Regulation of the Cell Death Program by DFNA5

摘要 细胞凋亡和上睑下垂在生长、存活、动态平衡和先天免疫中起着关键的生理作用。 在所有哺乳动物中。这些通路的失调可能会导致许多疾病，包括自身免疫， 炎症性、动脉粥样硬化性和恶性疾病。而细胞凋亡是一种免疫上的“沉默”形式 程序性细胞死亡通过激活效应器caspase3、6和7执行，上睑下垂是一种 炎症性形式的程序性细胞死亡，通过激活炎性半胱氨酸蛋白酶1、4和 5在人类中。这些炎性半胱氨酸酶切割Gasdermin蛋白GSDMD，产生一个毛孔- 形成使质膜通透性的N-末端片段，导致细胞肿胀、渗透 细胞裂解和胞内内容物渗漏。如果凋亡细胞没有被清除，如在 体外培养条件或体内吞噬功能受损的条件下，它们会进展为溶解和炎症 称为继发性坏死期。继发性坏死与下垂有几个共同的特征，包括 质膜通透性、肿胀和溶解，因此它可以代表一种形式的下垂。 事实上，我们最近发现，激活的caspase-3在 细胞凋亡产生一段坏死的DFNA5-N片段，靶向质膜，使其通透 导致继发性坏死/下垂。有趣的是，与WT细胞不同的是，DFNA5缺失的细胞不会发生农达 并肿胀，但广泛碎裂成小的凋亡小体，表明DFNA5是细胞内的一种调节因子 在细胞凋亡过程中的解体。因为细胞在凋亡过程中的解体可能会影响死亡细胞的清除 通过吞噬细胞，我们提出了DFNA5在凋亡过程中裂解诱导血管形成毛孔的假设。 释放“找到我”和其他吞噬细胞激活信号的质膜，以清除凋亡细胞和 以缓和凋亡细胞分解成小的凋亡小体的过程。在本申请中，我们提出了AIMS 为了进一步了解DFNA5在体内的生理功能，以及它在自身免疫中的作用， 炎症和先天免疫。我们将研究DFNA5缺陷小鼠对凋亡细胞的清除 并进一步评估DFNA5缺乏在糖尿病发生发展中的病理生理后果。 自身免疫力。我们还将描述DFNA5的另一个新功能，该功能参与调节 线粒体凋亡途径及其活性如何影响细胞增殖和对凋亡的敏感性 刺激物。最后，我们将研究DFNA5在宿主对病毒病原体的天然免疫中的作用， 感染性休克和炎症诱导的恶性肿瘤。这项研究的结果应该会对 这增加了我们对细胞凋亡过程中激活的信号通路的理解 导致细胞通透性及其对组织动态平衡、自身免疫和炎症的影响 疾病。

Abstract Apoptosis and pyroptosis play key physiological roles in growth, survival, homeostasis and innate immunity of all mammals. Disregulation of these pathways could lead to many diseases including autoimmune, inflammatory, atherosclerotic and malignant diseases. While apoptosis is an immunologically “silent” form of programmed cell death executed by activation of the effector caspases 3, 6 and 7, pyroptosis is an inflammatory form of programmed cell death executed by activation of the inflammatory caspases 1, 4, and 5 in humans. These inflammatory caspases cleave the gasdermin protein, GSDMD, to generate a pore- forming N-terminal fragment that permeabilizes the plasma membrane leading, to cellular swelling, osmotic cell lysis and leakage of intracellular contents. If apoptotic cells are not scavenged, such as seen under in vitro culture conditions or conditions of impaired phagocytosis in vivo, they progress to a lytic and inflammatory phase called secondary necrosis. Secondary necrosis shares several features with pyroptosis including plasma membrane permeabilization, swelling, and lysis, and therefore it could represent a form of pyroptosis. Indeed, we discovered recently that activated caspase-3 cleaves the gasdermin-related protein DFNA5 during apoptosis to generate a necrotic DFNA5-N fragment that targets the plasma membrane to permeabilize it and induce secondary necrosis/pyroptosis. Interestingly, unlike WT cells, DFNA5-deficient cells do not roundup and swell, but extensively fragment into small apoptotic bodies suggesting that DFNA5 is a regulator of cellular disassembly during apoptosis. As cellular disassembly during apoptosis may impact clearance of dying cells by phagocytes, we propose to test the hypothesis that DFNA5 cleavage during apoptosis induces pores in the plasma membrane to release “find me” and other phagocyte activation signals to clear apoptotic cells and to temper down disassembly of apoptotic cells into small apoptotic bodies. In this application we propose aims to gain further insight into the physiological function of DFNA5 in vivo, and its role in autoimmunity, inflammation and innate immunity. We will study the clearance of apoptotic cells in DFNA5-deficient mice and further assess the pathophysiological consequences of DFNA5 deficiency in the development of autoimmunity. We will also characterize another novel function of DFNA5 involved in the regulation of the mitochondrial apoptotic pathway and how this activity might impact cell proliferation and sensitivity to apoptotic stimuli. Finally, we will investigate the role of DFNA5 in the host innate immunity against viral pathogens, septic shock and inflammation-induced malignancy. Results from this research should have a high impact on the field and increase our understanding of the signaling pathways activated during the apoptotic program leading to cell permeabilization and their impact on tissue homeostasis, and autoimmune and inflammatory diseases.