Degradation of Glycogen and Necrotic Cell Death
PYGL (glycogen phosphorylase) catalyzes degradation of glycogen and therefore allows it to be used as an energy source. The researchers were unable to obtain PYGL antibodies to further study the RIP3/PYGL interaction in necrosis. However, the interaction was induced by TNF but not zVAD. They incubated flag RIP3 missing glutathione S-transferase, which did not enhance PYGL activity and could only determine that RIP3 activates PYGL. They measured PYGL activity in N cells, RIP1 knockdown and RIP3 knockdown without and without TNF or TNF and zVAD combination treatment. RIP3 and RIP1 were both required in TNF or TNF and zVAD induced increased PYGL activity. Expression of RIP3 enhances PYGL activity in 293T cells. They also wanted to determine if PYGL is involved in necrosis so they knocked the PYGL gene out with siRNA. The knockdown of PYGL partially inhibited TNF and zVAD induced cell death. PYGL is a contributor to necrosis.
GLUL (glutamate-ammonia ligase) is an enzyme present in cytosol. It catalyzes the condensation of the amino acid glutamate (GLU) and ammonia in formation of glutamine (GLN). Glutamine can enter mitochondria and be utilized as an energy source. GLUD1 (glutamate dehydrogenase 1) is an enzyme in mitochondrial matrix that converts glutamate to ?-ketoglutarate. Both of the enzymes are essential for utilization of glutamate and glutamine to form ATP (adenosine triphosphate) by oxidative phosphorylation in mitochondria. Mitochondrial oxidation of glutamine is possibly sensitizing the mitochondria. The researchers observed increased interaction of RIP3, which was endogenous to the cell, and GLUL and GLUD1 in N cells treated with TNF or TNF and zVAD combination. Functional RIP3 increases GLUL activity. They were unable to detect the activity of endogenous GLUD1 and GLUL in N cells. They used siRNA to knockdown GLUL or GLUD1. TNF and zVAD combination induced the cell death in this case, suggesting that the enzymes utilize glutamine and glutamate as energy sources during necrosis.
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ROS production is required in necrotic cell death in cell lines. The authors hypothesized, that RIP3 will increase energy metabolism required for the production of ROS. They confirmed that ROS is require for necrosis but not for apoptosis. They observed that the depletion of RIP3 lowers ROS concentration and the enhancement of RIP3 increases the ROS concentration. The amount of ROS present was always directly proportional to the amount of necrotic cell death. When the 3 main enzymes, PYGL, GLUL and GLUD1, were depleted by siRNA knockdown, the amount of ROS was directly proportional to the number of the cell death. They conducted experiments focused on energy metabolism depletion and the results suggested the mitochondrial ROS has a role in TNF cytotoxicity. Increasing glucose and increasing energy source suggested that the switch from apoptosis to necrosis initiated by RIP3 would be partially caused by increased metabolism due to ROS production.
zVAD enhances the functions of RIP1 and RIP3 because the enzymes are shown to be cleaved by caspases. The ROS production increased energy metabolism could be responsible for RIP3 function as the mediator of necrosis. RIP3 determines the type of the cell death and therefore the energy metabolism also has effects on cell death.
At the very end, the authors suggest that inhibition of RIP1 can have a positive effect on patients with ischemic brain injury and other possible diseases in animals. The inhibition of RIP3 could have a positive effect on patients with diseases that are necrosis associated. Necrosis associated diseases include diabetes and cerebral ischemia. Knockdown of RIP3 had a positive effect on acute pancreatitis in mice. RIP3 has a potential to become an important drug target in all the diseases associated with necrosis.
The paper cleared the last unknown in the necrotic death pathway by discovery of RIP3. The authors observed the interaction of RIP3 and key enzymes involved in the energy metabolism associated with necrotic cell death. The discovery of RIP3 was simultaneously done by two other groups. , The paper also advanced the knowledge of the ROS involvement in necrosis. Important potential drug targets were identified.