Lipid Ligands of SPLUNC1 Proteins

SPLUNC1 蛋白的脂质配体

• 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
• 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

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）：自十多年前发现该家族以来，PLP 10（腭、肺和鼻上皮克隆）蛋白家族的同源脂质配体的身份一直难以确定。该家族最具特征的成员，短腭、肺和鼻上皮克隆1（SPLUNC 1）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，其在免疫系统中的功能已引起极大的兴趣。SPLUNC 1在宿主防御病原体感染中起着至关重要的作用。SPLUNC 1对肺炎支原体具有杀菌活性，并阻止铜绿假单胞菌的生长; N-末端结构域抑制上皮钠通道（ENaC），并且还报道SPLUNC 1充当表面活性剂以降低气道表面张力并干扰病原体的生物膜形成。然而，没有明确的证据表明SPLUNC 1的功能与BPI相似，包括对大多数革兰氏阴性细菌或革兰氏阳性细菌没有明显的杀菌活性，没有LPS中和作用，也没有调理素活性。此外，关于SPLUNC 1和LPS之间的结合存在争议的报道。为了更好地了解SPLUNC 1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SPLUNC 1的高分辨率结构。令我们惊讶的是，BPI和SPLUNC 1之间存在巨大的结构差异。整体结构与屋尘螨过敏原Der p 7相似。特别地，SPLUNC 1的表面覆盖有带负电荷的斑块，与BPI的带正电荷的表面相反，这对于结合LPS的带负电荷的头部基团是必不可少的。我们的体外结合测定显示SPLUNC 1和LPS之间没有结合。为了鉴定可能与SPLUNC 1结合的潜在脂质，我们对来源于人293细胞的SPLUNC 1进行质谱分析，发现SPLUNC 1主要被鞘磷脂（SM）饱和，少量被磷脂酰胆碱（PC）饱和。体外结合实验证明SPLUNC 1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，DPPC，肺中最丰富的脂质表面活性剂，结合SPLUNC 1。此外，DPPC是来自人和小鼠BAL液的脂质提取物中唯一结合SPLUNC 1的脂质，而SM是不可检测的。有趣的是，SPLUNC 1是DPPC的第一个鉴定的蛋白受体，DPPC是能够将表面张力降低到接近零的肺表面活性剂的唯一活性表面组分。我们假设DPPC可能是SPLUNC 1的同源脂质配体，而不是LPS。因此，本提案的第一个目标是鉴定、验证和确认SPLP 1的同源脂质配体。另 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。我们已经对SPLUNC 1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部基团，而且还取决于脂肪酸链。因此，该提议的第二个目标是阐明SPLUNC 1和脂质之间特异性决定簇的结构基础。基于所确定的脂质的结构特征和SPLUNC 1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 1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。这项提案将调查SPLUNC 1蛋白，一个成员的特点不佳的磷脂酶AMPs家族。SPLUNC 1在患有哮喘、COPD和囊性纤维化疾病的患者中显著下调。DPPC在哮喘中高度升高。鞘脂合成受损导致气道高反应性，而SM是鞘脂代谢过程的主要产物。确定SPLUNC 1在气道中的调节和功能的新机制将提高我们对过敏性气道中先天免疫受损的理解。通过操纵SPLUNC 1、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）中的细菌或其他病原体感染提供机会。我们在排除LPS的同时发现DPPC是SPLUNC 1的同源受体，这也将在磷脂酰肌醇领域带来巨大的影响，可能会彻底改变该领域的研究热点方向。