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，可以通过三种不同的途径（经典途径、凝集素途径和替代途径）激活，导致逐渐放大的炎症级联反应。补体系统的激活通常受到一系列下调因子的严格控制，以尽量减少宿主组织的损伤。然而，当补体激活不受调节时，它会导致多种炎症性疾病过程中的组织损伤，包括心肌梗塞、类风湿性关节炎和移植排斥。该研究计划的长期目标是阐明 C1q（经典途径）和 MBL（凝集素途径）复合物初始识别配体时补体激活的分子基础，这是开发减轻或预防治疗方法的先决条件不受调节的补体激活破坏宿主组织。我们之前发现人星状病毒的外壳蛋白（CP）分别在 C1q 和 MBL 水平有效抑制补体的经典途径和凝集素途径。最近，已鉴定出一种源自野生型 CP 的 30 个氨基酸肽，可介导此活性。此外，初步数据表明，这种外壳蛋白肽 (CPP) 衍生物与 C1q 结合，抑制与 C1q/MBL 受体钙网蛋白 (CRT/cC1qR) 的相互作用。 C1q 和 MBL 激活小分子抑制剂的发现为破译这些复合物的功能提供了一种新试剂。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以及 CRT/cC1qR 相互作用所需的相关丝氨酸蛋白酶之间的相互作用至关重要。本应用的实验重点是确定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后果。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同源丝氨酸蛋白酶激活的功能影响：(a) 通过结合 (ELISA) 测定、功能测定和利用 CPP 的合成衍生物进行质谱蛋白质足迹分析，(b) 通过表面等离子共振和等温滴定量热法表征 CPP 结合 C1q/MBL 的动力学， (c) 通过利用我们实验室开发的竞争性结合测定法 (ELISA) 测定 CPP 与 C1q 的相互作用改变与其同源丝氨酸蛋白酶复合物的相互作用的程度。具体目标 2 将通过以下方式评估 CPP 改变与人中性粒细胞上的 C1q-CRT/cC1qR 相互作用的程度及其对细胞功能的影响： (a) 利用以下方法确定 CPP 抑制 C1q 与中性粒细胞上的 CRT/cC1qR 结合的能力(b) 使用流式细胞术表征 CPP 破坏 C1q-CRT/cC1qR 相互作用对中性粒细胞呼吸爆发活动的生物学后果SOD 抑制性铁细胞色素 C 还原测定。 公共健康相关性：该研究项目的成功执行将明确 CPP 抑制 C1 和 MBL 激活补体级联的机制，以及阻碍 C1q-CRT/cC1qR 相互作用对中性粒细胞功能的影响。补体激活失调会导致人类炎症性疾病，例如类风湿性关节炎、心肌梗塞和移植排斥，但目前可用于治疗用途的补体抑制剂很少。了解这种新型肽补体抑制剂的作用机制将为其开发为预防补体介导的免疫病理学的治疗化合物铺平道路。