Lipid Ligands of SPLUNC1 Proteins

SPLUNC1 蛋白的脂质配体

基本信息

批准号：
8771137
负责人：
GONGYI ZHANG
金额：
$ 23.78万
依托单位：
NATIONAL JEWISH HEALTH
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8771137
关键词：
A549 Address Affinity Allergic Asthma Bacteria Binding Biological Assay Bronchoalveolar Lavage Fluid Cell Line Cells Charge Chickens Choline Chronic Obstructive Airway Disease Chronic lung disease Complex Cystic Fibrosis Data Dermatophagoides pteronyssinus antigen p 7 Disease Epithelial Epithelial Cells Eukaryota Family Family member Fatty Acids Goals Gram-Negative Bacteria Gram-Positive Bacteria Growth Head Host Defense House Dust Mite Allergens Human Immune response Immune system In Vitro Infection Knowledge Lead Ligand Binding Ligands Lipid Binding Lipids Lipopolysaccharides Lung Lung diseases Mass Spectrum Analysis Membrane Proteins Metabolism Microbial Biofilms Molecular Structure Mus Mycoplasma pneumoniae Names Nasal Epithelium Natural Immunity Opsonin Palate Pathologic Patients Pharmaceutical Preparations Physicians Play Process Protein Family Proteins Pseudomonas aeruginosa Pulmonary Surfactants Regulation Reporting Research Research Personnel Resolution Role Sodium Channel Specificity Sphingolipids Sphingomyelins Structure Surface Surface Tension Tail Therapeutic Time Translating Work airway hyperresponsiveness antimicrobial peptide bactericidal permeability increasing protein bactericide base combat drug development egg epithelial Na+ channel improved inorganic phosphate interest killings member novel pathogen protein folding public health relevance receptor screening success surfactant therapeutic development weapons

项目摘要

DESCRIPTION (provided by applicant): The identity of the cognate lipid ligands for the PLUNC (palate, lung, and nasal epithelium clone) protein family have remained elusive since the family was discovered more than one decade ago. The most characterized member of the family, the short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is abundantly expressed by normal airway epithelial cells. Due to its distribution in allergic airways and sequence similarity to BPI (bactericidal/permeability increasing protein), its function in the innae immune system has become of great interest. SPLUNC1 plays crucial roles in host defense against pathogen infections. SPLUNC1 has bactericidal activity against Mycoplasma pneumoniae and arrests the growth of Pseudomonas aeruginosa; the n-terminal domain inhibits the epithelial sodium channel (ENaC), and it is also reported that SPLUNC1 acts as surfactant to reduce airway surface tension and interfere with biofilm formation by pathogens. However, no definitive evidence has shown that SPLUNC1 functions similarly to BPI, including no apparent bactericidal activity against most Gram-negative bacteria or Gram-positive bacteria, no neutralization of LPS, and no opsonin activity. Furthermore, there are controversial reports about the binding between SPLUNC1 and LPS. To better understand the function roles of SPLUNC1 in host defense and innate immune system, we recently determined the high-resolution structure of SPLUNC1. To our surprise, there are dramatic structural differences between BPI and SPLUNC1. The overall structure is similar to that of Der p 7, a house-dust mite allergen. In particular, the surface of SPLUNC1 is covered with negatively charged patches, in contrast to the positively charged surface of BPI, which is essential to bind the negatively charged head group of LPS. Our in vitro binding assays showed no binding between SPLUNC1 and LPS. To identify potential lipids that might bind to SPLUNC1, we subjected SPLUNC1 derived from human 293 cells to mass spectrometry analysis and found that SPLUNC1 was saturated mainly with Sphingomyelin(SM) and minorly with Phosphatidyl choline (PC). In vitro binding assays proved that SPLUNC1 could bind Sphingomyelin but not the most common PC (POPC from chicken egg). Lipid binding screen revealed that DPPC, the most rich lipid surfactant in lung, binds to SPLUNC1. Furthermore, DPPC is the only lipid of lipid extracts from human and mouse BAL fluid that binds SPLUNC1 while SM is undetectable. Interestingly, SPLUNC1 is the first identified protein receptor of DPPC, which is the only active surface component of lung surfactant capable of lowering surface tension to near zero. We hypothesize that DPPC could be the cognate lipid ligand for SPLUNC1 but not LPS. Thus, the first goal of this proposal is to identify, verify, and confirm the cognate lipid ligands of SPLUNC. On the other hand, information regarding the specific binding between proteins and lipids is also limited. We have carried out systemic screening of potential targets for SPLUNC1. Our preliminary data showed that the specificity between ligand and protein is not only determined by the head group of the lipid but also the by the fatty acid chains. Accordingly, the second goal of this proposal i to elucidate the structural basis of the specificity determinants between SPLUNC1 and lipid(s). Based on structural features of the lipid identified and the general specific interaction determinants of SPLUNC1 with lipids, we will proceed with investigating potential lipid ligands for other PLUNC family members. Antimicrobial peptides (AMP) are key weapons by which eukaryotes protect themselves against infection and represent a major component of the innate immune system. This proposal will investigate the SPLUNC1 protein, a member of the poorly characterized PLUNC family of AMPs. SPLUNC1 is dramatically down regulated in patients with Asthma, COPD, and Cystic Fibrosis diseases. DPPC is highly elevated in Asthma. Impaired sphingolipid synthesis causes airway hyperreactivity while SM is a major product of sphingolipid metabolism process. Determining the novel mechanisms of SPLUNC1 regulation and function in the airways will improve our understanding of impaired innate immunity in allergic airways. By translating our research discoveries into therapies through manipulation of SPLUNC1, DPPC, and SM, our proposed work will ultimately provide opportunities to treat bacterial or other pathogen infections in chronic lung diseases such as asthma and other pulmonary diseases. Our discovery of DPPC as the cognate receptor of SPLUNC1 while ruling out LPS will also bring great impact in the PLUNC field, which may completely change the direction of the research focus of the field.

描述（由申请人提供）：自从十多年前发现PLUNC（腭、肺和鼻上皮克隆）蛋白家族以来，该家族的同源脂质配体的身份一直难以捉摸。该家族中最具特征的成员是短腭、肺和鼻上皮克隆 1 (SPLUNC1) 蛋白，在正常气道上皮细胞中大量表达。由于其在过敏性气道中的分布以及与 BPI（杀菌/通透性增加蛋白）的序列相似性，其在内免疫系统中的功能引起了人们的极大兴趣。 SPLUNC1 在宿主防御病原体感染方面发挥着至关重要的作用。 SPLUNC1对肺炎支原体具有杀菌活性，并能抑制铜绿假单胞菌的生长； N端结构域抑制上皮钠通道（ENaC），据报道，SPLUNC1充当表面活性剂，降低气道表面张力并干扰病原体生物膜的形成。然而，没有明确的证据表明SPLUNC1的功能与BPI类似，包括对大多数革兰氏阴性菌或革兰氏阳性菌没有明显的杀菌活性，对LPS没有中和作用，也没有调理素活性。此外，关于 SPLUNC1 和 LPS 之间的结合也存在争议。为了更好地了解SPLUNC1在宿主防御和先天免疫系统中的功能作用，我们最近确定了SPLUNC1的高分辨率结构。令我们惊讶的是，BPI 和 SPLUNC1 之间存在显着的结构差异。整体结构与屋尘螨过敏原 Der p 7 相似。特别是，SPLUNC1 的表面覆盖着带负电的斑块，与 BPI 带正电的表面相反，BPI 的表面对于结合 LPS 带负电的头基至关重要。我们的体外结合测定显示 SPLUNC1 和 LPS 之间没有结合。为了鉴定可能与 SPLUNC1 结合的潜在脂质，我们对源自人 293 细胞的 SPLUNC1 进行了质谱分析，发现 SPLUNC1 主要被鞘磷脂 (SM) 饱和，少量被磷脂酰胆碱 (PC) 饱和。体外结合测定证明SPLUNC1可以结合鞘磷脂，但不能结合最常见的PC（来自鸡蛋的POPC）。脂质结合筛选显示，肺中最丰富的脂质表面活性剂 DPPC 与 SPLUNC1 结合。此外，DPPC 是人类和小鼠 BAL 液脂质提取物中唯一与 SPLUNC1 结合的脂质，而 SM 无法检测到。有趣的是，SPLUNC1是第一个被识别的DPPC蛋白受体，DPPC是肺表面活性剂中唯一能够将表面张力降低至接近零的活性表面成分。我们假设 DPPC 可能是 SPLUNC1 的同源脂质配体，但不是 LPS。因此，该提案的首要目标是鉴定、验证和确认 SPLUNC 的同源脂质配体。另一方面另一方面，有关蛋白质和脂质之间特异性结合的信息也很有限。我们对SPLUNC1的潜在靶点进行了系统筛选。我们的初步数据表明，配体和蛋白质之间的特异性不仅取决于脂质的头基，还取决于脂肪酸链。因此，该提案的第二个目标是阐明 SPLUNC1 和脂质之间特异性决定因素的结构基础。基于所鉴定的脂质的结构特征以及 SPLUNC1 与脂质的一般特异性相互作用决定因素，我们将继续研究其他 PLUNC 家族成员的潜在脂质配体。抗菌肽（AMP）是真核生物保护自身免受感染的关键武器，也是先天免疫系统的主要组成部分。该提案将研究 SPLUNC1 蛋白，它是尚未充分表征的 AMP PLUNC 家族的成员。 SPLUNC1 在哮喘、慢性阻塞性肺病和囊性纤维化疾病患者中显着下调。哮喘患者中 DPPC 升高。鞘脂合成受损会导致气道高反应性，而 SM 是鞘脂代谢过程的主要产物。确定 SPLUNC1 在气道中调节和功能的新机制将提高我们对过敏性气道先天免疫受损的理解。通过操纵 SPLUNC1、DPPC 和 SM 将我们的研究发现转化为治疗方法，我们提出的工作最终将为治疗慢性肺部疾病（如哮喘和其他肺部疾病）的细菌或其他病原体感染提供机会。我们发现DPPC作为SPLUNC1的同源受体，同时排除LPS，也将给PLUNC领域带来巨大的影响，这可能会彻底改变该领域的研究重点方向。