Regulation of necrotic cell death by protein kinase A and cylindromatosis

描述(由申请人提供)：程序性坏死是一种形态和分子上与细胞凋亡截然不同的细胞死亡形式。细胞因坏死而死亡，会向组织环境释放内源性佐剂或“危险信号”，从而引发炎症和刺激免疫反应。程序性坏死在抗病毒免疫中的作用得到了最近发现的病毒抑制剂的支持，这些病毒抑制剂可以阻断细胞死亡的这一途径。从机制上讲，程序性坏死需要丝氨酸/苏氨酸蛋白激酶RIP1，当半胱氨酸天冬氨酸酶被抑制时，程序性坏死被最佳地诱导。这些结果突显了程序性坏死在宿主防御感染性病原体中的可能作用。为了进一步了解调控程序性坏死的分子机制，我们通过小干扰RNA(SiRNA)激酶和可能参与这一非凋亡性细胞死亡途径的癌症相关基因进行筛选。我们最近描述了从屏幕上发现的另一个RIP家族成员，RIP3，在肿瘤坏死因子诱导和病毒诱导的程序性坏死中的关键作用。除RIP3外，RNAi筛查还发现蛋白激酶A(PKA)催化亚单位b和肿瘤抑制因子圆柱瘤病(CyLD)对程序性坏死至关重要。在这个应用中，我们将研究PKA-C2和CyLD调节RIP1/RIP3依赖的程序性坏死的分子机制。我们将研究它们在促坏死性信号复合体中的招募，以及它们如何调控促坏死性信号复合体的形成。我们将确定CyLD的脱泛素酶活性是否是其功能所必需的，并探索CyLD在程序性坏死过程中可能的底物。最后，我们将以体外痘苗病毒感染为模型，检验PKA-C2和CyLD在病毒感染中的生理学相关性。 公共卫生相关性：细胞坏死导致的死亡会引起炎症，并可能极大地影响免疫反应的质量。然而，人们对调控这一过程的信号知之甚少。我们最近确定了两个分子，PKA-C2和CyLD，在坏死性细胞死亡中是必不可少的。在这个提案中，我们将阐明这两个分子通过坏死控制细胞死亡的机制。此外，我们将评估这两个分子在病毒感染过程中如何影响细胞死亡反应。这些研究将使我们能够更好地理解控制细胞死亡和炎症的信号。最终，从这些研究中获得的知识将有助于制定控制细胞损伤引起的炎症性疾病的策略。

DESCRIPTION (provided by applicant): Programmed necrosis is a morphologically and molecularly distinct form of cell death from apoptosis. Cell death by necrosis releases endogenous adjuvants or "danger signals" into the tissue milieu, which can trigger inflammation and stimulate immune responses. A role for programmed necrosis in anti-viral immunity is supported by recent discoveries of viral inhibitors that block this pathway of cell death. Mechanistically, programmed necrosis requires the serine/threonine protein kinas RIP1 and is optimally induced when caspases are inhibited. These results highlight a possible role for programmed necrosis in host defense against infectious agents. In order to further understand the molecular mechanism that regulates programmed necrosis, we screened by small interference RNA (siRNA) kinases and cancer-related genes that may participate in this non-apoptotic cell death pathway. We recently described the critical role of another RIP family member identified from the screen, RIP3, in TNF-induced and virus-induced programmed necrosis. In addition to RIP3, protein kinase A (PKA) catalytic subunit b and the tumor suppressor cylindromatosis (CYLD) were also identified from the RNAi screen as crucial for programmed necrosis. In this application, we will examine the molecular mechanisms by which PKA-C2 and CYLD regulates RIP1/RIP3-dependent programmed necrosis. We will examine their recruitment to the pro-necrotic signaling complex and how they might regulate the formation of the pro-necrotic signaling complex. We will determine whether deubiquitinase activity of CYLD is required for its function and explore the possible substrates for CYLD during programmed necrosis. Finally, we will test the physiological relevance of PKA-C2 and CYLD in virus infections using in vitro vaccinia virus infection as a model. PUBLIC HEALTH RELEVANCE: Cell death by necrosis causes inflammation and can greatly impact the quality of an immune response. However, the signals that regulate this process are poorly understood. We have recently identified two molecules, PKA-C2 and CYLD, to be essential for necrotic cell death. In this proposal, we will elucidate the mechanisms by which these two molecules control cell death by necrosis. Furthermore, we will evaluate how these two molecules may affect the cell death response during virus infections. These studies will allow us to better understand the signals that control cell death and inflammation. Eventually, the knowledge gained from these studies will aid the development of strategies to control inflammatory diseases caused by cell injury.