Regulation of necrotic cell death by protein kinase A and cylindromatosis

描述（申请人提供）：程序性坏死是一种形态学和分子上与细胞凋亡不同的细胞死亡形式。细胞坏死导致的死亡释放内源性佐剂或“危险信号”进入组织环境，从而引发炎症并刺激免疫反应。程序性坏死在抗病毒免疫中的作用被最近发现的阻断细胞死亡途径的病毒抑制剂所支持。从机制上讲，程序性坏死需要丝氨酸/苏氨酸蛋白激酶RIP1，当半胱天冬酶被抑制时，程序性坏死最容易被诱导。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。为了进一步了解调控程序性坏死的分子机制，我们筛选了可能参与这一非凋亡细胞死亡途径的小干扰RNA （siRNA）激酶和癌症相关基因。我们最近描述了从筛选中发现的另一个RIP家族成员RIP3在tnf诱导和病毒诱导的程序性坏死中的关键作用。除了RIP3外，蛋白激酶A （PKA）催化亚基b和肿瘤抑制因子圆筒状病（CYLD）也从RNAi筛选中被鉴定为程序性坏死的关键。在本研究中，我们将研究PKA-C2和CYLD调控RIP1/ rip3依赖性程序性坏死的分子机制。我们将研究它们在促坏死信号复合体中的募集，以及它们如何调节促坏死信号复合体的形成。我们将确定CYLD的去泛素酶活性是否需要其功能，并探索程序性坏死期间CYLD可能的底物。最后，我们将以体外牛痘病毒感染为模型，检验PKA-C2和CYLD在病毒感染中的生理相关性。

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.