散发型帕金森病蛋白酶体功能抑制SH-SY5Y细胞模型的分子伴侣蛋白质组学研究
发布时间：2019-05-19 19:16
【摘要】：[背景]在散发型帕金森病(sporadic Parkinson's, sPD)病理机制的泛素-蛋白酶体系统(ubiquitin-proteasome system, UPS)障碍通路中,分子伴侣蛋白质高表达被认为是蛋白质分解应激反应的保护性反馈机制。[目的]为了揭示分子伴侣蛋白质在sPD病理机制UPS功能障碍通路中的表达规律,本研究进行了sPD蛋白酶体功能抑制SH-SY5Y细胞模型的分子伴侣蛋白质组学分析。[方法]10 μmol/L全反式维甲酸和80 nmol/L佛波醇12-十四酸酯13-乙酸酯,相继作用SH-SY5Y细胞各72 h将SH-SY5Y细胞诱导为分化细胞(对照组),10 μmol/L人工蛋白酶体抑制剂继续作用对照组24 h建立了sPD蛋白酶体功能抑制SH-SY5Y细胞模型(实验组)；对照组、实验组蛋白质样品的差异胶内电泳获得了106个差异表达蛋白质,其中17个差异表达蛋白质变化最明显并且被指定为目的蛋白质：实验组蛋白质样品的制备胶二维电泳获得了目标蛋白质；蛋白质胰蛋白酶酶解产物的基质辅助激光解析电离-飞行时间质谱获得了目标蛋白质的肽质量指纹(或质谱数据),质谱数据在线检索从SwissProt、NCBInr两个蛋白质数据库中获得了目标蛋白质的17各候选蛋白质；同源蛋白质的生物信息学数据分析确认候选蛋白质的生物学功能及其类别；实验组基因转录物相对定量分析进一步验证了其中的一个目标蛋白质。[结果]首先,这17个目标蛋白质的候选蛋白质分别为：热休克蛋白质-27 (27-kDa heat shock protein, HSP-27)、热休克蛋白质-32 (heat shock protein 32, HSP-32)、葡萄糖调节蛋白质-58 (58-kDa glucose regulated protein, GRP-58)、热休克蛋白质-70 (70-kDa heat shock protein, HSP-70)、热休克关联蛋白质-70 (70-kDa heat shock cognate protein, HSC-70)、葡萄糖调节蛋白质-75 (75-kDa glucose regulated protein, GRP-75)、X热休克蛋白质-105 (105-kDa heat shock protein, HSP-105)、氧调节蛋白质-150 (150-kDa oxygen regulated protein, ORP-150)、钙离子结合蛋白质-1 (calcium-binding protein 1, CaBP-1)、CBP-50蛋白质(CBP-50 protein, CBP-50)、热休克蛋白质-60 (60-kDa mitochondrial heat shock protein 60 mHSP-60),脯氨酰羟化酶-p多肽(prolyl-4-hydroxylase beta polypeptide, P4HB);应激诱导磷蛋白质-1 (stress induced phosphoprotein-1或stress inducible phosphoprotein-1, STIP-1)、14-3-3蛋白质ε (14-3-3zeta,14-3-3ξ),含T-复合体多态-1蛋白质p亚基(T-complex polypeptide 1 beta subunit, TCP-1β)、含T-复合体多肽-1蛋白质ε亚基(T-complex polypeptide 1 epsilon subunit, TCP-1ε)、含缬酪肽蛋白质(Valosin-containing protein, VCP);其次,它们被归纳为属于如下4个分子伴侣蛋白质类别：HSP-27、HSP-32、HSP-70、HSC-70、HSP-105、GRP-58、GRP-75、ORP150和VCP为9个伴侣蛋白质,P4HB、14-3-3ξ、CaBP-1和CBP-50为4个类伴侣蛋白质,STIP-1为共伴侣蛋白质,mHSP-60、TCP-1β和TCP-1ε为3个伴侣素。[结论]上述结果提示,HSP-70等17个分子伴侣蛋白质在sPD病理机制UPS功能障碍通路中参与了蛋白质分解应激反应。
[Abstract]:[background] in the ubiquitin proteasome system (ubiquitin-proteasome system, UPS) disorder pathway), which is the pathological mechanism of sporadic Parkinson's disease (sporadic Parkinson's, sPD), The high expression of molecular chaperone protein is considered to be the protective feedback mechanism of protein decomposition stress response. [objective] in order to reveal the expression of molecular chaperone protein in UPS dysfunction pathway, the molecular chaperone proteome analysis of sPD proteasome function inhibition SH-SY5Y cell model was carried out. [methods] SH-SY5Y cells were induced into differentiated cells by 10 渭 mol/L all-trans retinoic acid and 80 nmol/L phorbol 12-decetetraate 13-acetate for 72 hours each (control group). 10 渭 mol/L artificial proteasome inhibitor continued to act on the control group for 24 hours to establish the SH-SY5Y cell model of sPD proteasome function inhibition (experimental group). In the control group, 106 differentially expressed proteins were obtained by differential gel electrophoresis in the experimental group. Among them, 17 differentially expressed proteins had the most obvious changes and were designated as the target proteins: the target proteins were obtained by two-dimensional gel electrophoresis of the protein samples in the experimental group. The peptide mass fingerprinting (or mass spectrometry data) of the target protein was obtained by matrix-assisted laser analytical ionization-time-of-flight mass spectrometry of protein trypsin hydrolysates. The mass spectrometry data were searched online from SwissProt,. 17 candidate proteins of the target protein were obtained from the two protein databases of NCBInr. The bioinformatics data analysis of homologous proteins confirmed the biological function and categories of candidate proteins, and the relative quantitative analysis of gene transcripts in the experimental group further verified one of the target proteins. [results] first of all, the candidate proteins of these 17 target proteins were heat shock protein-27 (27-kDa heat shock protein, HSP-27), heat shock protein-32 (heat shock protein 32, HSP-32). Glucose regulated protein-58 (58-kDa glucose regulated protein, GRP-58), heat shock protein-70 (70-kDa heat shock protein, HSP-70), heat shock associated protein-70 (70-kDa heat shock cognate protein, HSC-70), Glucose regulated protein-75 (75-kDa glucose regulated protein, GRP-75), X heat shock protein-105 (105-kDa heat shock protein, HSP-105), oxygen-regulated protein-150 (150-kDa oxygen regulated protein, ORP-150), Calcium binding protein-1 (calcium-binding protein 1, CaBP-1), CBP-50 protein (CBP-50 protein, CBP-50), heat shock protein-60 (60-kDa mitochondrial heat shock protein 60 mHSP-60), Prolyl hydroxylase-p polypeptide (prolyl-4-hydroxylase beta polypeptide, P4HB); Stress induced phosphorus protein-1 (stress induced phosphoprotein-1 or stress inducible phosphoprotein-1, STIP-1), 14 鈮，
本文编号：2480996
[Abstract]:[background] in the ubiquitin proteasome system (ubiquitin-proteasome system, UPS) disorder pathway), which is the pathological mechanism of sporadic Parkinson's disease (sporadic Parkinson's, sPD), The high expression of molecular chaperone protein is considered to be the protective feedback mechanism of protein decomposition stress response. [objective] in order to reveal the expression of molecular chaperone protein in UPS dysfunction pathway, the molecular chaperone proteome analysis of sPD proteasome function inhibition SH-SY5Y cell model was carried out. [methods] SH-SY5Y cells were induced into differentiated cells by 10 渭 mol/L all-trans retinoic acid and 80 nmol/L phorbol 12-decetetraate 13-acetate for 72 hours each (control group). 10 渭 mol/L artificial proteasome inhibitor continued to act on the control group for 24 hours to establish the SH-SY5Y cell model of sPD proteasome function inhibition (experimental group). In the control group, 106 differentially expressed proteins were obtained by differential gel electrophoresis in the experimental group. Among them, 17 differentially expressed proteins had the most obvious changes and were designated as the target proteins: the target proteins were obtained by two-dimensional gel electrophoresis of the protein samples in the experimental group. The peptide mass fingerprinting (or mass spectrometry data) of the target protein was obtained by matrix-assisted laser analytical ionization-time-of-flight mass spectrometry of protein trypsin hydrolysates. The mass spectrometry data were searched online from SwissProt,. 17 candidate proteins of the target protein were obtained from the two protein databases of NCBInr. The bioinformatics data analysis of homologous proteins confirmed the biological function and categories of candidate proteins, and the relative quantitative analysis of gene transcripts in the experimental group further verified one of the target proteins. [results] first of all, the candidate proteins of these 17 target proteins were heat shock protein-27 (27-kDa heat shock protein, HSP-27), heat shock protein-32 (heat shock protein 32, HSP-32). Glucose regulated protein-58 (58-kDa glucose regulated protein, GRP-58), heat shock protein-70 (70-kDa heat shock protein, HSP-70), heat shock associated protein-70 (70-kDa heat shock cognate protein, HSC-70), Glucose regulated protein-75 (75-kDa glucose regulated protein, GRP-75), X heat shock protein-105 (105-kDa heat shock protein, HSP-105), oxygen-regulated protein-150 (150-kDa oxygen regulated protein, ORP-150), Calcium binding protein-1 (calcium-binding protein 1, CaBP-1), CBP-50 protein (CBP-50 protein, CBP-50), heat shock protein-60 (60-kDa mitochondrial heat shock protein 60 mHSP-60), Prolyl hydroxylase-p polypeptide (prolyl-4-hydroxylase beta polypeptide, P4HB); Stress induced phosphorus protein-1 (stress induced phosphoprotein-1 or stress inducible phosphoprotein-1, STIP-1), 14 鈮，
本文编号：2480996
本文链接：https://www.wllwen.com/yixuelunwen/shenjingyixue/2480996.html
最近更新
教材专著
