Lipid Ligands of SPLUNC1 Proteins

描述(申请人提供)：自从十多年前该家族被发现以来，PLUNC(腭部、肺和鼻部上皮克隆)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是短腭、肺和鼻上皮克隆1(SPLUN1)蛋白，正常呼吸道上皮细胞大量表达该蛋白。由于其在过敏性呼吸道中的分布和与BPI(杀菌/通透性增加蛋白)序列的相似性，其在机体免疫系统中的作用已引起人们的极大兴趣。SPLunc1在宿主抵抗病原体感染的防御中发挥着重要作用。SPLunc1对肺炎支原体具有杀菌活性，抑制铜绿假单胞菌的生长；其N-端结构域抑制上皮钠通道(ENaC)，也有报道SPLunc1作为表面活性物质降低呼吸道表面张力，干扰病原菌生物被膜的形成。然而，没有确凿的证据表明SPLunc1的功能类似于BPI，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，没有中和内毒素，也没有调理活性。此外，关于SPLunc1与内毒素的结合也有争议的报道。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的高分辨结构。令我们惊讶的是，公众宣传局和SPLUN1之间存在着巨大的结构性差异。它的整体结构类似于屋尘螨变应原Der p 7。特别是，SPLuc1的表面覆盖着带负电荷的斑块，这与BPI的带正电的表面形成对比，BPI的表面对于结合带负电的内毒素头基是必不可少的。我们的体外结合实验表明，SPLunc1与内毒素没有结合。为了确定可能与SPLunc1结合的潜在脂类，我们对来自人293细胞的SPLunc1进行了质谱分析，发现SPLunc1主要与鞘磷脂(SM)饱和，其次是磷脂酰胆碱(PC)。体外结合实验表明，SPLunc1能与鞘磷脂结合，但不能与最常见的卵磷脂结合。脂质结合筛选显示，肺中最丰富的脂质表面活性物质DPPC与SPLunc1结合。此外，DPPC是人和小鼠BAL液脂提物中唯一与SPLunc1结合的脂类，而SM则检测不到。有趣的是，SPLunc1是第一个被发现的DPPC的蛋白受体，它是肺表面活性物质中唯一能够将表面张力降低到接近零的活性表面成分。我们推测DPPC可能是SPLuc1的同源脂类配体，但不是内毒素。因此，这项提议的第一个目标是鉴定、验证和确认SPLUNC的同源脂类配体。另一方面 另一方面，关于蛋白质和脂类之间的特定结合的信息也是有限的。我们已经系统地筛选了SPLunc1的潜在目标。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类的头部基团，而且还取决于脂肪链。因此，这项建议的第二个目标是阐明SPLUN1和脂质之间特异性决定因素的结构基础(S)。根据已鉴定的脂类的结构特征和SPLunc1与脂类的一般特异性相互作用决定因素，我们将继续研究其他PLUNC家族成员的潜在脂类配体。抗菌肽(AMP)是真核生物保护自身免受感染的重要武器，是天然免疫系统的重要组成部分。这项提案将研究SPLunc1蛋白，它是特征不佳的PLUNC AMP家族的成员。在哮喘、慢性阻塞性肺疾病和囊性纤维化患者中，SPLunc1的表达显著下调。哮喘患者DPPC显著升高。鞘磷脂合成受损导致气道高反应性，而SM是鞘磷脂代谢过程的主要产物。确定SPLUN1在呼吸道中的调节和功能的新机制将有助于我们更好地了解过敏性呼吸道先天免疫受损的情况。通过操纵SPLuc1、DPPC和SM将我们的研究发现转化为治疗方法，我们拟议的工作最终将提供治疗慢性肺部疾病(如哮喘和其他肺部疾病)中的细菌或其他病原体感染的机会。我们在排除内毒素的同时发现了DPPC作为SPLUN1的同源受体，这也将给PLUNC领域带来巨大的影响，这可能会彻底改变该领域研究重点的方向。

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.