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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Cellular and Molecular Immunology - A Study Section (CMIA)
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Leitner, Wolfgang W
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University of Massachusetts Medical School Worcester
Schools of Medicine
United States
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Moquin, David M; McQuade, Thomas; Chan, Francis Ka-Ming (2013) CYLD deubiquitinates RIP1 in the TNF?-induced necrosome to facilitate kinase activation and programmed necrosis. PLoS One 8:e76841
Chan, Francis Ka-Ming; Moriwaki, Kenta; De Rosa, María José (2013) Detection of necrosis by release of lactate dehydrogenase activity. Methods Mol Biol 979:65-70
McQuade, Thomas; Cho, Youngsik; Chan, Francis Ka-Ming (2013) Positive and negative phosphorylation regulates RIP1- and RIP3-induced programmed necrosis. Biochem J 456:409-15
Kurtulus, Sema; Hildeman, David (2013) Assessment of CD4(+) and CD8 (+) T cell responses using MHC class I and II tetramers. Methods Mol Biol 979:71-9
Li, Jixi; McQuade, Thomas; Siemer, Ansgar B et al. (2012) The RIP1/RIP3 necrosome forms a functional amyloid signaling complex required for programmed necrosis. Cell 150:339-50
Chan, Francis Ka-Ming; Baehrecke, Eric H (2012) RIP3 finds partners in crime. Cell 148:17-8
Chan, Francis Ka-Ming (2012) Fueling the flames: Mammalian programmed necrosis in inflammatory diseases. Cold Spring Harb Perspect Biol 4:
Cho, YoungSik; McQuade, Thomas; Zhang, Haibing et al. (2011) RIP1-dependent and independent effects of necrostatin-1 in necrosis and T cell activation. PLoS One 6:e23209
Challa, Sreerupa; Chan, Francis Ka-Ming (2010) Going up in flames: necrotic cell injury and inflammatory diseases. Cell Mol Life Sci 67:3241-53
Moquin, David; Chan, Francis Ka-Ming (2010) The molecular regulation of programmed necrotic cell injury. Trends Biochem Sci 35:434-41