Evolution of effector caspase conformational landscapes

半胱天冬酶是一类古老的半胱氨酸蛋白酶，其对细胞凋亡和细胞增殖至关重要。 分化，但很少有人知道半胱天冬酶活性是如何调节凋亡与非凋亡。 凋亡事件。我们在这个提议中的主要目标是从一个 常见的支架，导致现存的半胱天冬酶簇的活性调节。凋亡半胱氨酸蛋白酶 从一个共同的祖先进化成两个不同的亚科，要么是单体（启动子 半胱天冬酶）或二聚体（效应物半胱天冬酶）。在生物学上，引发剂半胱天冬酶的二聚化是重要的生物学机制。 细胞命运决定特性;相反，二聚体效应器半胱天冬酶进化出独特的变构 微调活动的机制。我们的长期目标是将进化生物学与严格的 生物化学研究来确定半胱天冬酶的进化轨迹。这些研究将激发新的 细胞凋亡调控机制，蛋白质寡聚化， 并开发出特异性改变的酶。这项资助的目的是描述突变的特征 这导致了两个不同的凋亡半胱天冬酶亚家族。的 中心假设是胱天蛋白酶-血红蛋白酶支架中的有限突变建立了折叠 和构象景观> 6.5亿年前，现存的半胱天冬酶进化不同 通过差异化修改常见交互网络来获得属性。我们的理论基础是， 重建的祖先蛋白质表明，在一个弱的祖先二聚体突变导致两个 具有不同寡聚特性的亚家族。提供了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 目的将测试以下假设：（Aim 1）弱二聚体共同祖先提供了一个平台， 二聚体和单体caspase亚家族的进化;（目的2）一个混杂的共同祖先 提供了一个平台，最小的修改，导致现代酶的选择;（目标3） 一个共同的变构网络的进化变化导致了独特的调节机制， 天然系综中不同构象的选择。这一贡献意义重大，因为它将 建立了底物特异性和变构调节的关键特征，这些特征在数百年来一直被保留下来， 数百万年的进化，而其他监管功能是现代和集群特定的。的 这项研究是创新的，因为我们建立了一个数据库（CaspBase），其中包含6600多个 从353个分类群的caspase序列，以推断祖先序列和重建祖先 蛋白质，这是用来表征进化轨迹。对胱天蛋白酶进化的深入了解是 影响力，因为在祖先的支架中保留了7亿多年的特性， 用于产生具有改变的特异性和变构调节的新酶。

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.