Design of Histatin 5 Variants for Improved Proteolytic Stability

用于提高蛋白水解稳定性的组氨酸 5 变体的设计

• 批准号: 10063508
• 负责人: Amy J Karlsson
• 金额: $ 15.04万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-12-01 至 2023-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10063508
• 关键词: Acquired Immunodeficiency Syndrome; Amino Acids; Antifungal Agents; Arginine; Aspartic Endopeptidases; Biochemical; Biological Assay; Biophysical Process; Candida albicans; Cations; Cell Wall; Cells; Charge; Cleaved cell; Collaborations; Complex; Data; Engineering; Environment; Environment Design; Enzymes; Exhibits; Family; Generations; Goals; Histidine; Human; Immune; Immune system; Individual; Innate Immune Response; Kinetics; Knowledge; Lead; Leucine; Location; Lysine; Microbiology; Modification; Oral; Oral candidiasis; Oral cavity; Organism; Parents; Patients; Peptide Hydrolases; Peptides; Play; Positioning Attribute; Predisposition; Production; Proteolysis; Resistance; Role; Running; Saliva; Salivary; Structure-Activity Relationship; Testing; Therapeutic; Therapeutic Agents; Variant; Work; analog; antimicrobial peptide; base; chemotherapy; design; experimental study; fungus; histatin 5; histidine-rich proteins; improved; in vivo; innate immune mechanisms; insight; member; microbial; molecular modeling; mouse model; opportunistic pathogen; oral commensal; oral infection; pathogen; peptide drug; prevent; prophylactic; protease C; protein aminoacid sequence; salivary histatins

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.

项目概要 白色念珠菌是一种口腔共生生物和机会致病菌。白色念珠菌是一种常见的 免疫系统受损患者（包括艾滋病患者和患者）口腔感染的原因 正在接受化疗的患者以及唾液水平低的患者。帮助防止负担 C. 为了避免白色念珠菌数量过高，健康个体的唾液中会产生多种免疫分子。之一 这些分子中最重要的是组氨酸 5 (Hst-5)，它是富含组氨酸的组氨酸家族的成员 抗菌肽。 Hst-5 是一种 24 个氨基酸的肽，具有最强的念珠菌活性 组氨酸肽。尽管唾液组蛋白能够有效杀死口腔中的白色念珠菌细胞，但 病原体还具有逃避 Hst-5 和其他肽杀死的机制。白色念珠菌产生一个家族 分泌型和细胞壁锚定的蛋白酶，称为分泌型天冬氨酸蛋白酶 (Saps)，能够 降解 Hst-5 并使其失活。先前的研究表明 Sap 酶在赖氨酸处裂解 Hst-5 残留物，在白色念珠菌细胞的裂解中也观察到这种情况。此外，人类唾液中还含有人类 以及也可以降解 Hst-5 的微生物蛋白酶。减少 Hst-5 的蛋白水解裂解并提高其 为了在口腔环境中发挥治疗潜力，我们设计了几种带有赖氨酸残基的 Hst-5 变体 被亮氨酸或精氨酸残基取代。最初的蛋白水解测试显示具有抗性的变异 由 Sap2 和 Sap9 酶及其变体进行的裂解，具有放大的裂解，突出了裂解的复杂性 SAPS 与 Hst-5 的相互作用。变体中的修改改变了 Sap 裂解的位置 酶并改变了降解肽的抗真菌活性。重要的是，肽具有改进的 与 Sap 酶一起孵育后，对裂解的抗性仍保持显着的抗真菌活性。 这些取代通常不会导致完整肽的抗真菌活性丧失，并且一些 肽表现出更强的活性。为了更好地理解 Hst-5 和 口腔中的蛋白水解酶并能够设计改进的肽疗法，我们建议 完成这项工作的两个目标。第一个目的是表征唾液蛋白酶和 具有 Hst-5 变体的其他 SAP，以了解导致裂解的结构-功能关系（以及 缺乏裂解）和抗真菌活性。第二个目标是利用通过我们获得的知识 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 肽序列对蛋白水解敏感性的影响。这个项目将加深我们对 Hst-5 与白色念珠菌 Sap 酶和人唾液中的酶相互作用，提供必要的 设计可作为治疗或预防口腔念珠菌病的药物的肽的知识。