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寄生菌，是一种条件致病菌。C.白色念珠菌是常见的 导致口腔感染的免疫系统受损的患者，包括艾滋病患者和 正在接受化疗的患者，以及唾液分泌量低的患者。为了防止C的负担。 为了防止白色念珠菌的浓度过高，健康人的唾液中会产生几种免疫分子。之一 这些分子中最重要的是组胺素5（Hst-5），它是富含组氨酸的组胺素家族的一员， 抗菌肽hst-5是一种24个氨基酸的肽，具有最强的杀念珠菌活性， 组胺素肽虽然唾液组胺素对C.口腔中的白色念珠菌细胞， 病原体还具有逃避Hst-5和其它肽的杀死的机制。C.白色念珠菌产生一个家族 分泌型和细胞壁锚定蛋白酶称为分泌型天冬氨酸蛋白酶（SAP），能够 降解Hst-5并使其失活。先前的工作表明，Sap酶在赖氨酸处切割Hst-5 残基，这也观察到在切割由C。白色念珠菌细胞此外，人类唾液中含有人类 以及也可以降解Hst-5的微生物蛋白酶。减少Hst-5的蛋白水解切割并提高其活性 作为口腔环境中的治疗剂的潜力，我们设计了几种具有赖氨酸残基的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， 通过Sap2和Sap9酶的切割和具有扩增切割的变体，突出了 Saps与Hst-5的相互作用变异体中的修饰改变了Sap切割的位置， 酶和改变降解肽的抗真菌活性。重要的是，具有改善的 在与Sap酶一起孵育后，对切割的抗性保持了显著的抗真菌活性。 这些取代通常不会导致完整肽的抗真菌活性的丧失，并且一些取代也会导致抗真菌活性的丧失。 肽表现出更强的活性。为了更好地了解Hst-5和 口腔中的蛋白水解酶，并能够设计改进的肽治疗剂，我们提出， 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变体的额外SAP以理解导致切割的结构-功能关系（以及 缺乏切割）和抗真菌活性。第二个目标是利用我们从中学到的知识， 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影响。这个项目将提高我们对 Hst-5与C.白色念珠菌Sap酶和酶在人类唾液中，提供必要的 知识，以设计肽，可以作为治疗或预防口腔念珠菌病的治疗剂。