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.

项目摘要 白色念珠菌是一种口腔共生生物和机会性病原体。白色念珠菌是一个频繁的 免疫系统受损的患者口腔感染的原因，包括艾滋病患者和患者 接受化学疗法，以及产生唾液水平较低的患者。帮助防止C的负担。 白色疾病变得过高，健康个体在其唾液中产生几种免疫分子。之一 这些分子中最重要的是Hestatin 5（HST-5），这是富含组氨酸的组蛋白家族的成员 抗菌肽。 HST-5是一种24-氨基酸肽，具有最强的念珠化活性 组蛋白肽。尽管唾液组蛋白可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。 C.白色唱片人产生一个家庭 分泌和细胞壁锚定的蛋白酶，称为分泌的天冬氨酸（SAPS），能够 降解HST-5并使其不活跃。先前的工作表明，SAP酶在赖氨酸上切割HST-5 残基，白色念珠菌细胞在裂解中也观察到。另外，人类唾液包含人类 和微生物蛋白酶，也可能降解HST-5。减少HST-5的蛋白水解裂解并改善其 在口腔环境中作为治疗性的潜力，我们设计了几种HST-5变体的赖氨酸残基 用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体 SAP2和SAP9酶的切割以及具有放大裂解的变体，突出了该酶的复杂性 SAPS与HST-5的相互作用。变体的修改使SAP改变了裂解的位置 酶并改变了降解肽的抗真菌活性。重要的是，改善的肽 对裂解的抗性在与SAP酶孵育后保持显着的抗真菌活性。 替代通常不会导致完整肽的抗真菌活性丧失，有几个 肽表现出更强的活性。更好地了解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 使用HST-5变体的其他SAPS了解导致裂解的结构 - 功能关系（和 缺乏切割）和抗真菌活性。第二个目的是利用通过我们的知识 初步数据并旨在设计和测试第二代HST-5变体，以进一步探索 肽序列对蛋白水解易感性的影响。该项目将提高我们对 HST-5与白色念珠菌SAP酶和人类唾液中的酶的相互作用，提供必要的 知识设计可以用作治疗或预防口服念珠菌病的肽。