Dimerization mechanisms of two procaspase subfamilies

描述(由申请人提供)：导致原天冬氨酸氨基转移酶激活的机制在细胞凋亡和炎症的调节中发挥核心作用。对于所有的前天冬氨酸酶，二聚体的形成是加工过程中的一个关键事件，因为酶的活性部位由两个二聚体单体贡献的环组成。然而，caspase亚家族在二聚化和成熟度方面存在着根本的差异，这种差异是调控细胞凋亡的一个关键方面。几种炎性和启动型前天冬氨酸蛋白酶在细胞中以非活性单体形式存在，但二聚后这些蛋白具有相对较高的酶活性。对于这些半胱氨酸天冬氨酸酶，链断裂只是稳定活性部位。相反，刽子手proaspase-3是一个稳定的二聚体，但它的酶活性非常低。在这种情况下，成熟依赖于启动子原天冬氨酸酶的切割。我们的长期目标是了解原天冬氨酸酶的二聚化、激活和酶活性是如何耦合的。总体而言，我们想确定为什么proaspase-1是单体，但活性位点在二聚时形成正确。相反，我们想确定为什么proaspase-3的稳定二聚体不活跃。我们假设proaspase二聚体以两种主要构象存在，其中只有一种是活性的，而在proaspase-3的情况下，非活性形式最稳定。此外，我们认为，二聚体界面上的变构位置影响两种形式之间的构象转换，并将二聚作用耦合到活性中心的形成。我们已经证明，蛋白质的三个区域可能在影响寡聚体状态和稳定原天冬氨酸酶的非活性形式方面发挥重要作用。我们建议使用生化和生物物理分析、蛋白质折叠研究和X射线结晶学研究来研究蛋白质的这三个区域。原天冬氨酸酶的激活机制具有重要的生物学意义，因为它们允许在细胞死亡过程中对半胱氨酸天冬氨酸酶的活性进行严格的调节，从维持启动子原天冬氨酸酶的单体状态到稳定二聚体效应性原天冬氨酸酶的非活性形式。了解caspase的激活过程为制定干预其在细胞中的活动的策略提供了关键。公共卫生相关性：了解caspase二聚化和激活有可能影响一些自身免疫性疾病、心脏病和癌症的治疗策略。学会选择性地操纵细胞凋亡的水平是治疗中的重要一步，因为细胞凋亡的失调是这些疾病的共同因素。

DESCRIPTION (provided by applicant): The mechanisms that lead to the activation of procaspases play central roles in the regulation of apoptosis and inflammation. For all procaspases, formation of a dimer is a critical event in processing because the active sites of the enzyme are comprised of loops contributed by both monomers of the dimer. However, a fundamental difference exists in the caspase subfamilies regarding dimerization and maturation, and this difference is a key aspect for regulating apoptosis. Several inflammatory and initiator procaspases exist as inactive monomers in the cell, but the proteins have relatively high enzymatic activity upon dimerization. For those caspases, chain cleavage simply stabilizes the active site. In contrast, the executioner procaspase-3 is a stable dimer, but it has very little enzymatic activity. In this case, maturation is dependent on cleavage by the initiator procaspases. Our long-term goals are to understand how dimerization, activation and enzymatic activity are coupled for procaspases. Overall, we want to determine why procaspase-1 is a monomer, but yet the active sites form properly upon dimerization. In contrast, we want to determine why the stable dimer of procaspase-3 is not active. We hypothesize that the procaspase dimer exists in two primary conformations, only one of which is active, and that the inactive form is most stable in the case of procaspase-3. Furthermore, we suggest that an allosteric site in the dimer interface affects the conformational switch between the two forms and couples dimerization to active site formation. We have shown that three regions of the protein may be important in affecting the oligomeric state and in stabilizing the inactive form of the procaspase. We propose to use biochemical and biophysical assays, protein folding studies, and X-ray crystallographic studies to examine these three regions of the protein. The procaspase activation mechanisms are important biologically because they allow for the tight regulation of caspase activity in cell death, from maintaining the monomeric state of initiator procaspases to stabilizing the inactive form of dimeric effector procaspases. Understanding the caspase activation process provides a key t develop strategies to intervene in their activities in the cell. PUBLIC HEALTH RELEVANCE: Understanding caspase dimerization and activation has the potential to affect therapeutic strategies for a number of autoimmune diseases, heart disease, and cancers. Learning to selectively manipulate the level of apoptosis is an important step in treatment because the dysregulation of apoptosis is a common factor to these diseases.