Molecular Basis of Human Neutrophil a-Defensin Function

描述(申请人提供)：人类(-防御素是一个3-4 kDa、阳离子和富含半胱氨酸的抗菌蛋白家族，主要在白细胞和上皮细胞中表达。它们可能通过破坏带负电荷的微生物膜杀死广泛的微生物，在吞噬和粘膜保护免受入侵病原体方面发挥关键作用。作为天然免疫的重要组成部分，人防御素通过趋化单核细胞、T淋巴细胞亚群和未成熟树突状细胞，在适应性免疫中发挥有效的免疫调节作用。最近，三种N端氨基酸残基不同的人中性粒细胞防御素(HNP1-3)被证明在体外可以抑制HFV-1感染。我们已经发现，远亲的人类中性粒细胞α-防御素第四成员(HNP4)和两个人类肠道α-防御素(HD5和HD6)也是外周血单核细胞中HIV-1复制的有效抑制物。 尽管α-防御素在先天免疫和获得性免疫中发挥着重要作用，并且在治疗感染性疾病方面具有潜在的治疗价值，但这些抗菌蛋白的结构/功能关系尚未被探索。支配α-防御素各种生物学功能的序列规则和结构决定因素及其作用机制仍然知之甚少。由于重组防御素固有的抗生素和膜溶解特性，生产重组防御素的难度在一定程度上阻碍了这方面的研究。另一方面，尽管α-防御素的体积很小，但由于与氧化折叠有关的已知问题，化学合成在技术上是具有挑战性的。因此，大量已发表的关于α-防御素的工作主要是基于从中性粒细胞中纯化的HNP1-3的研究。由于其他三种天然α-防御素的回收数量很少(HNP4)、较小(HD5)或零(HD6)，因此人们对它们的性质知之甚少。 我们实验室最近开发了一种强大的合成方法和一种有效的折叠方案，用于高纯度和高产量地生产所有六种人α-防御素，首次实现了对这些抗菌蛋白的系统和比较结构/功能关系的研究。这项研究不仅将更好地了解防御素在分子水平上的作用，更重要的是，将为设计基于防御素的新型治疗剂以对抗感染微生物提供有价值的见解。本申请设想的具体目标如下。具体目的1：阐明其前肽对HNPs的抑制机制。具体目标2：剖析影响α-防御素抗菌活性和特异性的分子决定因素。具体目标3：破译HNP齐聚的生理相关性和功能重要性。

DESCRIPTION (provided by applicant): Human (-defensins are a family of 3-4 kDa, cationic and Cys-rich antimicrobial proteins expressed predominantly in leukocytes and epithelial cells. They kill a broad range of microbes presumably through disruption of the negatively charged microbial membrane, playing critical roles in phagocytosis and in mucosal protection against invading pathogens. While an important component of innate immunity, human (-defensins also function as effective immune modulators in adaptive immunity by chemoattracting monocytes, subsets of T lymphocytes and immature dendritic cells. Recently, three human neutrophil (-defensins (HNP1-3), differing from each other by a single amino acid residue at the N-terminus have been shown to suppress HFV-1 infection in vitro. We have found that the distantly related fourth member of human neutrophil a-defensins (HNP4) and two human intestinal alpha-defensins (HD5 and HD6) are also effective inhibitors of HIV-1 replication in peripheral blood mononuclear cells. Despite the prominent roles alpha-defensins play in both innate and adaptive immunity and their potential therapeutic value in the treatment of infectious diseases, the structure/function relationships for these antimicrobial proteins have not yet been explored. The sequence rules and structural determinants that govern the great variety of biological functions of alpha-defensins and mechanisms of their action continue to remain poorly understood. Such study has been hindered, in part, by the difficulty producing recombinant defensins due to their inherent antibiotic and membranolytic properties. On the other hand, chemical synthesis of alpha-defensins, in spite of their small size, is technically challenging due to known problems associated with oxidative folding. Consequently, a significant body of the published work on alpha-defensins has been based largely on studies of HNP1-3 purified from neutrophils. As the other three native alpha-defensins have been recovered in amounts that are small (HNP4), smaller (HD5), or nil (HD6), considerably less is known about their properties. Our laboratory has recently developed a robust synthetic approach to, and an efficient folding protocol for, the production of all six human alpha-defensins in high purity and yield, enabling for the first time a systematic and comparative structure/function relationship study on these antimicrobial proteins. Such study will not only provide a better understanding of how defensins function at the molecular level, but more importantly, yield valuable insights into designing defensin-based novel therapeutic agents to combat infectious microbes. Specific aims envisioned in this application are as follows. Specific Aim 1: Elucidate the mechanism of inhibition of HNPs by their pro peptides. Specific Aim 2: Dissect the molecular determinants for the antimicrobial activity and specificity of alpha-defensins. Specific Aim 3: Decipher the physiological relevance and functional importance of HNP oligomerization.