Proteolysis and Skin Antimicrobials

描述（由申请人提供）：该提案旨在继续对皮肤和微生物蛋白酶如何改变抗菌肽功能进行成功的研究。先前的数据表明，抗菌素抗菌肽的激活依赖于丝氨酸蛋白酶，包括激肽释放酶 5 和 7。底物抗菌素前体 hCAP 18 的替代处理可产生多种具有替代功能的成熟肽。该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可作为改变正常宿主免疫力的毒力机制。最近的数据表明，cathelicidin 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 对于防御 A 组链球菌引起的皮肤感染至关重要。这些发现表明皮肤上皮的核酸识别具有新的作用。我们提案的重点是定义导管素加工控制模式的蛋白水解产物。这种方法还将扩展到评估微生物如何影响这些事件。具体目标 1. 定义抗菌素肽与角质形成细胞响应 CpG 和基因组 DNA 的能力之间的结构-功能关系。我们将生成一个抗菌素肽库并测试它们影响 TLR9 激活的能力。该筛选将定义肽的关键结构元件，并确定导致这些产品生成的关键酶促步骤。具体目标 2. 表征对 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确定目标 1 中确定的肽的生理相关性，检查替代性导管素肽刺激的角质形成细胞产物如何改变 T 细胞极化，并确定这些发现在体内皮肤炎症反应中的作用。具体目标3。确定细菌病原体的蛋白酶和核酸酶表达如何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抗菌素肽的大小和活性特征。我们将研究嗜中性粒细胞和肥大细胞产生的基于 DNA 的细胞外陷阱 (ET) 中的导管素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表达如何影响这些相互作用，以及抗菌素本身如何促进 ET 的产生。 公共卫生相关性：酶的作用通过改变免疫系统组成部分的功能方式来影响免疫功能。我们发现皮肤细胞产生的肽需要特定的酶来激活。该提案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.