Park 7 gene encodes a conserved protein called DJ-1 protein, which involves autophagy stress, but the mechanism is unclear. Therefore, it is necessary to explore the mechanism of DJ-1 regulation PC-12 autophagical str...Park 7 gene encodes a conserved protein called DJ-1 protein, which involves autophagy stress, but the mechanism is unclear. Therefore, it is necessary to explore the mechanism of DJ-1 regulation PC-12 autophagical stress. Using CRISPR/Cas9 technique to construct DJ-1 knockout PC-12 cell lines, we culture wild-type and DJ-1 knockout PC-12 cell lines, establish oxidative stress cell model by MPP+, and divide them into wild-type control group (WT), wild-type intervention group (WT + MPP+), DJ-1 knockout control group (KO) and DJ-1 knockout intervention group (KO + MPP+), and explore the role of DJ-1 in regulating neuronal autophagy stress by cell viability assay, immunofluorescence, confocal, western blotting and electron microscopy. The results show that the growth ability of DJ-1 knockout cells is inferior to that of normal cells, and DJ-1 knockout cells are more sensitive to oxidative stress and more vulnerable to damage than wild-type cells. Exposing to MPP+, DJ-1 proteins undergo oxidative responses at Cys-106 sites, while DJ-1 knockout PC-12 cells do not show similar responses. The wild-type PC-12 cells have the confocal in both anti-oxidant DJ-1 antibody and anti-C-Raf phosphorylation antibody. The activated DJ-1 induces the phosphorylation of C-Raf at Ser338 sites to activate directly C-Raf, and subsequently activates ERK1/2 signaling pathways to antagonize MPP+-induced neurotoxicity. Lack of DJ-1, oxidative stress can not promote C-Raf activation. Although the phosphorylation level of cell ERK is also increased, the increase of intranucleus pERK is not obvious. Wild type and DJ-1 knockout PC-12 cells can produce autophagical stress in the face of oxidative stress, but the proportion of autophagolysosomes produced in wild type PC-12 cells is larger than that in DJ-1 knockout cells. PD98059 can reduce autophagy stress in the state of oxidative stress in wild-type PC-12 cells, and the number of autophagolysosomes is similarly reduced, while sorafenib decreased slightly DJ-1 the autophagical stress, and the proportion of autophagolysosomes decreased more. Therefore, we can infer that activated DJ-1 directly phosphorylates C-Raf at Ser-338 sites, then activating C-Raf, subsequent activation of the MEK/ERK pathway. DJ-1 promotes autophagy maturation through the C-Raf/ERK pathway, thereby improving cell survival.展开更多
文摘Park 7 gene encodes a conserved protein called DJ-1 protein, which involves autophagy stress, but the mechanism is unclear. Therefore, it is necessary to explore the mechanism of DJ-1 regulation PC-12 autophagical stress. Using CRISPR/Cas9 technique to construct DJ-1 knockout PC-12 cell lines, we culture wild-type and DJ-1 knockout PC-12 cell lines, establish oxidative stress cell model by MPP+, and divide them into wild-type control group (WT), wild-type intervention group (WT + MPP+), DJ-1 knockout control group (KO) and DJ-1 knockout intervention group (KO + MPP+), and explore the role of DJ-1 in regulating neuronal autophagy stress by cell viability assay, immunofluorescence, confocal, western blotting and electron microscopy. The results show that the growth ability of DJ-1 knockout cells is inferior to that of normal cells, and DJ-1 knockout cells are more sensitive to oxidative stress and more vulnerable to damage than wild-type cells. Exposing to MPP+, DJ-1 proteins undergo oxidative responses at Cys-106 sites, while DJ-1 knockout PC-12 cells do not show similar responses. The wild-type PC-12 cells have the confocal in both anti-oxidant DJ-1 antibody and anti-C-Raf phosphorylation antibody. The activated DJ-1 induces the phosphorylation of C-Raf at Ser338 sites to activate directly C-Raf, and subsequently activates ERK1/2 signaling pathways to antagonize MPP+-induced neurotoxicity. Lack of DJ-1, oxidative stress can not promote C-Raf activation. Although the phosphorylation level of cell ERK is also increased, the increase of intranucleus pERK is not obvious. Wild type and DJ-1 knockout PC-12 cells can produce autophagical stress in the face of oxidative stress, but the proportion of autophagolysosomes produced in wild type PC-12 cells is larger than that in DJ-1 knockout cells. PD98059 can reduce autophagy stress in the state of oxidative stress in wild-type PC-12 cells, and the number of autophagolysosomes is similarly reduced, while sorafenib decreased slightly DJ-1 the autophagical stress, and the proportion of autophagolysosomes decreased more. Therefore, we can infer that activated DJ-1 directly phosphorylates C-Raf at Ser-338 sites, then activating C-Raf, subsequent activation of the MEK/ERK pathway. DJ-1 promotes autophagy maturation through the C-Raf/ERK pathway, thereby improving cell survival.
