Evolution of effector caspase conformational landscapes

效应器半胱天冬酶构象景观的进化

• 批准号: 9902492
• 负责人: ALLAN CLAY CLARK
• 金额: $ 29.4万
• 依托单位: UNIVERSITY OF TEXAS ARLINGTON
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9902492
• 关键词: Affect; Allosteric Regulation; Amino Acid Sequence; Apoptosis; Apoptotic; Area; Arthritis; Autoimmune Diseases; Binding; Biochemical; Biochemistry; Biological; Biological Models; Biology; Biophysics; CASP3 gene; CASP6 gene; CASP8 gene; Caspase; Cell Differentiation process; Cells; Charge; Data; Databases; Development; Diabetes Mellitus; Dimerization; Enzymes; Equilibrium; Event; Evolution; Family; Goals; Grant; Homeostasis; Hydrophobicity; Individual; Kinetics; Lead; Malignant Neoplasms; Manuals; Modernization; Modification; Molecular Conformation; Mutation; Neurodegenerative Disorders; Pathway interactions; Peptide Hydrolases; Property; Proteins; Reporting; Research; Specificity; Structure; Substrate Specificity; Testing; base; biophysical analysis; dimer; effective therapy; enzyme activity; enzyme substrate; human disease; innovation; insight; monomer; scaffold

Caspases are an ancient class of cysteinyl proteases that are critical to apoptosis and cell differentiation, but little is known about how caspase activity is modulated for apoptotic versus non- apoptotic events. Our primary goal in this proposal is to determine evolutionary trajectories from a common scaffold that resulted in modulation of activity in extant caspase clusters. The apoptotic caspases evolved from a common ancestor into two distinct subfamilies that are either monomers (initiator caspases) or dimers (effector caspases). Biologically, dimerization of initiator caspases is an important cell-fate determining property; conversely, dimeric effector caspases evolved unique allosteric mechanisms to fine-tune activity. Our long-term goal is to integrate evolutionary biology with rigorous biochemical studies to define the evolutionary trajectories in caspases. These studies will stimulate new areas for evolutionary biochemical studies on apoptotic regulatory mechanisms, protein oligomerization, and developing enzymes with altered specificities. The objective of this grant is to characterize mutations that occurred in the common ancestor that resulted in two distinct subfamilies of apoptotic caspases. The central hypothesis is that limited mutations in the caspase-hemoglobinase scaffold established the folding and conformational landscapes >650 million years ago and that extant caspases evolved different properties through differentially modifying common interaction networks. Our rationale is that studies of reconstructed ancestral proteins suggest that mutations in a weak ancestral dimer resulted in two subfamilies with different oligomeric properties. Neofunctionalization of the two subfamilies provided distinct enzyme substrate selection as well as allosteric mechanisms to modulate activity. Our specific aims will test the following hypotheses: (Aim1) A weakly dimeric common ancestor provided a platform for evolution of dimeric and monomeric caspase subfamilies; (Aim 2) A promiscuous common ancestor provided a platform for minimal modifications that resulted in modern enzyme selection; (Aim 3) Evolutionary changes in a common allosteric network resulted in unique regulatory mechanisms through selection of different conformations in the native ensemble. This contribution is significant since it will establish key features of substrate specificity and allosteric regulation that were retained through hundreds of millions of years of evolution, while other regulatory features are modern and cluster-specific. The proposed research is innovative because we established a database (CaspBase) containing over 6,600 caspase sequences from 353 taxa in order to infer ancestral sequences and reconstruct ancestral proteins, which are used to characterize evolutionary trajectories. Insight into caspase evolution is impactful because properties retained for more than 700 million years in the ancestral scaffold can be used to generate new enzymes with altered specificities and allosteric regulation.

胱天蛋白酶是一类古老的半胱氨酸蛋白酶，对细胞凋亡至关重要 分化，但对caspase活性如何调节凋亡与非 - 非 - 凋亡事件。该提案中我们的主要目标是确定从一个 常见的支架导致了现有caspase簇中活性的调节。凋亡的胱天蛋白酶 从共同祖先演变成两个不同的亚家族，它们是单体（引发剂） caspase）或二聚体（效应子caspase）。从生物学上讲，引发液的二聚化是重要的 命令确定特性；相反，二聚体效应子caspases进化出独特的变构 微调活性的机制。我们的长期目标是将进化生物学与严格的 生化研究以定义胱天蛋白酶中的进化轨迹。这些研究将刺激新的 凋亡调节机制，蛋白质寡聚化的进化生化研究领域 并开发具有改变特异性的酶。这笔赠款的目的是表征突变 这发生在共同祖先中，导致了两个不同的凋亡胱天蛋白酶亚家族。这 中心假设是caspase-hemogomoglobinase支架中有限的突变建立了折叠 构象景观> 6.5亿年前，现存的caspase进化了不同 通过差异修改通用交互网络的属性。我们的理由是对 重建的祖传蛋白表明，弱祖先二聚体中的突变导致两个 具有不同寡聚特性的亚家族。提供的两个亚家族的新功能化 不同的酶底物选择以及调节活性的变构机制。我们的具体 AIMS将检验以下假设：（ AIM1）一个虚弱的二聚体共同祖先为 二聚体和单体caspase亚家族的进化； （目标2）一个混杂的共同祖先 为最小修饰提供了一个平台，从而导致现代酶选择； （目标3） 通用变构网络的进化变化导致通过 在天然合奏中选择不同构象。这种贡献很重要，因为它将 建立通过数百个保留的底物特异性和变构调节的关键特征 数百万年的进化，而其他监管特征则是现代的且特定于集群的。这 拟议的研究具有创新性，因为我们建立了一个包含6,600多个数据库（CASPBASE） 来自353个分类单元的caspase序列，以推断祖先序列并重建祖先 蛋白质，用于表征进化轨迹。对胱天蛋白酶进化的洞察力是 有影响力，因为在祖先脚手架中保留了超过7亿年的财产可以是 用于生成具有改变特异性和变构调节的新酶。