Reference number : GC-2017-Q1-01538, amendment number : , agreement type : G, recipient legal name : Sad, Subash (University of Ottawa)|Sad, Subash (Université d’Ottawa), recipient country : CA, recipient province : nan, recipient city : Ontario, recipient postal code : nan, program name english : Discovery Grants Program - Individual, program name french : Programme de subventions à la découverte - individuelles, program purpose english : Apoptosis is a predominant form of cell death that is orchestrated by intrinsic and extrinsic mechanisms that lead to activation of caspases, which cleave substrates that are important for cell survival. In contrast, cell necrosis is an alternative mechanism of cell death that results in membrane rupture and release of intracellular contents to the external milieu. The molecular mechanisms involved in necrosis signaling are unclear. 
Alternative cell death signaling is induced when RipK1 is dysengaged from the TNFR complex, which results in interaction of RipK1 with RipK3, FADD and Casp-8 in the ripoptosome complex that results in cell death by Casp-8. When Casp-8 activity is inhibited, RipK3 interacts with and phosphorylates MLKL in the necrosome complex, which leads to trimerization of MLKL and its relocation to cell membrane, resulting in cell death by necroptosis that is identical to necrosis. 
 Preliminary results: Since there is substantial overlap in ripoptosome and necrosome signaling (RipK1, RipK3, FADD and Casp-8), unravelling of the initial steps that lead to necrosome activation will be incomplete without understanding of the ripoptosome complex. Our results have revealed an interesting dichotomy in ripoptosome and necrosome signaling during differentiation of monocytes to macrophages. Monocytes are highly susceptible to ripoptosome, but resistant to necrosome signaling. Upon differentiation to macrophages, they acquire resistance to ripoptosome, but susceptibility to necrosome signaling. The mechanisms behind this switch in susceptibility of monocytes/macrophages to ripoptosome versus necrosome signaling appears to involve XIAP- and MAPK- signaling. Our preliminary results have also uncovered a previously unrecognized mechanism that causes premature degradation of the necrosome, which appears to involve the scaffold function of RipK3 that results in K48-ubiquitination of interacting proteins. 
 Hypothesis: MK2 and RipK3 regulate necrosome signaling through distinct mechanisms. Inhibition of these regulatory mechanisms results in potent necrosome signaling. 
 Aims: 1) Evaluate how differentiation of monocytes impacts ripoptosome and necrosome signaling 2) determine how MK2 regulates ripoptosome/necrosome signaling in macrophages 3) decipher how RipK3-scaffold causes premature degradation of the necrosome. 
 Approach: We apply rigorous methods such as cell imaging, immunoprecipitations, bioinformatics and western blotting with primary cells (WT, knockout and knock-in). We use knockout cells, inhibitors, siRNA or CRISPR approach to decipher specific mechanisms. 
 Significance: Deciphering how necrosome signaling is induced and regulated at the molecular level will provide novel insights into the cell biology of this important pathway and help us identify various downstream target genes that can be targeted for therapeutic intervention., program purpose french : Apoptosis is a predominant form of cell death that is orchestrated by intrinsic and extrinsic mechanisms that lead to activation of caspases, which cleave substrates that are important for cell survival. In contrast, cell necrosis is an alternative mechanism of cell death that results in membrane rupture and release of intracellular contents to the external milieu. The molecular mechanisms involved in necrosis signaling are unclear. 
Alternative cell death signaling is induced when RipK1 is dysengaged from the TNFR complex, which results in interaction of RipK1 with RipK3, FADD and Casp-8 in the ripoptosome complex that results in cell death by Casp-8. When Casp-8 activity is inhibited, RipK3 interacts with and phosphorylates MLKL in the necrosome complex, which leads to trimerization of MLKL and its relocation to cell membrane, resulting in cell death by necroptosis that is identical to necrosis. 
 Preliminary results: Since there is substantial overlap in ripoptosome and necrosome signaling (RipK1, RipK3, FADD and Casp-8), unravelling of the initial steps that lead to necrosome activation will be incomplete without understanding of the ripoptosome complex. Our results have revealed an interesting dichotomy in ripoptosome and necrosome signaling during differentiation of monocytes to macrophages. Monocytes are highly susceptible to ripoptosome, but resistant to necrosome signaling. Upon differentiation to macrophages, they acquire resistance to ripoptosome, but susceptibility to necrosome signaling. The mechanisms behind this switch in susceptibility of monocytes/macrophages to ripoptosome versus necrosome signaling appears to involve XIAP- and MAPK- signaling. Our preliminary results have also uncovered a previously unrecognized mechanism that causes premature degradation of the necrosome, which appears to involve the scaffold function of RipK3 that results in K48-ubiquitination of interacting proteins. 
 Hypothesis: MK2 and RipK3 regulate necrosome signaling through distinct mechanisms. Inhibition of these regulatory mechanisms results in potent necrosome signaling. 
 Aims: 1) Evaluate how differentiation of monocytes impacts ripoptosome and necrosome signaling 2) determine how MK2 regulates ripoptosome/necrosome signaling in macrophages 3) decipher how RipK3-scaffold causes premature degradation of the necrosome. 
 Approach: We apply rigorous methods such as cell imaging, immunoprecipitations, bioinformatics and western blotting with primary cells (WT, knockout and knock-in). We use knockout cells, inhibitors, siRNA or CRISPR approach to decipher specific mechanisms. 
 Significance: Deciphering how necrosome signaling is induced and regulated at the molecular level will provide novel insights into the cell biology of this important pathway and help us identify various downstream target genes that can be targeted for therapeutic intervention., agreement title english : Activation and regulation of necrosis in macrophages, agreement title french : Activation and regulation of necrosis in macrophages, agreement number : RGPIN, agreement value : 170000.0, agreement start date : 2017-05-10, agreement end date : nan, description english : nan, description french : nan, expected results english : nan, expected results french : nan, additional information english : Grant or Award spanning more than one fiscal year. (2017-2018 to 2022-2023), additional information french : Subvention ou bourse octroyée s'appliquant à plus d'un exercice financier. (2017-2018 à 2022-2023), owner organization : nserc-crsng, owner organization title : Natural Sciences and Engineering Research Council of Canada | Conseil de recherches en sciences naturelles et en génie du Canada