Uncovering the biological roles of inflammatory caspases through chemical approaches

项目摘要 炎症性半胱天冬酶（半胱氨酸依赖性，天冬氨酸特异性蛋白酶）与蛋白质 称为炎性体的复合物，通过产生炎症在先天免疫应答中发挥作用 和细胞死亡来应对病原体和危险信号炎症反应的失调是 与脓毒症和从自身免疫性和神经退行性疾病到癌症的疾病状态相关。 由于其在人类疾病中的广泛作用，炎性半胱天冬酶是许多药物发现的焦点 程序.该提案的长期目标是开发用于评估抑制的工具， 炎症半胱天冬酶，并允许确定每种酶在炎症反应中的作用。这 通过开发用于活性测定的肽底物， 与炎症性半胱天冬酶有关到目前为止，炎症性半胱天冬酶的底物的产生， 难以捉摸产生选择性的先前方法集中于改变氨基酸N-末端以 天冬氨酸残基PI已经表明，可以通过以下方式改变天冬氨酸的C端氨基酸： 将报告发色团作为非天然氨基酸的侧链并入，并观察到显著的 炎症性半胱天冬酶与不同的肽反应的不同动力学， 酸caspase-4或caspase-5在炎症小体形成中的作用机制尚不清楚， 可用的是半胱天冬酶-4。为了解决这些酶的个体生物学作用，结构研究 炎性小体的形成必须在C-4和C-5上独立进行。以前的研究已经 使用无活性的突变酶而不是化学上更相关的生物活性形式进行 灭活这一建议的假设是，生物学作用和激活机制的每一个 炎性半胱天冬酶可以通过使用化学方法研究每种酶的活性来揭示。 以下具体目标将解决这一假设：1）开发和验证具有100- 200个氨基酸的肽底物。 通过改变天冬氨酸蛋白酶C-末端侧的氨基酸， 酸残基，并确定产生最大信噪比所需的发色团对。（二） 用诱变和化学方法确定caspase-4和caspase-5的激活机制。 使用多种高信噪比染料对开发炎性半胱天冬酶的选择性底物 将允许通过独特的荧光报告物评估每种胱天蛋白酶的活性。创建这些 底物将积极影响药物开发领域，以对抗炎性疾病和败血症。 研究炎性小体形成的化学方法将允许炎性半胱天冬酶在炎症中的作用。 先天性免疫反应被去卷积，有助于我们理解的进步， 炎症该提案将为本科生提供跨学科的研究经验， 使他们接触化学生物学和生物物理学在改善人类健康方面的应用。

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.