Microbicidal Proteins from Platelets

描述（由申请人提供）：我们发现了人血小板杀微生物蛋白（pmp）是杀微生物的趋化因子，称为人血小板杀微生物蛋白（hPKs），以反映其杀微生物和趋化因子的功能。hPKs在人类血液中具有先前未被识别的杀微生物活性，但在结构上与细胞毒性防御素样肽明显不同。因此，hPKs具有独特的结构-活性，与授予的杀微生物活性相关，而不伴随细胞毒性。特定的hPK结构域赋予直接的抗菌活性，并在模拟酸性吞噬溶酶体的环境中放大了杀微生物活性。不同的hPK结构域促进中性粒细胞吞噬和病原体的细胞内杀伤。我们假设hPKs中的特定决定因素支配这些独立但互补的抗菌功能。然而，hPKs协调宿主防御作用的结构决定因素尚不清楚。我们建议可以识别这些决定因素，定义其抗菌特性，并解决结构-活性特征。我们的补充具体目标将探讨这些假设：具体目标1：确定控制hPKs直接杀微生物功能的结构决定因素。我们将评估关键hPKs的肽库，以确定赋予相关杀微生物功能的结构域。战略突变肽和合成类似物将用于分离精确的结构决定因素，负责不同hPKs共享或独特的杀微生物谱。特定目标2：确定特定hPK结构域在关键中性粒细胞抗菌功能中的作用。我们将在体外使用多色流式细胞术解剖增强中性粒细胞吞噬、氧化破裂或细胞内杀伤的hPK结构域。这些研究将确定哪些hPK结构域直接增强中性粒细胞的关键抗菌机制，或通过增强这些细胞的固有能力。特异性目标3：解决hPK抗菌决定因子的结构-活性关系（SARs）。我们将利用互补的NMR、CD和FTIR研究来解决hPK决定因素中的SAR主题，以指导分子建模。我们独特的研究将是马赛克肽，旨在验证和交换不同hPKs中功能特征的作用。这些方法将确定特异性的SARs和功能决定因子，这些决定因子可能是不同的hPKs或其他杀肌毒素所特有或共有的。我们的发现创造了一个独特的机会来定义在先天免疫和适应性免疫中控制其多种抗菌功能的hPKs的分子特征。反过来，这些见解将加速开发预防或治疗严重感染的新策略，特别是由抗生素耐药病原体引起的感染。

DESCRIPTION (provided by applicant): We have discovered human platelet microbicidal proteins (PMPs) that are microbicidal chemokines, termed human platelet kinocidins (hPKs) to reflect their microbicidal and chemokine functions. hPKs have previously unrecognized microbicidal activity in human blood, but differ markedly in structure from cytotoxic defensin-like peptides. Thus, hPKs have unique structure-activity correlates to confer microbicidal activity without concomitant cytotoxicity. Specific hPK domains confer direct antimicrobial activity, and have amplified microbicidal activity in environments simulating acidic phagolysosomes. Distinct hPK domains promote neutrophil phagocytosis and intracellular killing of pathogens. We hypothesize that specific determinants in hPKs govern these independent but complementary antimicrobial functions. Yet, structural determinants responsible for the coordinated host defense roles of hPKs are unknown. We propose these determinants can be identified, their antimicrobial features defined, and structure-activity signatures resolved. Our complementary Specific Aims will explore these hypotheses: Specific Aim 1: Identify the structural determinants governing direct microbicidal functions of hPKs. We will assess peptide libraries of key hPKs to identify domains that confer relevant microbicidal functions. Strategic mutant peptides and synthetic analogues will be used to isolate precise structural determinants responsible for the shared or unique microbicidal profiles of distinct hPKs. Specific Aim 2: Define the roles of specific hPK domains on key neutrophil antimicrobial functions. We will dissect hPK domains that potentiate neutrophil phagocytosis, oxidative burst, or intracellular killing in vitro using multicolor flow cytometry. These studies will define which hPK domains enhance the key antimicrobial mechanisms in neutrophils directly, or by augmenting the inherent capabilities of these cells. Specific Aim 3: Resolve structure-activity relationships (SARs) in hPK antimicrobial determinants. We will resolve SAR themes in hPK determinants using complementary NMR, CD, and FTIR studies to guide molecular modeling. Unique to our studies will be mosaic peptides designed to validate and interchange the roles of functional signatures in distinct hPKs. These approaches will identify the specific SARs and functional determinants that may be specific to or shared among distinct hPKs or other kinocidins. Our discoveries create a unique opportunity to define molecular signatures in hPKs that govern their multiple antimicrobial functions in innate and adaptive immunity. In turn, these insights will accelerate development of novel strategies to prevent or treat serious infections, particularly those caused by antibiotic-resistant pathogens.