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: 8318091
• 负责人: NEEL KUMAR KRISHNA
• 金额: $ 18.31万
• 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 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8318091
• 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.

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分，其可通过三种不同途径（经典、凝集素和替代）激活，导致炎症级联反应逐渐放大。补体系统的激活通常由一系列下调因子严格控制，以使宿主组织损伤最小化。然而，当发生不受调节的补体激活时，它在广泛的炎性疾病过程中导致组织损伤，包括心肌梗死、类风湿性关节炎和移植排斥。该研究项目的长期目标是阐明补体激活的分子基础，即C1 q（经典途径）和MBL（凝集素途径）复合物对配体的初始识别，作为开发治疗方法的先决条件，以减轻或预防不受调节的补体激活对宿主组织的破坏。我们先前发现，人星状病毒的外壳蛋白（CP）分别在C1 q和MBL水平有效地抑制补体的经典途径和凝集素途径。最近，已经鉴定了来自野生型CP的介导该活性的30个氨基酸的肽。此外，初步数据表明，这种外壳蛋白肽（CPP）衍生物结合C1 q，抑制与C1 q/MBL受体钙网蛋白（CRT/cC 1 qR）的相互作用。C1 q和MBL激活的小分子抑制剂的发现提供了一种新的试剂来解释这些复合物的功能。我们的具体假设是，CPP直接结合C1 q和MBL的胶原样区域，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之间的相互作用以及CRT/cC 1 qR相互作用至关重要。本申请的实验重点是确定抑制补体激活所需的CPP和C1 q/MBL之间的精确相互作用以及CPP抑制C1 q/MBL-CRT/cC 1 qR相互作用对细胞功能的功能后果。具体目标1将表征CPP和C1 q/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活化的功能效应：（a）利用CPP的合成衍生物，通过结合（ELISA）测定、功能测定和质谱蛋白足迹法来确定CPP/C1 q和CPP/MBL的相互作用结构域，（B）通过表面等离子体共振和等温滴定量热法表征CPP结合C1 q/MBL的动力学，和（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C1 q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将通过以下方式评价CPP改变与人中性粒细胞上C1 q-CRT/cC 1 qR相互作用的程度及其对细胞功能的影响：（a）利用流式细胞术测定CPP抑制C1 q与嗜中性粒细胞上的CRT/cC 1 qR结合的能力，和（B）利用SOD-酶活性测定法表征CPP破坏C1 q-CRT/cC 1 qR相互作用对嗜中性粒细胞中呼吸爆发活性的生物学后果。铁细胞色素C还原试验。

项目成果

Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway.

临床低温通过经典途径增加补体激活和细胞破坏。

• DOI: 10.1186/1479-5876-12-181
• 发表时间: 2014-06-24
• 期刊: Journal of translational medicine
• 影响因子: 7.4
• 作者: Shah TA;Mauriello CT;Hair PS;Sandhu A;Stolz MP;Bass WT;Krishna NK;Cunnion KM
• 通讯作者: Cunnion KM