Regulation of necrotic cell death by protein kinase A and cylindromatosis

描述（由申请方提供）：程序性坏死是一种形态学和分子学上与细胞凋亡不同的细胞死亡形式。坏死导致的细胞死亡向组织环境释放内源性佐剂或“危险信号”，这可引发炎症并刺激免疫反应。最近发现的阻断这种细胞死亡途径的病毒抑制剂支持了程序性坏死在抗病毒免疫中的作用。从机制上讲，程序性坏死需要丝氨酸/苏氨酸蛋白激酶RIP 1，并且当半胱天冬酶被抑制时最佳诱导。这些结果突出了程序性坏死在宿主防御感染因子中的可能作用。为了进一步了解调节程序性坏死的分子机制，我们通过小干扰RNA（siRNA）激酶和癌症相关基因筛选可能参与这种非凋亡细胞死亡途径的基因。我们最近描述了另一个RIP家族成员的关键作用，从屏幕上确定，RIP 3，在TNF诱导和病毒诱导的程序性坏死。除了RIP 3之外，蛋白激酶A（PKA）催化亚基B和肿瘤抑制因子圆柱瘤病（CYLD）也从RNAi筛选中被鉴定为对程序性坏死至关重要。在本申请中，我们将研究PKA-C2和CYLD调节RIP 1/RIP 3依赖性程序性坏死的分子机制。我们将研究它们对促坏死信号复合物的募集以及它们如何调节促坏死信号复合物的形成。我们将确定CYLD的去泛素化酶活性是否是其功能所必需的，并探索程序性坏死过程中CYLD的可能底物。最后，我们将使用体外牛痘病毒感染作为模型来测试PKA-C2和CYLD在病毒感染中的生理相关性。 公共卫生相关性：坏死引起的细胞死亡会导致炎症，并会极大地影响免疫反应的质量。然而，调控这一过程的信号却知之甚少。我们最近已经确定了两种分子，PKA-C2和CYLD，是坏死细胞死亡所必需的。在这个提议中，我们将阐明这两种分子通过坏死控制细胞死亡的机制。此外，我们将评估这两种分子如何影响病毒感染期间的细胞死亡反应。这些研究将使我们更好地了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.