| Lamprey,the most primitive jawless vertebrate extant on earth,is considered as the most ideal model organism among the existing jawless vertebrates and is an important bridge linking invertebrates and vertebrates.Prohibitin(PHB)is a highly conserved protein that is widely distributed in a variety of biological cells.The PHB family contains two members(PHB1 and PHB2)with high homology between the two members.The PHB2 protein consists of three components: the N-terminus of a hydrophilic transmembrane ? helix(the LXXLL sequence at the N-terminus,which binds to the estrogen receptor),and a conserved PHB region(for protein interactions and interaction of lipid rafts plays an important role)and the C-terminus used to form complexes with PHB1.The PHB1 of the homo PHB protein family contains 7 exons,consisting of 275 amino acids,encoding a 32 kDa protein;the PHB2 protein contains 10 exons and consists of 316 amino acids,encoding a 33.3 kDa protein.The PHB1 and PHB2 proteins form a heterodimer and 12-16 pairs of such heterodimers polymerize to form a rosette palisade complex with a diameter of 20-25 nm,anchoring the inner mitochondrial membrane,maintaining mitochondrial structure and function stability.Reactive oxygen species(ROS)is an active oxygen species produced during cell metabolism.Superoxide radical(02-),hydrogen peroxide(H2O2)and hydroxyl radical(-OH)are common.The proper amount of ROS is beneficial to the vital activity of the cells and helps to promote the cell proliferation and cell differentiation.However,excess ROS can cause oxidative damage to lipids,proteins and DNA in cells.Compared with normal cells,tumor cells are mostly in a state of high oxidative stress,which is more sensitive to the increase of ROS concentration.In addition,ROS can also activate downstream signaling pathways(such as MAPK pathway)to induce early apoptosis of cells.Long-term oxidative stress can cause the body to suffer from Parkinson's disease,heart disease and Alzheimer's disease and other related diseases.In this study,based on the previous work of the research group,the expression of Lm-PHB2 in human embryonic kidney epithelial cells(293T)and Chang liver cells(CHL)was further observed by laser confocal microscopy.The results show that Lm-PHB2 is mainly localized in the nucleus under normal conditions.When the cells are stimulated by hydrogen peroxide,Lm-PHB2 translocation from the cell nucleus to the mitochondria.To explore the mechanism of localized migration of Lm-PHB2 under oxidative stress conditions,truncated and amino acid mutants of the rLm-PHB2 amino acid sequence were constructed(Lm-PHB21-50 aa,Lm-PHB251-200 aa,Lm-PHB2201-299 aa and Lm-PHB21-57aaAAA)was observed by laser confocal microscopy for the expression and localization of the truncated and amino acid mutants.The results showed that only the truncated mutant Lm-PHB21-50 aa was able to specifically localize to mitochondria.The localization of Lm-PHB2 to mitochondria is determined by the N-terminal sequence of the protein,and the amino acids at positions 17,48 and 54 are the key amino acids that determine its localization and migration.Extraction of recombinant PHB2 proteins by Ni column purification,293 T cells were incubated with rLm-PHB2 proteins and stimulated by hydrogen peroxide,the rLm-PHB2 proteins influnced in ROS analyzed by flow cytometry.The results showed that the rLm-PHB2 proteins can significantly inhibit hydrogen peroxide-induced ROS generated,and increasing oxidative stress tolerance in 293 T cells.To further explore the mechanism of oxidative stress tolerance in Lm-PHB2 enhanced cells,the mitochondrial morphology and mitochondrial membrane potential of Lm-PHB2 induced by hydrogen peroxide were observed by laser confocal fluorescence microscopy.The results showed that Lm-PHB2 can participate in the maintenance of mitochondrial morphology and mitochondrial membrane potential stability.To investigate the mechanism of Lm-PHB2 involvement in maintaining mitochondrial morphology stability,the effect of Lm-PHB2 on the expression of OPA1 in 293 T cells before and after hydrogen peroxide stimulation analyzed by Q-PCR and Western blot.The results showed that pEGFP-N1 was transfected in 293 T cells the expression of OPA1 mRNA and protein was significantly increased after hydrogen peroxide stimulation.These results indicate that Lm-PHB2 participates in maintaining mitochondrial morphological and functional stability through interaction with OPA1 proteins.Then the used of immunofluorescence and co-immunoprecipitation methods to detect the interaction between the Lm-PHB2 and OPA1,the results show that when 293 T cells stimulated by hydrogen peroxide,OPA1 and Lm-PHB2 can interacted.To investigate whether Lm-PHB2 influences hydrogen peroxide-induced apoptosis,Q-PCR and Western blot methods were used to detect whether the pEGFP-N1-Lm-PHB2 plasmisd was transfected into 293 T cells with or without hydrogen peroxide.The expression of HAX1 at both mRNA and protein levels was significantly increased after hydrogen peroxide in 293 T cells after transfected with pEGFP-N1-Lm-PHB2 plasmids.Similarly,the results of immunofluorescence and co-immunoprecipitation showed that Lm-PHB2 interacts with HAX1 to resist hydrogen peroxide-induced apoptosis.In summary,based on the previous work,this paper initially revealed the molecular mechanism of localization and translocation of Lm-PHB2 in 293 T cells and CHL cells under oxidative stress conditions,Lm-PHB2 translocalization to mitochondria is determined by the N-terminal sequence,and amino acids 17,48,and 54 are the key amino acids;Lm-PHB2 was found to maintain mitochondrial morphology and inhibit the outbreak of ROS by interacting with mitochondrial inner membrane protein OPA1.This enhanced the oxidative stress tolerance of the cells;at the same time,Lm-PHB2 could interact with the anti-apoptotic protein HAX1 to inhibit hydrogen peroxide-induced apoptosis.This laid the foundation for the study of cellular oxidative stress mechanism and provided new clues for the treatment of diseases caused by oxidative stress. |
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