Abstract
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.
Highlights
Park 7 gene encodes a conserved protein called DJ-1, and is found to have an autosomal recessive gene deletion mutation in 2003, which can lead to early onset Parkinson’s disease (PD) [1]
The results show that DJ-1 knockout cells have more autophagy stress than wild-type PC-12 cells in the base stage, and both wild-type PC-12 cells and DJ-1 knockout cells can induce stronger autophagy stress in the face of oxidative stress (Figure 5(A))
In order to observe the effect of DJ-1 on cell survival, we start from the basic state to the oxidative stress state respectively, and found that DJ-1 knockout seriously affects the viability, which supports the above views
Summary
Park 7 gene encodes a conserved protein called DJ-1, and is found to have an autosomal recessive gene deletion mutation in 2003, which can lead to early onset Parkinson’s disease (PD) [1]. DJ-1 consists of 189 amino acids, containing three cysteine residues at sites 46, 53 and 106. Of the three cysteine residues, the 106-site cysteine residues are highly sensitive to oxidative stress [2] [3] [4]. The trigger of autophagy is precisely regulated by a series of waterfall events, including MAPK/ ERK and p38 signaling pathways. ERK nuclear translocation is associated with autophagy stress [7] [8] [9], but the mechanism of DJ-1 genes regulating autophagy stress is still not completely clear, especially the effect on autophagy maturation has not been reported, so it is necessary to explore deeply
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