Study On BPDE Protein Adducts In SH-SY5Y Cells

Objective:Identification of binding protein of 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene(BPDE)in neuroblastoma cells(SH-SY5Y)by drug affinity responsive target stability method,and analyzed by bioinformatics Screening of target proteins closely related to neurotoxicity,provides a basis for further study of the mechanism of action of benzo[a]pyrene(B[a]P)neurotoxicity.Methods:Culture and collect human neuroblastoma cells(SH-SY5Y),extract protein and adjust the protein concentration to 6g/L.the cell lysate was divided into two groups,a BPDE incubation group and a DMSO solvent control group.mix with BPDE stock solution or equal volume of DMSO,respectively,incubate for 2h at room temperature.Then add the thermolysin in proportion.after digesting,a part of the sample was separated by SDS-PAGE gel electrophoresis and stained,and the others were subjected to LC-MS/MS to detect differential proteins.Bioinformatics analysis of the identified binding proteins to select to the target proteins closely related to neurotoxicity.Western blot and cellular thermal shift assay were used to verify the binding of target protein to BPDE.Results:The results of SDS-PAGA electrophoresis showed that there was no significant difference in the protein bands between the BPDE incubation group and the DMSO vehicle group.However,after adding the thermolysin,observed there was visible different bands between the BPDE incubation group and the DMSO vehicle group.A total of 472 proteins were identified by non-labeled quantitative proteomics analysis,of which 69 proteins were only found in the DMSO vehicle group,and 298 proteins were only found in the BPDE incubation group.According to the difference of multiples > 2.0 times and the value of P < 0.05(including only one group of proteins present),428 binding proteins were identified,of which 81.3% of the binding proteins were significantly higher in the BPDE incubation group than in the vehicle group.GO analysis of 428 binding proteins revealed that: Biological processes mainly involved cell reaction and regulation,the classification of the largest number of proteins is mainly related to macromolecular,organic nitrogen,protein and other metabolic processes.The cellular components were mainly related to cell vesicle transport,the classification of the largest number of proteins is mainly involves organelles,vesicles,extracellular exosomes,and the like.Molecular function mainly involves the intermolecular binding,including protein binding,RNA binding,the classification of the largest number of proteins is mainly involves nucleic acid binding,nucleotide binding,enzyme binding and the like.Through KEGG analysis showed that: The top 10 KEGG signaling pathways with the most significant P values are proteasome,carbon metabolism,pyruvate metabolism,splices,protein processing in the endoplasmic reticulum,ribosomes,RNA trafficking,aminoacyl-tRNA biosynthesis,Epstein-Bar virus infection,glycolysis/gluconeogenesis.mainly involves energy metabolism,protein synthesis in the endoplasmic reticulum,lysosomes,ribosomes,and genetic information processing.Other neurotoxic-related metabolic pathways include PI3K-Akt signaling pathways involved in neuronal signaling,learning and memory,MAPK signaling pathways,long-term potentiation,cAMP signaling pathways,calcium signaling pathways,and repair-related nucleotide excision Repair,mismatch repair,homologous recombination,basal excision repair,synaptic vesicle cycle associated with synaptic function,dopaminergic synapses,glutamatergic synapses,serotoninergic synapses,etc.Protein interaction analysis found that proteins located in key parts of the center and closely related proteins mainly involved protein processing in the endoplasmic reticulum,neuronal energy metabolism,synaptic function,signaling between nerve cells and learning and memory,combined with GO analysis and KEGG.Analysis,screening to determine 40 S ribosomal protein S21,AP-2 complex subunit α-2,40 S ribosomal protein S5,casein kinase I isoform δ,casein kinase I isoform ε,AP-2 complex subunit α-1,ATP synthase subunit α,mitochondria,40 S ribosomal protein S10,40 S ribosomal protein S14,40 S ribosomal protein S6,transketol,UV-clearing repair protein RAD23 homolog A,protein disulfide bond isomerization Enzyme A3,glutamate receptor ion type NMDAR2 A,malate dehydrogenase,60 S ribosomal protein L27,60 S ribosomal protein L6,dihydrolipoyl dehydrogenase,glyceraldehyde-3-phosphate dehydrogenase,40 S Ribosomal protein S2,clathrin heavy chain,protein disulfide isomerase A4,neutral alpha-glucosidase AB,serine/threonine protein phosphatase 2A65 kDa regulatory subunit Aα isoform,fructose diphosphate aldehyde Reduced enzyme A,elongation factor 2,78 kDa glucose 26 proteins that may be located at critical points in neurotoxic damage-related signaling pathways as the BPDE-binding target protein of interest.The NMDAR2 A and RAD23 A proteins were selected for validation.Western blot results showed that there was no significant difference in the relative content of NMDAR2 A and RAD23 A protein in the BPDE-incubated group compared with the DMSO solvent control when no thermophilic protease digestion;but after the thermolysin digestion,the BPDE incubation group The relative contents of NMDAR2 A and RAD23 A proteins were significantly higher than those in the DMSO solvent control group(P<0.05).cellular thermal shift assay showed that ΔTm=(7.35±1.74)°C for NMDAR2 A protein and ΔTm=(9.64±2.42)°C for RAD23 A protein,the difference was statistically significant(P<0.05).The accuracy and reliability of the target identification technique of DARTS were verified,and it was confirmed that there was a direct effect between BPDE and NMDAR2 A protein and RAD23 A protein.Conclusion:BPDE binding can alter the sensitivity of binding proteins to thermolysin digestion,and drug affinity response target stability methods can be used to identify and screen BPDE Protein Adducts.A total of 428 SH-SY5 Y cell proteins that bind to BPDE were identified by this method.Among them,26 binding proteins such as NMDAR2 A and RAD23 A may be important target proteins related to BPDE neurotoxicity.