Proteolysis and Skin Antimicrobials

描述(申请人提供)：该提案寻求继续一条成功的研究路线，研究皮肤和微生物蛋白水解酶如何改变抗菌肽的功能。以往的数据表明，长春花素抗菌肽的激活依赖于包括激肽释放酶5和7在内的丝氨酸蛋白酶。底物长春花素前体Hcap18的替代处理可以产生各种成熟的具有不同功能的多肽。对这一过程的控制与皮肤病有关。酒渣鼻和牛皮癣。微生物蛋白酶的额外处理作为一种毒力机制来改变正常的宿主免疫。最近的数据显示，长春花素的替代酶处理使角质形成细胞能够通过TLR9识别自身DNA，并且TLR9对于抵御A组链球菌的皮肤感染是必不可少的。这些发现表明了皮肤上皮细胞识别核酸的新作用。我们建议的重点是定义青蒿素的蛋白水解物的加工控制模式。这种方法还将扩展到对微生物如何影响这些事件的评估。具体目的1.明确长春花素多肽与角质形成细胞对CpG和基因组DNA的反应能力之间的结构-功能关系。我们将建立一个长春新素多肽文库，并测试它们影响TLR9激活的能力。这一筛选将定义多肽的关键结构元素，并确定导致这些产品产生的关键酶步骤。具体目标2.确定对SA1中定义的产品的免疫反应，以确定其对皮肤炎症的影响。我们将通过使用小鼠皮肤炎症模型来建立AIM 1中确定的多肽的生理学相关性，研究替代长春花素多肽刺激的角质形成细胞产物如何改变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。特定目标3.确定细菌病原体的蛋白酶和核酸酶表达如何调节宿主皮肤的先天免疫反应。我们将研究细菌病原体表达的蛋白水解酶如何改变皮肤中肠毒素多肽的大小和活性。我们将研究放线菌素在中性粒细胞和肥大细胞产生的基于DNA的细胞外陷阱(ETS)中的表达及其对角质形成细胞TLR-9信号的影响。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，以及长春花素本身如何促进ET的产生。 与公共健康相关：酶的作用通过改变免疫系统各组成部分的功能来影响免疫功能。我们发现，皮肤细胞产生的多肽需要特定的酶来激活。这项提案将详细研究酶的特定多肽和核酸产物如何控制皮肤炎症和影响人类皮肤病。

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.