Internal death programs play significant roles in many diseases. Pathogenic effects can result from inefficient cell death or from inappropriate or excessive death such as that caused by the human immunodeficiency virus (HIV) during AIDS or the SAR-CoV virus during SARS. In this project, we are taking a multifaceted approach to studying molecular mechanisms of both apoptotic and nonapoptotic death programs in lymphocytes as well as other cell types. A major focus of our investigations are death-inducing cell surface receptors in the tumor necrosis factor receptor (TNFR) superfamily such as TNFR1 and CD95/Fas/APO-1. Both receptors play an important role in stimulating both apoptotic and nonapoptotic death of cells principally in immune processes. Little is known about how these alternative death pathways are entrained to receptor signaling. Interestingly, both receptors can have effects beside death such as the induction of transcription factors. We are trying to understand how these receptors stimulate the intracellular machinery that causes cell death in preference to other cellular outcomes. We have discovered that inhibition of caspase-8 in non-lymphoid cells can lead to another form of cell death exhibiting particular cytoplasmic double membrane structures called autophagy. Autophagy is an evolutionarily conserved process from humans to yeast by which cytoplasmic proteins and organelles are catabolized but very little was known about results at the end of autophagy when cells were selecting between autophagic cell death and survival. Mitochondria have a primary physiological role in producing ATP as an energy source, but also regulate cell death. In response to cellular stress, dysfunctional mitochondria produce ROS and other pro-death mediators to initiate programmed cell death pathways, like apoptosis or necropotosis. Mitophagy, a selective form of autophagy, can target dysfunctional mitochondria for lysosomal degradation and protect cells from oxidative damage. This is beneficial for the survival of terminally differentiated cells, like nerve and heart muscle cells. Several regulators of mitophagy, including PINK1, Nix (BNIP3L), and PARKIN have been identified. Mutations or deletions of those genes have been related with a variety of diseases, including ischemia injury in myocardial infarction and stroke, as well as neurodegenerative disease. Hence, understanding the detailed mechanism of mitophagy remains an important goal for improving the diagnosis and treatment of diseases involving mitochondria. Two autosomal recessive Parkinsons disease genes, PINK1 (PTEN induced putative kinase 1) and PARKIN, regulate mitophagic clearance of dysfunctional mitochondria. In healthy cells, PINK1 is constitutively degraded by mitochondrial proteases, such as mitochondria inner membrane protease Presenilin Associated, Rhomboid-Like (PARL) protein. Membrane depolarization of dysfunctional mitochondria inhibits PINK1 degradation, causing it to accumulate and promote mitophagy via recruitment of another familial Parkinson's protein, the E3 ubiquitin ligase PARKIN. However, the detailed mechanism of PINK1 degradation and stabilization remains unclear. We have studyPGAM5, paralog member 5 of a family of highly conserved phosphoglycerate mutases, a 32-kDa mitochondrial protein that apparently lacks phosphotransfer function on phosphoglycerates, but retains activity as a serine/threonine protein phosphatase that regulates the ASK1 kinase. The functions of PGAM5 are complex since it also serves as an anti-oxidant regulator in the Kelch ECH associating protein 1-nuclear factor-E2-related factor 2 (KEAP1-NRF2) signaling pathway (19, 20). Recently, PGAM5 was found as a downstream mitochondrial target of RIP3 in the necrosis pathway in cancer cells, by recruiting the RIP1-RIP3-MLKL necrosis """"""""attack"""""""" complex to mitochondria (3, 21). Interestingly, PGAM5 has also been reported as a genetic suppressor of PINK1 in Drosophila (22), as well as a substrate of PARL (23). Thus, it is important to establish the in vivo role of PGAM5 involving mitochondria. using a new strain of knockout mice, we found that PGAM5 is critical for PINK1 stabilization on damaged mitochondria to initiate mitophagy since loss of PGAM5 totally disables PINK1 stabilization. Cells deficient with PGAM5 showed elevated ROS originated from mitochondria, and exacerbated cell necrosis compared to control wild type cells. In stroke and cardiac ischemic-reperfusion injury models, PGAM5-deficient mice showed significantly increased severity of injuries in the brain and heart compared to wild-type mice, indicating that PGAM5 protects against ischemia-reperfusion-induced necrosis. Taken together, our data suggest that PGAM5 promotes PINK1-mediated mitophagy, which could be cytoprotective in ischemic injuries. Moreover, PGAM5 also provides a functional linkage between malfunction of mitophagy and the pathogenesis of necrosis.
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