Novel Roles for Effector Procaspases

描述（由申请人提供）：胱天蛋白酶是在所有真核细胞中发现的半胱氨酸脱氨酶，作为酶原，其需要诱导的接近和/或蛋白水解裂解以激活。激活的半胱天冬酶作为细胞死亡的调节者和执行者，响应于发育线索或环境应激（例如凋亡）和感染（例如焦亡和坏死性凋亡）。活化的半胱天冬酶可以通过细胞因子成熟控制炎症反应以及诱导伤口愈合反应中的细胞增殖和迁移（例如“凤凰升起”现象）而具有同样深刻的全身效应。因此，由于异常激活可能对细胞产生灾难性的后果，因此对caspase激活机制和活性caspase的功能进行了大量的研究。长期以来，人们一直认为半胱天冬酶（半胱天冬酶原）的酶原没有功能，并且仅存在于允许细胞快速和决定性的手段来诱导细胞死亡或炎症反应。在研究细胞色素c释放后效应器caspase-3和-7对线粒体功能的作用时，我们偶然发现了caspase-3和caspase-7的新功能，为细胞携带杀伤分子的原因提供了新的线索。小鼠胚胎成纤维细胞（MEFs）是缺乏半胱氨酸蛋白酶3显示增强的粘附和改变迁移速度，这是与增加纤连蛋白分泌。引入半胱天冬酶原-3或无催化活性的半胱天冬酶原-3逆转这种表型。Casp 7缺陷的MEFs改变了形态，失去了方向性持久迁移。这些事件似乎与改变肌动蛋白细胞骨架组织，也是独立的半胱天冬酶活性。因此，效应蛋白酶原不仅是稳定的杀伤分子，它们在活细胞中具有细胞功能。本申请的前两个特定目的旨在确定这些效应物前半胱氨酸蛋白酶如何调节细胞粘附、迁移和分泌。此外，我们发现，观察到的生存优势与半胱天冬酶3缺陷MEFs是依赖于细胞粘附。Casp 3缺陷型细胞不能抵抗与细胞外基质粘附的丧失（失巢凋亡）。这表明半胱天冬酶原-3的非凋亡功能也可以影响细胞对凋亡信号的反应。这将对目前的前馈回路模型产生重大影响，该模型已被提出来解释效应器半胱天冬酶如何影响半胱天冬酶激活上游发生的事件。因此，本申请的最终特定目的将重新检查Casp 3丢失改变细胞存活的机制。这些目标的完成将对我们如何看待半胱天冬酶原产生范式转变的影响，并可能需要重新解释半胱天冬酶缺陷细胞产生的数据。

DESCRIPTION (provided by applicant): Caspases are cysteine aspartases found in all eukaryotic cells as zymogens that require induced proximity and/or proteolytic cleavage for activation. Activated caspases function as both regulators and executioners of cell death in response to developmental cues or environmental stress (e.g. apoptosis) and infections (e.g. pyroptosis and necroptosis). Activated caspases can have equally profound systemic effects by controlling inflammatory responses through cytokine maturation as well as induction of cell proliferation and migration in the wound-healing response (e.g. the "phoenix rising" phenomena). Therefore there has been a significant amount of research on the mechanism of caspase activation and the function of active caspases since aberrant activation could have catastrophic consequences for the cell. It has long been presumed that the zymogens of caspases (procaspases) had no function and were only present to allow for a quick and decisive means for a cell to induce cell death or an inflammatory response. While studying the role of the effector caspases-3 and -7 on mitochondrial function following cytochrome c release, we serendipitously discovered novel functions for procaspase-3 and procaspase-7 that shed new light on why cells carry killer molecules. Mouse embryo fibroblasts (MEFs) that are deficient in casp3 display enhanced adhesion and altered migration velocity that is associated with increased fibronectin secretion. Introduction of procaspase-3 or a procaspase-3 that is catalytically inactive reverses this phenotype. Casp7-deficient MEFs have altered morphology and loss of directionally-persistent migration. These events appear to be associated with altered actin cytoskeleton organization and are also independent of caspase activity. Thus effector procaspases are not only poised killer molecules, they have cellular functions in viable cells. The first two Specific Aims of this application are designed to determine how these effector procaspases regulate cell adhesion, migration and secretion. Additionally we have found that the survival advantage observed with casp3-deficient MEFs is dependent on cell adhesion. Casp3-deficient cells are not protected against loss of adherence to extracellular matrix (anoikis). This suggests that the non-apoptotic functions of procaspase-3 can also influence the cellular response to apoptotic signals. This would have a significant impact on current models of feed-forward loops that have been proposed to explain how an effector caspase could influence events that occur upstream of caspase activation. Therefore the final Specific Aim of this application will re-examine the mechanism by which loss of casp3 alters cell survival. Completion of these Aims will have a paradigm-shifting effect on how we think about procaspases and may require a re-interpretation of data generated with caspase-deficient cells.