Novel Roles for Effector Procaspases

描述（由申请人提供）：半胱氨酸天冬氨酸酶是在所有真核细胞中发现的半胱氨酸天冬氨酸酶，作为酶原，需要诱导接近和/或蛋白水解裂解才能激活。激活的半胱天冬酶在响应发育线索或环境应激（如凋亡）和感染（如焦亡和坏死）时，既是细胞死亡的调节者，也是细胞死亡的执行者。激活的半胱天冬酶可以通过细胞因子成熟来控制炎症反应，并在伤口愈合反应中诱导细胞增殖和迁移（例如“凤凰升起”现象），从而具有同样深刻的全身效应。因此，人们对caspase的激活机制和活性caspase的功能进行了大量的研究，因为异常激活可能对细胞造成灾难性的后果。长期以来，人们一直认为半胱天冬酶（原半胱天冬酶）的酶原没有任何功能，只是为细胞诱导细胞死亡或炎症反应提供了一种快速而决定性的手段。在研究效应体caspase-3和-7在细胞色素c释放后对线粒体功能的作用时，我们偶然发现了procaspase-3和procaspase-7的新功能，这为细胞携带杀伤分子的原因提供了新的思路。缺乏casp3的小鼠胚胎成纤维细胞（mef）表现出粘附增强和迁移速度改变，这与纤维连接蛋白分泌增加有关。引入procaspase-3或无催化活性的procaspase-3可逆转这种表型。casp7缺失的mef改变了形态并失去了定向持续迁移。这些事件似乎与肌动蛋白细胞骨架组织的改变有关，也与半胱天冬酶活性无关。因此，效应原葡聚糖酶不仅是蓄势待发的杀伤分子，而且在活细胞中具有细胞功能。该应用程序的前两个特定目的旨在确定这些效应原胞浆酶如何调节细胞粘附，迁移和分泌。此外，我们发现casp3缺陷mef的生存优势依赖于细胞粘附。casp3缺陷细胞不受细胞外基质粘附丧失的保护（anoikis）。这表明procaspase-3的非凋亡功能也可以影响细胞对凋亡信号的反应。这将对目前提出的前馈循环模型产生重大影响，该模型用于解释效应caspase如何影响caspase激活上游发生的事件。因此，本应用程序的最终特定目标将重新检查casp3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.