【摘要】：[背景]在散發(fā)型帕金森病(sporadic Parkinson's, sPD)病理機制的泛素-蛋白酶體系統(tǒng)(ubiquitin-proteasome system, UPS)障礙通路中,分子伴侶蛋白質(zhì)高表達被認為是蛋白質(zhì)分解應(yīng)激反應(yīng)的保護性反饋機制。[目的]為了揭示分子伴侶蛋白質(zhì)在sPD病理機制UPS功能障礙通路中的表達規(guī)律,本研究進行了sPD蛋白酶體功能抑制SH-SY5Y細胞模型的分子伴侶蛋白質(zhì)組學分析。[方法]10 μmol/L全反式維甲酸和80 nmol/L佛波醇12-十四酸酯13-乙酸酯,相繼作用SH-SY5Y細胞各72 h將SH-SY5Y細胞誘導為分化細胞(對照組),10 μmol/L人工蛋白酶體抑制劑繼續(xù)作用對照組24 h建立了sPD蛋白酶體功能抑制SH-SY5Y細胞模型(實驗組)；對照組、實驗組蛋白質(zhì)樣品的差異膠內(nèi)電泳獲得了106個差異表達蛋白質(zhì),其中17個差異表達蛋白質(zhì)變化最明顯并且被指定為目的蛋白質(zhì)：實驗組蛋白質(zhì)樣品的制備膠二維電泳獲得了目標蛋白質(zhì)；蛋白質(zhì)胰蛋白酶酶解產(chǎn)物的基質(zhì)輔助激光解析電離-飛行時間質(zhì)譜獲得了目標蛋白質(zhì)的肽質(zhì)量指紋(或質(zhì)譜數(shù)據(jù)),質(zhì)譜數(shù)據(jù)在線檢索從SwissProt、NCBInr兩個蛋白質(zhì)數(shù)據(jù)庫中獲得了目標蛋白質(zhì)的17各候選蛋白質(zhì)；同源蛋白質(zhì)的生物信息學數(shù)據(jù)分析確認候選蛋白質(zhì)的生物學功能及其類別；實驗組基因轉(zhuǎn)錄物相對定量分析進一步驗證了其中的一個目標蛋白質(zhì)。[結(jié)果]首先,這17個目標蛋白質(zhì)的候選蛋白質(zhì)分別為：熱休克蛋白質(zhì)-27 (27-kDa heat shock protein, HSP-27)、熱休克蛋白質(zhì)-32 (heat shock protein 32, HSP-32)、葡萄糖調(diào)節(jié)蛋白質(zhì)-58 (58-kDa glucose regulated protein, GRP-58)、熱休克蛋白質(zhì)-70 (70-kDa heat shock protein, HSP-70)、熱休克關(guān)聯(lián)蛋白質(zhì)-70 (70-kDa heat shock cognate protein, HSC-70)、葡萄糖調(diào)節(jié)蛋白質(zhì)-75 (75-kDa glucose regulated protein, GRP-75)、X熱休克蛋白質(zhì)-105 (105-kDa heat shock protein, HSP-105)、氧調(diào)節(jié)蛋白質(zhì)-150 (150-kDa oxygen regulated protein, ORP-150)、鈣離子結(jié)合蛋白質(zhì)-1 (calcium-binding protein 1, CaBP-1)、CBP-50蛋白質(zhì)(CBP-50 protein, CBP-50)、熱休克蛋白質(zhì)-60 (60-kDa mitochondrial heat shock protein 60 mHSP-60),脯氨酰羥化酶-p多肽(prolyl-4-hydroxylase beta polypeptide, P4HB);應(yīng)激誘導磷蛋白質(zhì)-1 (stress induced phosphoprotein-1或stress inducible phosphoprotein-1, STIP-1)、14-3-3蛋白質(zhì)ε (14-3-3zeta,14-3-3ξ),含T-復合體多態(tài)-1蛋白質(zhì)p亞基(T-complex polypeptide 1 beta subunit, TCP-1β)、含T-復合體多肽-1蛋白質(zhì)ε亞基(T-complex polypeptide 1 epsilon subunit, TCP-1ε)、含纈酪肽蛋白質(zhì)(Valosin-containing protein, VCP);其次,它們被歸納為屬于如下4個分子伴侶蛋白質(zhì)類別：HSP-27、HSP-32、HSP-70、HSC-70、HSP-105、GRP-58、GRP-75、ORP150和VCP為9個伴侶蛋白質(zhì),P4HB、14-3-3ξ、CaBP-1和CBP-50為4個類伴侶蛋白質(zhì),STIP-1為共伴侶蛋白質(zhì),mHSP-60、TCP-1β和TCP-1ε為3個伴侶素。[結(jié)論]上述結(jié)果提示,HSP-70等17個分子伴侶蛋白質(zhì)在sPD病理機制UPS功能障礙通路中參與了蛋白質(zhì)分解應(yīng)激反應(yīng)。
[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，

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