Uncovering the biological roles of inflammatory caspases through chemical approaches

通过化学方法揭示炎症半胱天冬酶的生物学作用

• 批准号: 10047041
• 负责人: Caitlin E. Karver
• 金额: $ 43.79万
• 依托单位: DE PAUL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10047041
• 关键词: Active Sites; Address; Amino Acids; Aspartate; Aspartic Acid; Autoimmune Diseases; Autoimmune Process; Binding; Biological; Biological Assay; Biological Models; Biology; Biomedical Research; Biophysics; C-terminal; CASP5 gene; Caspase; Cessation of life; Chemicals; Cleaved cell; Conflict (Psychology); Cysteine; Development; Disease; Drug Targeting; Dyes; Energy Transfer; Enzymes; Fluorescence; Fluorescence Resonance Energy Transfer; Fluorogenic Substrate; Funding; Health; Human; Individual; Inflammasome; Inflammation; Inflammatory; Inflammatory Response; Innate Immune Response; Interdisciplinary Study; Ketones; Kinetics; Laboratories; Lipopolysaccharides; Location; Malignant Neoplasms; Methods; Micelles; Monitor; Mutagenesis; N-terminal; Neurodegenerative Disorders; Noise; Peptide Hydrolases; Peptides; Play; Property; Regulation; Reporter; Reporting; Research; Research Personnel; Role; Sepsis; Side; Signal Transduction; Site; Structure; Students; Substrate Specificity; Training; Universities; Variant; autoinflammatory; base; chromophore; combat; drug development; drug discovery; enzyme structure; experience; fluorophore; human disease; improved; inhibitor/antagonist; insight; mutant; novel; pathogen; programs; protein complex; quantum; response; therapeutic development; therapeutic lead compound; tool; tool development; undergraduate student; unnatural amino acids

Project Summary Inflammatory caspases (cysteine-dependent, aspartate specific proteases) are associated with protein complexes termed inflammasomes that play a role in the innate immune response by producing inflammation and cellular death in response to pathogens and danger signals. Dysregulation of the inflammatory response is associated with sepsis and disease states ranging from autoimmune and neurodegenerative disorders to cancer. Due to their role in a wide range of human diseases, inflammatory caspases are a focus for many drug discovery programs. The long term objective for this proposal is the development of tools for assessing inhibition of inflammatory caspases and allowing the role each enzyme in the inflammatory response to be determined. This objective can be best addressed by developing peptide substrates for activity assays that selectively interact with individual inflammatory caspases. To date, the creation of such substrates for inflammatory caspases has been elusive. Prior approaches to generating selectivity have focused on varying the amino acids N-terminal to the aspartate residue. The PIs have shown it is possible to vary the amino acids C-terminal to the aspartate by incorporating the reporting chromophores as side chains of non-natural amino acids and observed markedly different kinetics for an inflammatory caspase reacting with peptides that differed by a single C-terminal amino acid. The mechanism of inflammasome formation is not clear for caspase-4 or -5 and the majority of information available is for caspase-4. In order to resolve the individual biological roles of these enzymes, structural studies of inflammasome formation must be performed on both C-4 and C-5 independently. Previous studies have been performed using inactive mutant enzyme rather than a more biologically relevant active form that is chemically inactivated. The hypothesis of this proposal is that the biological roles and mechanism of activation of each inflammatory caspase can be uncovered by using a chemical approach to studying the activity of each enzyme. The following specific aims will address this hypothesis: 1) Develop and validate peptide substrates with 100- fold selectivity for each inflammatory caspase by varying the amino acids on the C-terminal side of the aspartic acid residue and determine the chromophore pair needed to produce the maximum signal-to-noise ratio. 2) Determine the mechanism of activation for caspase-4 and caspase-5 using mutagenic and chemical methods. The development of selective substrates for inflammatory caspases using multiple, high signal-to-noise dye pairs will allow the activity of each caspase to be assessed via a unique fluorescence reporter. The creation of these substrates will positively impact the field of drug development to combat inflammatory diseases and sepsis. Chemical approaches to studying inflammasome formation will allow the roles of inflammatory caspases in the innate immune response to be de-convolved, contributing to the advancement of our understanding of inflammation. This proposal will provide an interdisciplinary research experience for undergraduate students, exposing them to applications of chemical biology and biophysics in improving human health.

项目摘要 炎性胱天蛋白酶（半胱氨酸依赖性，天冬氨酸特异性蛋白酶）与蛋白质有关 复合物称为炎症，通过产生炎症在先天免疫反应中发挥作用 以及响应病原体和危险信号的细胞死亡。炎症反应的失调是 与败血症和疾病状态有关，从自身免疫和神经退行性疾病到癌症。 由于它们在各种人类疾病中的作用，炎症性胱天蛋白酶是许多药物发现的重点 程序。该提案的长期目标是开发用于评估抑制的工具 炎症性胱天蛋白酶酶并允许确定每个酶在炎症反应中的作用。这 通过开发肽基板进行活动分析，可以最好地解决目标 与个体的炎症胱天蛋白酶。迄今为止，为炎症性胱天蛋白酶创建此类底物具有 难以捉摸。先前的选择性方法的重点是将氨基酸N末端变化为 天冬氨酸残留物。 PIS已表明可以通过将氨基酸C末端变为天冬氨酸 将报告发色团纳入非天然氨基酸的侧链，并明显观察到 用于与单个C末端氨基不同的炎性胱天冬酶反应的炎症性caspase的不同动力学 酸。对于caspase -4或-5，炎症组形成的机制尚不清楚，大多数信息 可用于caspase-4。为了解决这些酶的个体生物学作用，结构研究 必须独立对C-4和C-5进行炎性体的形成。以前的研究是 使用非活性突变酶进行 灭活。该提议的假设是每个提议的生物学作用和激活机制 可以通过使用化学方法研究每种酶的活性来发现炎症性胱天蛋白酶。 以下具体目的将解决此假设：1）开发和验证具有100-的肽底物 通过改变天冬氨酸的C末端的氨基酸，对每种炎症性caspase的折叠选择性 酸残留物并确定产生最大信噪比所需的发色团对。 2） 使用诱变和化学方法确定caspase-4和caspase-5的激活机理。 使用多个高信号到噪声染料对开发炎症胱天蛋白酶的选择性底物 将允许每个caspase的活性通过唯一的荧光报道来评估。这些创造 底物将积极影响药物发育领域，以对抗炎症性疾病和败血症。 研究炎性体形成的化学方法将允许炎症性胱天蛋白酶在 先天免疫反应是撤消卷积的，有助于我们对理解的发展 炎。该建议将为本科生提供跨学科的研究经验， 将它们暴露于化学生物学和生物物理学在改善人类健康中的应用。