【摘要】：[背景]在散發(fā)型帕金森病(sporadic Parkinson's, sPD)病理機(jī)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(dá)被認(rèn)為是蛋白質(zhì)分解應(yīng)激反應(yīng)的保護(hù)性反饋機(jī)制。[目的]為了揭示分子伴侶蛋白質(zhì)在sPD病理機(jī)制UPS功能障礙通路中的表達(dá)規(guī)律,本研究進(jìn)行了sPD蛋白酶體功能抑制SH-SY5Y細(xì)胞模型的分子伴侶蛋白質(zhì)組學(xué)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(xì)胞各72 h將SH-SY5Y細(xì)胞誘導(dǎo)為分化細(xì)胞(對(duì)照組),10 μmol/L人工蛋白酶體抑制劑繼續(xù)作用對(duì)照組24 h建立了sPD蛋白酶體功能抑制SH-SY5Y細(xì)胞模型(實(shí)驗(yàn)組)；對(duì)照組、實(shí)驗(yàn)組蛋白質(zhì)樣品的差異膠內(nèi)電泳獲得了106個(gè)差異表達(dá)蛋白質(zhì),其中17個(gè)差異表達(dá)蛋白質(zhì)變化最明顯并且被指定為目的蛋白質(zhì)：實(shí)驗(yàn)組蛋白質(zhì)樣品的制備膠二維電泳獲得了目標(biāo)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(shí)間質(zhì)譜獲得了目標(biāo)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(gè)蛋白質(zhì)數(shù)據(jù)庫(kù)中獲得了目標(biāo)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(xué)數(shù)據(jù)分析確認(rèn)候選蛋白質(zhì)的生物學(xué)功能及其類(lèi)別；實(shí)驗(yàn)組基因轉(zhuǎn)錄物相對(duì)定量分析進(jìn)一步驗(yàn)證了其中的一個(gè)目標(biāo)蛋白質(zhì)。[結(jié)果]首先,這17個(gè)目標(biāo)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(dǎo)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(fù)合體多態(tài)-1蛋白質(zhì)p亞基(T-complex polypeptide 1 beta subunit, TCP-1β)、含T-復(fù)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(gè)分子伴侶蛋白質(zhì)類(lèi)別：HSP-27、HSP-32、HSP-70、HSC-70、HSP-105、GRP-58、GRP-75、ORP150和VCP為9個(gè)伴侶蛋白質(zhì),P4HB、14-3-3ξ、CaBP-1和CBP-50為4個(gè)類(lèi)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(gè)伴侶素。[結(jié)論]上述結(jié)果提示,HSP-70等17個(gè)分子伴侶蛋白質(zhì)在sPD病理機(jī)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，

本文編號(hào)：2480996