Structure and function analysis of C1/C1q and MBL using a novel peptide inhibitor

使用新型肽抑制剂对 C1/C1q 和 MBL 进行结构和功能分析

• 批准号: 8174301
• 负责人: NEEL KUMAR KRISHNA
• 金额: $ 21.98万
• 依托单位: EASTERN VIRGINIA MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-10 至 2013-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8174301
• 关键词: Amino Acids; Astrovirus; Attenuated; Binding; Binding Sites; Biological; Biological Assay; Biology; Calorimetry; Capsid Proteins; Cell physiology; Cell surface; Childhood; Cleaved cell; Collagen; Competitive Binding; Complement; Complement 1q; Complement Activation; Complement Inactivators; Complement component C1r; Complex; Data; Development; Disease; Dose; Enzyme-Linked Immunosorbent Assay; Flow Cytometry; Gastroenteritis; Goals; Graft Rejection; Human; In Vitro; Inflammatory; Kinetics; Laboratories; Lectin; Ligands; Measures; Mediating; Methods; Molecular; Myocardial Infarction; Natural Immunity; Outcome; Pathway interactions; Peptides; Process; Protein Binding; Protein Footprinting; Reagent; Research; Research Project Grants; Respiratory Burst; Rheumatoid Arthritis; Serine Protease; Serum Proteins; Structure; Surface Plasmon Resonance; Testing; Therapeutic; Therapeutic Uses; Tissues; Titrations; base; calreticulin; cell type; complement pathway; complement system; immunopathology; inhibitor/antagonist; neutrophil; novel; prevent; programs; receptor; research study; small molecule; therapeutic development

DESCRIPTION (provided by applicant): The complement system is a critical component of innate immunity comprised of serum proteins, which can be activated by three different pathways (classical, lectin and alternative) causing a progressively amplifying inflammatory cascade. Activation of the complement system is generally tightly controlled by an array of down- regulators to minimize host-tissue damage. However, when unregulated complement activation occurs, it contributes to tissue damage in a wide range of inflammatory disease processes including myocardial infarction, rheumatoid arthritis and transplant rejection. The long-range goal of this research program is to elucidate the molecular basis of complement activation upon initial recognition of ligand by the C1q (classical pathway) and MBL (lectin pathway) complex as a prerequisite to the development of therapeutic methods to attenuate or prevent host tissue destruction by unregulated complement activation. We previously discovered that the coat protein (CP) of human astrovirus potently suppresses both the classical and lectin pathways of complement at the level of C1q and MBL, respectively. Recently, a 30 amino acid peptide derived from the wild-type CP mediating this activity has been identified. In addition, preliminary data demonstrates that this coat protein peptide (CPP) derivative binds C1q to inhibit interaction with the C1q/MBL receptor, calreticulin (CRT/cC1qR). The discovery of a small molecule inhibitor of C1q and MBL activation provides a novel reagent to decipher how these complexes function. Our specific hypothesis is that CPP directly binds to the collagen-like region of C1q and MBL that is critical for interaction between the associated serine proteases required for initiation of classical and lectin pathway activity as well as CRT/cC1qR interaction. The experimental focus of this application is to define the precise interactions between CPP and C1q/MBL required to inhibit complement activation as well as the functional consequence of CPP inhibition of the C1q/MBL-CRT/cC1qR interaction on cellular function. Specific Aim 1 will characterize the interactions between the CPP and C1q/MBL and measure functional effects on cognate serine protease activation by: (a) defining the interaction domains of CPP/C1q and CPP/MBL via binding (ELISA) assays, functional assays and mass spectrometric protein footprinting utilizing synthetic derivatives of CPP, (b) characterizing the kinetics of CPP binding C1q/MBL by surface plasmon resonance and isothermal titration calorimetry and (c) assaying the extent to which CPP interaction with C1q alters interaction with its cognate serine protease complex by utilizing a competitive binding assay developed in our laboratory (ELISA). Specific Aim 2 will evaluate the extent to which CPP can alter the interaction with C1q-CRT/cC1qR on human neutrophils and its effect on cellular function by: (a) determining the ability of CPP to inhibit C1q binding to CRT/cC1qR on neutrophils utilizing flow cytometry and (b) characterizing the biological consequence of CPP disruption of the C1q-CRT/cC1qR interaction on respiratory burst activity in neutrophils using a SOD-inhibitable ferricytochrome C reduction assay. PUBLIC HEALTH RELEVANCE: Successful execution of this research project will define both the mechanism of CPP inhibition of C1 and MBL activation of the complement cascade and the consequence of impeding C1q-CRT/cC1qR interaction on neutrophil function. Dysregulated complement activation contributes to inflammatory diseases in humans such as rheumatoid arthritis, myocardial infarction and transplant rejection, however very few complement inhibitors are currently available for therapeutic use. Understanding the mechanism of action of this novel peptidic complement inhibitor will pave the way for its development as a therapeutic compound to prevent complement- mediated immunopathology.

描述（由申请人提供）：补体系统是由血清蛋白组成的先天免疫的关键组成部分，血清蛋白可以通过三种不同的途径（经典，凝集素和替代性）激活，从而逐渐扩大炎症级联反应。补体系统的激活通常由一系列下调器密切控制，以最大程度地减少宿主组织损伤。但是，当发生不调节的补体激活时，它会导致组织损害在广泛的炎症性疾病过程中，包括心肌梗塞，类风湿关节炎和移植排斥。该研究计划的远距离目标是阐明C1Q（经典途径）对配体初始识别配体时补体激活的分子基础，并将MBL（凝集素途径）复合物作为衰减或防止无受调节的补体互补互联构成宿主组织破坏的治疗方法的先决条件。我们先前发现，人星座病毒的外套蛋白（CP）分别在C1Q和MBL水平上分别抑制了补体的经典和凝集素途径。最近，已经鉴定出源自介导这种活性的野生型CP的30个氨基酸肽。此外，初步数据表明，这种外套蛋白肽（CPP）衍生物结合C1q，抑制与C1Q/MBL受体钙网蛋白（CRT/CC1QR）的相互作用。 C1Q和MBL活化的小分子抑制剂的发现提供了一种新型试剂，以破译这些复合物如何发挥作用。我们的具体假设是CPP直接与C1Q和MBL的胶原蛋白样区域结合，这对于启动经典和凝集素途径活性所需的相关丝氨酸蛋白酶以及CRT/CC1QR相互作用至关重要。该应用的实验重点是定义抑制补体激活所需的CPP和C1Q/MBL之间的精确相互作用，以及CPP抑制C1Q/MBL-CRT/CC1QR相互作用对细胞功能的功能结果。具体目标1将表征CPP和C1Q/MBL之间的相互作用，并通过以下方式测量对认知丝氨酸蛋白酶激活的功能效应： C1Q/MBL通过表面等离子体的共振和等温滴定量热法和（c）分析CPP与C1Q与CPP的相互作用的程度通过利用我们实验室（ELISA）中开发的竞争性结合测定法来改变与其同源丝氨酸蛋白酶复合物的相互作用。具体目标2将评估CPP可以在多大程度上改变与C1Q-CRT/CC1QR对人中粒细胞上的相互作用，以及通过以下方面的影响：（a）确定CPP能够抑制CC1Q与CRT/CC1QR结合的能力，该能力与中性粒细胞在使用CRT/CC1QR上使用CC1QR，以利用流式细胞膜和（B）表征CC1QR。 C1Q-CRT/CC1QR在中性粒细胞中使用SOD可抑制的铁色素C减少测定法对中性粒细胞的呼吸爆发活性的相互作用。 公共卫生相关性：该研究项目的成功执行将既定义CPP抑制C1的机制和补体级联反应的MBL激活的机制，以及阻碍中性粒细胞功能的C1Q-CRT/CC1QR相互作用的结果。补体失调激活导致人类炎症性疾病，例如类风湿关节炎，心肌梗死和移植排斥，但是目前很少有补体抑制剂可用于治疗。了解这种新型肽补体抑制剂的作用机理将为其作为一种治疗化合物的发育铺平道路，以防止补体介导的免疫病理学。