Proteolysis and Skin Antimicrobials

描述（由申请人提供）：本提案旨在继续成功研究皮肤和微生物蛋白酶如何修饰抗菌肽功能。先前的数据已经表明，凯萨林菌素抗微生物肽的活化依赖于丝氨酸蛋白酶，包括激肽释放酶5和7。底物凯萨林菌素前体hCAP 18的替代加工产生具有替代功能的多种成熟肽。这种处理的控制与皮肤病有关，即。红斑痤疮和牛皮癣。微生物蛋白酶的额外加工作为改变正常宿主免疫力的毒力机制。最近的数据显示，凯萨林菌素的替代酶促加工使角质形成细胞能够通过TLR 9识别自身DNA，并且TLR 9对于防御A组链球菌的皮肤感染是必不可少的。这些研究结果表明，皮肤上皮细胞的核酸识别的一个新的作用。我们建议的重点是确定哪些蛋白水解产物对凯萨林菌素的加工控制模式。这种方法也将扩展到评估微生物如何影响这些事件。具体目标1。定义cathelicidin肽与角质形成细胞对CpG和基因组DNA反应能力之间的结构-功能关系。我们将产生一个cathelicidin肽库，并测试它们影响TLR 9激活的能力。该筛选将确定肽的关键结构要素，并确定导致这些产物产生的关键酶促步骤。具体目标2。表征对SA 1中定义的产品的免疫应答，以确定其对皮肤炎症的影响。我们将通过使用皮肤炎症的小鼠模型来建立在目标1中鉴定的肽的生理相关性，检查由替代性凯萨林菌素肽刺激的角质形成细胞的产物如何改变T细胞极化，并建立这些发现在体内皮肤炎症反应中的作用。具体目标3.确定细菌病原体如何表达蛋白酶和核酸酶调节宿主皮肤先天免疫反应。我们将研究细菌病原体的蛋白酶表达如何改变皮肤中凯萨林菌素肽的大小和活性。我们将研究cathelicidin在由中性粒细胞和肥大细胞产生的基于DNA的细胞外陷阱（cathelicidin）中的表达及其对角质形成细胞TLR-9信号传导的影响。我们将研究细菌蛋白酶和核酸酶的表达如何影响这些相互作用，以及cathelicidins本身如何促进ET的产生。 公共卫生相关性： 酶的作用通过改变免疫系统功能的组成部分来影响免疫功能。我们发现皮肤细胞产生的肽需要特定的酶来激活。本计划将详细研究酶的特定肽和核酸产物如何控制皮肤炎症并影响人类皮肤疾病。

DESCRIPTION (provided by applicant): This proposal seeks to continue a successful line of investigation into how skin and microbial proteases modify antimicrobial peptide function. Previous data have shown that activation of cathelicidin antimicrobial peptides is dependent on serine proteases including Kallikrein 5 and 7. Alternative processing of the substrate cathelicidin precursor hCAP 18 results in a variety of mature peptides with alternative function. Control of this processing is relevant to skin diseases ie. rosacea and psoriasis. Additional processing by microbial proteases serves as a virulence mechanism to alter normal host immunity. Recent data have shown alternative enzymatic processing of cathelicidin enables keratinocytes to recognize self-DNA through TLR9 and that TLR9 is essential for defense against skin infection by Group A Streptococcus. These findings suggest a novel role for nucleic acid recognition by skin epithelium. The focus of our proposal is to define what proteolytic products of cathelicidin processing control pattern. This approach will also extend to evaluation of how microbes influence these events. Specific Aim 1. Define the structure-function relationships between cathelicidin peptides and the capacity of keratinocytes to respond to CpG and genomic DNA. We will generate a library of cathelicidin peptides and test their ability to influence TLR9 activation. This screen will define the critical structural elements of the peptide and identify key enzymatic steps that result in the generation of these products. Specific Aim 2. Characterize the immune response to products defined in SA1 to determine the consequences of this to skin inflammation. We will establish the physiological relevance of peptides identified in aim 1 by using mouse models of skin inflammation, examine how the products of keratinocytes stimulated by alternative cathelicidin peptides alter T cell polarization, and establish the role of these findings in the in vivo skin inflammatory response. Specific Aims3. Determine how protease and nuclease expression by a bacterial pathogen modulates host cutaneous innate immune responses. We will study how protease expression by a bacterial pathogen changes the size and activity profile of cathelicidin peptides in skin. We will investigate cathelicidin expression in DNA-based extracellular traps (ETs) produced by neutrophils and mast cells and their effect on keratinocyte TLR-9 signaling. We will study how bacterial protease and nuclease expression influences these interactions, and how cathelicidins themselves may promote ET production. PUBLIC HEALTH RELEVANCE: The action of enzymes influences immune function by changing the way components of the immune system function. We have discovered that peptides produced by skin cells require specific enzymes for activation. This proposal will study in detail how specific peptide and nucleic acid products of enzymes control skin inflammation and influence human skin diseases.