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Browsing by Author "Green, Douglas R."
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Item Generation of Casp8FL122/123GG Mice Using CRISPR-Cas9 Technology(Cell Press, 2020-12-18) Pelletier, Stephane; Tummers, Bart; Green, Douglas R.; Medical and Molecular Genetics, School of MedicineThe purpose of this protocol is to describe the generation of missense mutations in mice using CRISPR-Cas9 technology. The current protocol focuses on the generation of a Casp8 FL122/123GG missense mutation, but it can be adapted to introduce any missense or nonsense mutation. For complete details on the use and execution of this protocol, please refer to Tummers et al. (2020).Item Metabolic activation of CaMKII by coenzyme A(Elsevier, 2013-11-07) McCoy, Francis; Darbandi, Rashid; Lee, Hoi Chang; Bharatham, Kavitha; Moldoveanu, Tudor; Royappa, Grace; Dodd, Keela; Lin, Wenwei; Chen, Si-Ing; Tangallapally, Rajendra P.; Kurokawa, Manabu; Lee, Richard E.; Shelat, Anang; Chen, Taosheng; Green, Douglas R.; Harris, Robert A.; Lin, Sue-Hwa; Fissore, Rafael A.; Colbran, Roger J.; Nutt, Leta K.; Biochemistry & Molecular Biology, School of MedicineActive metabolism regulates oocyte cell death via calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated phosphorylation of caspase-2, but the link between metabolic activity and CaMKII is poorly understood. Here we identify coenzyme A (CoA) as the key metabolic signal that inhibits Xenopus laevis oocyte apoptosis by directly activating CaMKII. We found that CoA directly binds to the CaMKII regulatory domain in the absence of Ca(2+) to activate CaMKII in a calmodulin-dependent manner. Furthermore, we show that CoA inhibits apoptosis not only in X. laevis oocytes but also in Murine oocytes. These findings uncover a direct mechanism of CaMKII regulation by metabolism and further highlight the importance of metabolism in preserving oocyte viability.Item The interaction between RIPK1 and FADD controls perinatal lethality and inflammation(Elsevier, 2024) Rodriguez, Diego A.; Tummers, Bart; Shaw, Jeremy J. P.; Quarato, Giovanni; Weinlich, Ricardo; Cripps, James; Fitzgerald, Patrick; Janke, Laura J.; Pelletier, Stephane; Crawford, Jeremy Chase; Green, Douglas R.; Medical and Molecular Genetics, School of MedicinePerturbation of the apoptosis and necroptosis pathways critically influences embryogenesis. Receptor-associated protein kinase-1 (RIPK1) interacts with Fas-associated via death domain (FADD)-caspase-8-cellular Flice-like inhibitory protein long (cFLIPL) to regulate both extrinsic apoptosis and necroptosis. Here, we describe Ripk1-mutant animals (Ripk1R588E [RE]) in which the interaction between FADD and RIPK1 is disrupted, leading to embryonic lethality. This lethality is not prevented by further removal of the kinase activity of Ripk1 (Ripk1R588E K45A [REKA]). Both Ripk1RE and Ripk1REKA animals survive to adulthood upon ablation of Ripk3. While embryonic lethality of Ripk1RE mice is prevented by ablation of the necroptosis effector mixed lineage kinase-like (MLKL), animals succumb to inflammation after birth. In contrast, Mlkl ablation does not prevent the death of Ripk1REKA embryos, but animals reach adulthood when both MLKL and caspase-8 are removed. Ablation of the nucleic acid sensor Zbp1 largely prevents lethality in both Ripk1RE and Ripk1REKA embryos. Thus, the RIPK1-FADD interaction prevents Z-DNA binding protein-1 (ZBP1)-induced, RIPK3-caspase-8-mediated embryonic lethality, affected by the kinase activity of RIPK1.