Lipid Ligands of SPLUNC1 Proteins

SPLUNC1 蛋白的脂质配体

• 批准号: 8895832
• 负责人: GONGYI ZHANG
• 金额: $ 19.81万
• 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 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8895832
• 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; Health; 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; receptor; screening; success; surfactant; therapeutic development; weapons; 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; Health; 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; 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被发现以来，该家族被发现以来，plunc（pa，肺和鼻上皮克隆）的同源脂质配体的身份一直难以捉摸。该家族中最有特征的成员，短pa，肺和鼻上皮克隆1（SPLUNC1）蛋白质由正常气道上皮细胞大量表达。由于其在过敏性气道中的分布和与BPI的序列相似性（杀菌/渗透性增加蛋白质），因此其在INNAE免疫系统中的功能已引起人们的极大关注。 SPLUNC1在针对病原体感染的宿主防御中起着至关重要的作用。 Splunc1具有针对支原体肺炎的杀菌活性，并阻止了铜绿假单胞菌的生长； N末端结构域抑制上皮钠通道（ENAC），还报道了Splunc1充当表面活性剂，可减少气道表面张力并通过病原体干扰生物膜形成。然而，没有明确的证据表明，Splunc1的作用类似于BPI，包括对大多数革兰氏阴性细菌或革兰氏阳性细菌的明显杀菌活性，没有中和LPS的中和，也没有反素活性。此外，关于Splunc1和LPS之间的结合存在有争议的报告。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之间存在巨大的结构差异。整体结构与Der P 7相似，Der P 7是一种内鞭螨过敏原。特别是，Splunc1的表面被带负电荷的斑块覆盖，而BPI的正电荷表面则是结合带负电荷的LPS的负电荷头组的必不可少的。我们的体外结合测定法在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主要用鞘磷脂（SM）饱和，而磷脂酰胆碱（PC）略微饱和。体外结合测定法证明，Splunc1可以结合鞘磷脂，而不是最常见的PC（来自鸡蛋的POPC）。脂质结合筛选显示，DPPC是肺中最丰富的脂质表面活性剂，与Splunc1结合。此外，DPPC是唯一来自人和小鼠BAL流体的脂质提取物的脂质，在SM无法检测的同时结合Splunc1的脂质。有趣的是，Splunc1是DPPC的第一个鉴定出的蛋白质受体，这是能够将表面张力降低到零的肺表面活性剂的唯一活性表面成分。我们假设DPPC可能是Splunc1而不是LP的同源脂质配体。因此，该提案的第一个目标是识别，验证和确认Splunc的同源脂质配体。另一方面 手，有关蛋白质和脂质之间特定结合的信息也受到限制。我们已经对Splunc1的潜在目标进行了全身筛选。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组，而且还由脂肪酸链确定。因此，该提案I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。基于确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）是主要武器，真核生物可以保护自己免受感染并代表先天免疫系统的主要组成部分。该提案将研究Splunc1蛋白，这是AMP的斑块家族较差的成员。 SPLUNC1在患有哮喘，COPD和囊性纤维化疾病的患者中受到了巨大调节。 DPPC在哮喘中高度升高。鞘脂合成受损会导致气道过度反应性，而SM是鞘脂代谢过程的主要产物。确定Splunc1调控和功能在气道中的新机制将改善我们对过敏气道先天免疫受损的理解。通过操纵SPLUNC1，DPPC和SM，通过将我们的研究发现转化为疗法，我们提出的工作最终将提供治疗细菌或其他病原体感染的机会，例如哮喘和其他肺部疾病。我们将DPPC作为Splunc1的同源受体的发现，同时排除了LPS也将对Plunc Field产生巨大影响，这可能会完全改变该领域研究重点的方向。