitutions D299G and/or T399I in the TLR4 ectodomain on surface expression and function of TLR4; and 3) the effects of variables in E (lipid A) structure and presentation on induction of MyD88- vs. TRIF-dependent signaling by activated TLR4. The two pathways are linked to different cellular responses so a better understanding of their differential regulation could have important implications for the design of compounds such as adjuvants with selective effects on the type and duration of TLR4 signaling. The studies proposed here should significantly increase understanding of the regulation of TLR4 function and produce insights applicable to the design and testing of novel TLR4-directed immune-modulators. Knowledge of the molecular and cellular rules regulating TLR4 function contributes to the understanding of what determines the nature and the strength of TLR4-dependent responses in settings including, but not limited to, GNB infection.

描述（由申请人提供）： 抽象的Toll样受体对保守和结构上独特的微生物分子的分子识别，以快速动员先天免疫效应器系统，后来诱导适应性免疫。 Toll样受体4（TLR4）对于识别革兰氏阴性细菌（GNB）表面上大量存在的独特糖脂的识别至关重要，这些糖脂构成了对许多GNB的宿主反应。对E的有效反应需要几种细胞外和细胞表面宿主蛋白的顺序作用，包括脂多糖结合蛋白（LBP），CD14，MD-2，以递送和激活TLR4。通过TLR4引起的内毒素依赖性反应导致MyD88-和TRIF依赖性信号通路的激活。内毒素结构的变化 - 正如GNB之间自然发生的那样 - 与E结合和信号转导相关的宿主细胞蛋白水平的变化使E.对E的宿主细胞反应的巨大多样化可能会导致对E的保护性反应，这可能会导致宿主病理的严重宿主病理，而当局部感染不受局部感染而不受GNB SepsIS的局部感染而加重全身性炎症反应。不适当的信号传导，无论是太广泛还是太谦虚，都可能导致各种传染病和炎症性疾病，包括败血症，动脉粥样硬化，自身免疫性综合征，肝病和急性肺损伤。 我们开发的新型试剂和实验方法允许在生理相关条件下对特定的高亲和力（PM）E-蛋白和TLR4相互作用进行检测和定量分析，从而允许确定TLR4激活的几种自然和合成调节剂的作用机理。 E.MD-2的稳定，水溶性单体复合物的发现和纯化，取决于结合E或MD-2的结构，可以作为PM浓度作为TLR4激动剂或拮抗剂作用，以探测内毒素触发的TLR4激活和结构氧化物的剩余奥秘的剩余之元，并源于Endox and的结构限制。越来越多的证据表明，TLR4的生理和病理生理学作用扩展了宿主对入侵GNB的反应。因此，对调节TLR4功能的分子和细胞规则的知识应提供新的见解，以了解确定TLR4依赖性反应的性质和强度，包括但不限于GNB感染。我们工作的长期目标是更好地了解TLR4依赖性细胞对GNB内毒素和宿主衍生的“危险分子”的机制。更具体地说，该工作的重点是理解1）E与MD-2相互作用指定TLR4的激活或拮抗作用的机制以及对TLR4的HEMIN激活的对比鲜明的结构和分子需求； 2）天然发生的多态性变体取代D299G和/或T399i对TLR4胞外域对TLR4的表面表达和功能的影响； 3）E（脂质A）结构中变量的影响和呈现对激活的TLR4诱导MyD88-与TRIF依赖性信号的影响。这两种途径与不同的细胞反应有关，因此更好地理解其差异调节可能对化合物的设计（例如佐剂）具有重要影响，例如对TLR4信号传导的类型和持续时间有选择性影响。这里提出的研究应显着提高对TLR4功能调节的理解，并产生适用于新型TLR4定向免疫调节剂的设计和测试的见解。对调节TLR4功能的分子和细胞规则的知识有助于理解确定TLR4依赖性响应的性质和强度，包括但不限于GNB感染。