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.

项目总结 本项目的目标是确定肺表面活性物质蛋白B(SP-B)的分子机制。 在呼吸中的作用，并开发一种优化的表面活性剂混合物，最终可用作 治疗急性呼吸窘迫综合征(ARDS)。SP-B是唯一的表面活性剂 呼吸所必需的蛋白质。它是在肺泡II型细胞中作为前体产生的，含有三个相关的 结构域，然后被蛋白质降解处理成单独的结构域(SP-BN，SP-BM，SP-BC)，与 中间结构域(SP-BM)目前被认为是“成熟”蛋白。表面活性物质起源于 板层小体分泌(Lb)，膜片密集堆积。出口的脂质双分子膜 然后在空气-水界面形成一层脂单分子层，降低表面张力，促进呼吸。 SP-BM和其他SP-B结构域的确切功能尚不清楚。基于晶体结构， 经过生化和细胞生物学实验，我们发现此前被人们忽视的SP-BN是一种 肺中的非特异性脂转移蛋白。我们还发现纯化的SP-BM重组为脂质体 结果产生了与人类LBS惊人相似的结构，这表明整个细胞器可以 是由单一蛋白质产生的。进一步的初步结果表明，气管内给药 纯化的SP-BN与脂质体一起在ARDS小鼠模型中具有良好的效果。基于这些 根据初步结果，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，并检测纯化的SP-BC 增强SP-BN的活性。CRISPR和对腺相关病毒(AAV)的感染将是 用于在小鼠身上测试SP-BN是否对呼吸和LB的形成有作用。我们将使用支气管镜检查 对ARDS患者和对照组进行灌洗液中SP-BN的检测。 具体目标2：SP-BM的功能是什么？ 我们将讨论SP-BM使用2D结晶和 生化技术。我们将建立SP-BM的表达和纯化系统，该系统已经被 这是该领域的一个主要瓶颈，并通过X射线结晶学或低温电子显微镜确定SP-BM的结构。我们 将在体外测试SP-BN和SP-BC是否与SP-BM协同作用形成类LB型结构。 具体目标#3：我们能否生产出具有治疗价值的表面活性物质？ 我们将测试SP-BN、SP-BM，可能还有SP-BC是否共同构成活性胞外 表面活性剂。气管内注射纯化蛋白的混合物将测试表达SP-1的小鼠是否 B在可诱导启动子下，在饮食中没有诱导剂的情况下保持正常的肺功能。这种混合物 也将在ARDS模型中进行测试，在ARDS模型中，肺损伤是由内毒素或酸诱导的。