Function and application of lung surfactant proteins

肺表面活性蛋白的功能及应用

• 批准号: 10556427
• 负责人: Tom A Rapoport
• 金额: $ 46.08万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-15 至 2025-01-21
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10556427
• 关键词: Acids; Acute; Acute Lung Injury; Acute Respiratory Distress Syndrome; Address; Air; Biochemical; Biochemistry; Biological Assay; Biological Models; Biological Process; Biological Testing; Breathing; Bronchoalveolar Lavage Fluid; Bronchoscopy; C-terminal; Cell Line; Cellular biology; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Cryoelectron Microscopy; Crystallization; Dependovirus; Diet; Disease; Electron Microscopy; Goals; Hospitals; Human; Hydrophobicity; In Vitro; Individual; Infection; Injury; Irrigation; Knockout Mice; Knowledge; Lipid Bilayers; Lipids; Lipopolysaccharides; Liposomes; Liquid substance; Lung; Lysosomes; Membrane; Methods; Microtomy; Modeling; Molecular; Mus; N-terminal; Organelles; Patients; Phospholipid Transfer Proteins; Phospholipids; Process; Protein Secretion; Proteins; Pulmonary Surfactant-Associated Protein B; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Recombinants; Respiration; Role; Saposins; Stress; Structure; Surface Tension; Syndrome; System; Techniques; Testing; Therapeutic; Trachea; Water; Woman; X-Ray Crystallography; alveolar epithelium; alveolar lamellar body; alveolar type II cell; design; dimer; experimental study; extracellular; improved; in vivo; lung injury; monolayer; mouse model; mutant; non-specific lipid transfer protein; promoter; protein aggregation; protein purification; pulmonary function; reconstitution; respiratory; structural biology; surfactant

PROJECT SUMMARY The goal of this project is to define the molecular mechanism by which lung surfactant protein B (SP-B) functions in respiration, and to develop an optimized surfactant mixture that could ultimately be used as a therapeutic for the treatment of Acute Respiratory Distress Syndrome (ARDS). SP-B is the only surfactant protein essential for breathing. It is made in alveolar type II cells as a precursor containing three related domains and is then proteolytically processed into the individual domains (SP-BN, SP-BM, SP-BC), with the middle domain (SP-BM) currently considered to be the “mature” protein. Surfactant originates from the secretion of lamellar bodies (LB), in which membrane sheets are densely stacked. The exported lipid bilayers then form a lipid monolayer at the air-water interface, which reduces surface tension and facilitates breathing. The exact functions of SP-BM and the other SP-B domains are unknown. Based on crystal structures, biochemical and cell biology experiments, we discovered that SP-BN, which had been largely ignored, is a non-specific lipid transfer protein in lungs. We also found that reconstitution of purified SP-BM into liposomes results in structures that have a striking resemblance to human LBs, suggesting that the entire organelle can be generated from a single protein. Further preliminary results indicate that intratracheal administration of purified SP-BN together with liposomes has a beneficial effect in a mouse model of ARDS. Based on these preliminary results, we will address the following questions: Specific aim #1: What are the functions of SP-BN and SP-BC? We will use purified SP-BN to elucidate the mechanism of lipid transfer and test whether purified SP-BC augments the activity of SP-BN. CRISPR and infection with adeno-associated virus (AAV) constructs will be used to test in mice whether SP-BN has a role in respiration and LB formation. We will use bronchoscopy of ARDS patients and control individuals to test for the presence of SP-BN in lavage fluid. Specific aim #2: What is the function of SP-BM? We will address the mechanism by which SP-BM forms LB-like structures using 2D crystallization and biochemical techniques. We will establish an expression and purification system for SP-BM, which has been a major bottleneck in the field, and determine structures of SP-BM by X-ray crystallography or cryo-EM. We will test whether SP-BN and SP-BC act synergistically with SP-BM to form LB-like structures in vitro. Specific aim #3: Can we generate a surfactant with therapeutic value? We will test whether SP-BN, SP-BM, and possibly SP-BC, together constitute the active extracellular surfactant. Intratracheal administration of a mixture of purified proteins will test whether mice expressing SP- B under an inducible promoter retain normal lung function in the absence of inducer in the diet. The mixture will also be tested in models of ARDS, in which lung injury is induced by either lipopolysaccharide or acid.

项目概要 该项目的目标是确定肺表面活性蛋白 B (SP-B) 的分子机制 呼吸作用，并开发一种优化的表面活性剂混合物，最终可用作 SP-B 是治疗急性呼吸窘迫综合征 (ARDS) 的唯一表面活性剂。 呼吸必需的蛋白质，由 II 型肺泡细胞制成，作为含有三种相关蛋白的前体。 结构域，然后通过蛋白水解加工成各个结构域（SP-BN、SP-BM、SP-BC），其中 中间结构域（SP-BM）目前被认为是“成熟”的蛋白质。 层状体（LB）的分泌，其中膜片密集堆叠，输出脂质双层。 然后在空气-水界面形成脂质单层，降低表面张力并促进呼吸。 根据晶体结构，SP-BM 和其他 SP-B 结构域的确切功能尚不清楚。 通过生化和细胞生物学实验，我们发现一直被忽视的SP-BN是一种 我们还发现将纯化的 SP-BM 重构到脂质体中。 结果产生与人类 LB 惊人相似的结构，这表明整个细胞器可以 进一步的初步结果表明，气管内给药。 基于这些，纯化的 SP-BN 与脂质体一起对 ARDS 小鼠模型具有有益的作用。 初步结果，我们将解决以下问题： 具体目标#1：SP-BN 和 SP-BC 的功能是什么？ 我们将使用纯化的SP-BN来阐明脂质转移的机制并测试纯化的SP-BC是否 增强 SP-BN 的活性并用腺相关病毒 (AAV) 构建体进行感染。 用于测试小鼠 SP-BN 是否在呼吸和 LB 形成中发挥作用，我们将使用支气管镜检查。 ARDS 患者和对照个体检测灌洗液中 SP-BN 的存在。 具体目标#2：SP-BM 的功能是什么？ 我们将探讨 SP-BM 使用 2D 结晶形成类 LB 结构的机制 我们将建立SP-BM的表达和纯化系统，该系统已被开发出来。 该领域的一个主要瓶颈，并通过 X 射线晶体学或冷冻电镜确定 SP-BM 的结构。 将测试SP-BN和SP-BC是否与SP-BM协同作用，在体外形成LB样结构。 具体目标#3：我们能否产生具有治疗价值的表面活性剂？ 我们将测试 SP-BN、SP-BM 和可能的 SP-BC 是否一起构成活性细胞外 气管内施用纯化蛋白质的混合物将测试小鼠是否表达 SP-。 在饮食中缺乏诱导剂的情况下，诱导型启动子下的 B 保留正常的肺功能。 还将在 ARDS 模型中进行测试，其中脂多糖或酸诱导肺损伤。