Design of Histatin 5 Variants for Improved Proteolytic Stability

项目总结 白色念珠菌是一种口腔共生微生物，也是一种条件致病菌。白色念珠菌是一种常见的 免疫系统受损患者口腔感染的原因，包括艾滋病患者和患者 正在接受化疗，以及唾液分泌水平较低的患者。为了帮助防止C。 白念珠菌变得过高，健康的人会在唾液中产生几种免疫分子。其中之一 这些分子中最重要的是组蛋白5(HST-5)，它是富含组氨酸的组蛋白家族的成员 抗菌肽。HST-5是一种由24个氨基酸组成的多肽，具有最强的杀菌活性。 组蛋白多肽。虽然唾液组织蛋白能有效杀灭口腔中的白色念珠菌细胞，但 病原菌也有逃避HST-5和其他多肽杀伤的机制。白念珠菌产生一个家庭 一种被称为分泌型天冬氨酸蛋白酶(SAP)的分泌型和细胞壁锚定的蛋白酶，能够 降解HST-5并使其失活。以前的工作表明，SAP酶在赖氨酸上裂解HST-5 残留物，在白色念珠菌细胞的裂解过程中也能观察到。此外，人类唾液中含有人类 以及也能降解HST-5的微生物蛋白酶。减少HST-5的蛋白水解性切割并改进其 在口腔环境中潜在的治疗作用，我们设计了几个含有赖氨酸残基的HST-5变体 用亮氨酸或精氨酸残基取代。初步蛋白分解测试显示变种具有抗药性 由Sap2和Sap9酶及其变异体通过扩增的切割作用，突出了 SAP与HST-5的相互作用。变异体中的修饰改变了SAP的切割位置 酶活性，并改变降解多肽的抗真菌活性。重要的是，具有改进的多肽 在与SAP酶孵育后，对切割的抗性保持了显著的抗真菌活性。 取代一般不会导致完整多肽的抗真菌活性丧失，以及几个 多肽表现出较强的活性。为了更好地了解HST-5和HST-5之间的复杂相互作用 口腔中的蛋白水解酶，并使改进的多肽疗法的设计成为可能，我们建议 完成这项工作的两个目标。第一个目标是描述唾液蛋白水解酶和唾液蛋白的相互作用。 带有HST-5变体的额外SAP，以了解导致切割的结构-功能关系(和 缺乏卵裂)和抗真菌活性。第二个目标是利用通过我们的 初步数据和目标1设计和测试第二代HST-5变体，进一步探索 多肽序列对蛋白水解性的影响。这个项目将提高我们对 HST-5与白色念珠菌SAP酶和人唾液中的酶的相互作用，提供必要的 了解设计可用作治疗或预防口腔念珠菌病的多肽。

PROJECT SUMMARY Candida albicans is an oral commensal organism and an opportunistic pathogen. C. albicans is a frequent cause of oral infection in patients with compromised immune systems, including AIDS patients and patients undergoing chemotherapy, and in patients who produce low levels of saliva. To help prevent the burden of C. albicans from becoming too high, healthy individuals produce several immune molecules in their saliva. One of the most important of these molecules is histatin 5 (Hst-5), a member of the histatin family of histidine-rich antimicrobial peptides. Hst-5 is a 24-amino acid peptide and has the strongest candidacidal activity of the histatin peptides. Although salivary histatin is effective at killing C. albicans cells in the oral cavity, the pathogen also has a mechanism for evading killing by Hst-5 and other peptides. C. albicans produces a family of secreted and cell-wall anchored proteases called secreted aspartic proteases (Saps) that are capable of degrading Hst-5 and making it inactive. Previous work showed that Sap enzymes cleave Hst-5 at lysine residues, which is also observed in cleavage by C. albicans cells. Additionally, human saliva contains human and microbial proteases that can also degrade Hst-5. To reduce proteolytic cleavage of Hst-5 and improving its potential as a therapeutic in the oral environment, we designed several Hst-5 variants with the lysine residues substituted with a leucine or arginine residue. Initial proteolysis testing showed variants with resistance to cleavage by Sap2 and Sap9 enzymes and variants with amplified cleavage, highlighting the complexity of the interaction of the Saps with Hst-5. The modifications in the variants shifted the location of cleavage by the Sap enzymes and altered the antifungal activity of the degraded peptides. Importantly, peptides with improved resistance to cleavage maintained significant antifungal activity following incubation with the Sap enzymes. The substitutions generally did not result in loss of antifungal activity for the intact peptide, and several peptides exhibited stronger activity. To better understand the complex interaction between the Hst-5 and proteolytic enzymes in the oral cavity and enable design of improved peptide therapeutics, we propose to complete two aims in this work. The first aim is to characterize the interaction of salivary proteases and additional Saps with Hst-5 variants to understand the structure-function relationships that lead to cleavage (and lack of cleavage) and antifungal activity. The second aim is to use the knowledge gained through our preliminary data and Aim 1 to design and test a second generation of Hst-5 variants that further explore the effect of peptide sequence on proteolytic susceptibility. This project will improve our understanding of the interaction of Hst-5 with C. albicans Sap enzymes and enzymes in human saliva, providing the necessary knowledge to design peptides that could function as therapeutics to treat or prevent oral candidiasis.