翟穎真　丁　笛 　侯陳建 　陶麗麗 　劉秀萍 ,3 △

(1復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院病理學(xué)系　上?！?00032; 2復(fù)旦大學(xué)附屬中山醫(yī)院病理科　上?！?00032;3復(fù)旦大學(xué)附屬上海市第五人民醫(yī)院病理科　上海　200240; 4北京大學(xué)深圳醫(yī)院病理科　深圳　518000)

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活化的大鼠肝星狀細(xì)胞賴氨酸乙?；鞍踪|(zhì)組學(xué)分析

翟穎真1丁笛2侯陳建1陶麗麗4劉秀萍1,3 △

(1復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院病理學(xué)系上海200032;2復(fù)旦大學(xué)附屬中山醫(yī)院病理科上海200032;3復(fù)旦大學(xué)附屬上海市第五人民醫(yī)院病理科上海200240;4北京大學(xué)深圳醫(yī)院病理科深圳518000)

目的對活化的大鼠肝星狀細(xì)胞(hepatic stellate cells,HSCs)進(jìn)行賴氨酸乙?；?lysine acetylation,Kac)蛋白質(zhì)組學(xué)分析。方法于清潔級雄性SD 大鼠中提取并培養(yǎng)原代HSCs,分為對照組(培養(yǎng)<2天)和活化組(培養(yǎng)≥10天)。對兩組細(xì)胞進(jìn)行裂解,iTRAQ/TMT標(biāo)記后的成對肽段樣品采用乙?；贵w進(jìn)行親和富集,應(yīng)用高分辨率LC-MS/MS技術(shù)進(jìn)行質(zhì)譜鑒定。最后對質(zhì)譜鑒定的乙?；鞍踪|(zhì)進(jìn)行生物信息學(xué)分析。結(jié)果應(yīng)用LC-MS/MS技術(shù)、GO注釋和KEGG通路等生物信息學(xué)分析方法,共確定309種蛋白質(zhì)中的470個(gè)Kac位點(diǎn)。對其中150個(gè)蛋白質(zhì)的231個(gè)Kac位點(diǎn)進(jìn)行肽段定量研究,與靜息的HSCs相比,在活化的HSCs中有109個(gè)蛋白質(zhì)的乙酰化肽段數(shù)目增加,18個(gè)蛋白質(zhì)的乙?；亩螖?shù)目減少,并涉及多種GO分類和KEGG通路。乙酰化蛋白質(zhì)廣泛定位于胞質(zhì)(38.1%)和線粒體(30.2%)。基于GO富集的聚類分析顯示Kac位點(diǎn)主要與能量代謝相關(guān);基于KEGG通路的聚類分析顯示這些蛋白質(zhì)廣泛參與三羧酸循環(huán),與線粒體關(guān)系密切。結(jié)論首次對HSCs的Kac蛋白質(zhì)進(jìn)行質(zhì)譜分析,并對發(fā)生乙?；牡鞍踪|(zhì)位點(diǎn)進(jìn)行量化鑒定,為揭示蛋白質(zhì)乙酰化在HSCs活化過程中的作用提供了一定的基礎(chǔ),并為抑制肝纖維化提供了潛在的分子靶標(biāo)。

肝星狀細(xì)胞;賴氨酸乙酰化;LC-MS/MS;翻譯后修飾;大鼠

賴氨酸乙?；?lysine acetylation,Kac)是一種蛋白質(zhì)中普遍存在、可逆的翻譯后修飾(posttranslational modification,PTM),首次于組蛋白中發(fā)現(xiàn)。之前對于Kac的研究主要集中于組蛋白,已經(jīng)確定的多種乙?；揎椆δ芡瑯釉醋杂诮M蛋白研究。組蛋白在乙?；D(zhuǎn)移酶和去乙酰基酶作用下,產(chǎn)生不同的乙?；揎棽⒄{(diào)節(jié)多種生物學(xué)功能[1],包括基因表達(dá)調(diào)節(jié)[2]、染色體重構(gòu)[3-4]及細(xì)胞壽命[5]。目前,越來越多的研究已發(fā)現(xiàn)非組蛋白同樣具有Kac現(xiàn)象,并調(diào)節(jié)多種重要的細(xì)胞生理功能[6-7]。肝星狀細(xì)胞(hepatic stellate cells,HSCs)活化是肝纖維化發(fā)生的中心環(huán)節(jié),經(jīng)常作為潛在的治療靶點(diǎn)[8-9]。最近的研究表明,蛋白質(zhì)乙?；揎椏赡茉贖SCs活化過程中起到重要作用[10-12];不同類型的去乙酰基酶抑制劑(如曲古霉素A[11]、丙戊酸[10]及Givinostat[13])可以抑制穩(wěn)定狀態(tài)的HSCs向纖維母細(xì)胞的轉(zhuǎn)化,并且可以抑制平滑肌肌動(dòng)蛋白和Ⅰ型膠原蛋白的表達(dá)。

目前蛋白質(zhì)組學(xué)注釋多通過生物信息學(xué)預(yù)測得出,對相關(guān)蛋白質(zhì)的注釋可能不全面或存在遺漏,而且尚未見HSCs乙?；鞍踪|(zhì)組學(xué)的相關(guān)研究。本研究以HSCs為研究對象,應(yīng)用同位素標(biāo)記的相對和絕對定量/串聯(lián)質(zhì)譜標(biāo)簽(isobaric tag for relative absolute quantitation/tandem mass tags,iTRAQ/TMT)技術(shù),結(jié)合液相二級質(zhì)譜(liquid chromatography-tandem mass spectrometry,LC-MS/MS)進(jìn)行高通量Kac質(zhì)譜分析。為進(jìn)一步探析蛋白質(zhì)乙?；贖SCs激活過程中是否存在相關(guān)作用提供基礎(chǔ),同時(shí)也為進(jìn)一步完善HSCs蛋白組學(xué)信息及挖掘潛在肝纖維化治療靶點(diǎn)提供有價(jià)值的數(shù)據(jù)。

材 料 和 方 法

大鼠原代HSCs分離取成年雄性SD大鼠,體質(zhì)量250～350 g,由復(fù)旦大學(xué)實(shí)驗(yàn)動(dòng)物部提供。通過鏈蛋白酶、膠原酶兩步體內(nèi)門靜脈灌注,消化大鼠肝臟組織,經(jīng)Histodenz密度梯度離心分離HSCs。臺盼藍(lán)拒染實(shí)驗(yàn)鑒定細(xì)胞活力,α-SMA、Ⅰ型膠原蛋白、結(jié)蛋白免疫熒光細(xì)胞化學(xué)染色(Desmin染色)鑒定細(xì)胞純度。HSCs得率為6.25×106個(gè)/g。細(xì)胞存活率在90%以上。光鏡(×200)下觀察,Desmin染色陽性細(xì)胞原代培養(yǎng)達(dá)90%以上,傳代培養(yǎng)達(dá)95%以上。

TMT標(biāo)記樣本細(xì)胞經(jīng)蛋白質(zhì)提取及胰蛋白酶消化后,進(jìn)行TMT(美國Thermo公司),最小肽段長度為7個(gè)氨基酸。在0.5 mol/L TEAB中,將來自于兩份樣本中的肽段與混合的TMT反應(yīng)物重新組合(不同的標(biāo)記物標(biāo)記不同樣本),室溫孵育1 h。標(biāo)記后合并兩份肽段混合物樣本,室溫孵育2 h,最后用Strata X C18 SPE柱 (美國Phenomenex公司)萃取脫鹽,采用真空離心法干燥。

HPLC分餾與親和富集通過Agilent 300 Extend C18柱(顆粒直徑5 μm,柱直徑4.6 mm,長度250 mm)的高pH反相HPLC,將干燥的肽段分餾為不同的小片段。為了富集Kac肽段,將胰蛋白酶分解形成的肽段溶入NETM緩沖液(含100 mmol/L NaCl、1 mmol/L EDTA、50 mmol/L Tris-HCl和0.5% NP-40,pH=8.0),并用預(yù)洗抗體珠(美國PTM Biolabs公司)孵育,置于低速搖床,4 ℃過夜。將抗體珠用NETN緩沖液沖洗4次,ddH2O沖洗2次,用0.1%TFA將綁定的肽段從抗體珠上洗脫。洗脫的肽段進(jìn)行組合,真空干燥。最終得到的多肽用C18 ZipTips(美國Millipore公司)進(jìn)行清洗,經(jīng)LC-MS/MS分析。

LC-MS/MS分析肽段溶解于0.1%甲酸,直接加樣于反相前置柱(Acclaim PepMap 100,美國Thermo公司)。應(yīng)用反相分析柱(Acclaim PepMap RSLC,美國Thermo公司)進(jìn)行肽段分離。經(jīng)溶劑B(0.1% 甲酸+98% 乙腈)在梯度6%～22%持續(xù)24 min,22%～36%持續(xù)8 min,之后4 min內(nèi)攀升到80%,并在80%維持至少4 min。整個(gè)過程于EASY-nLC 1000 超高相液相色譜(UPLC)系統(tǒng)中進(jìn)行,恒定流速為280 L/min(0 ℃,1個(gè)標(biāo)準(zhǔn)大氣壓)。

采用Q Exactive-TMplus hybrid quadrupole-Orbitrap質(zhì)譜儀(美國Thermo公司)對分離出的肽段進(jìn)行分析。

MS/MS數(shù)據(jù)分析MS/MS數(shù)據(jù)結(jié)果采用Max Quant分析軟件與Andromeda 搜索引擎(v.1.4.1.2)聯(lián)合分析。串聯(lián)質(zhì)譜結(jié)合反向誘餌數(shù)據(jù)庫對大鼠數(shù)據(jù)進(jìn)行搜索。胰蛋白酶/P規(guī)定為切斷酵素最高丟失值為3,每個(gè)肽段具有4個(gè)修飾位點(diǎn)和5個(gè)電荷。蛋白質(zhì)、肽段、修飾位點(diǎn)的錯(cuò)誤發(fā)現(xiàn)率(false discovery rate,FDR)規(guī)定為1%。最小肽段長度設(shè)置為7。采用TMT-6-plex定量評值方法,Max Quant軟件中的其他參數(shù)設(shè)為默認(rèn)值,位點(diǎn)定位可能性設(shè)為>0.75。

生物信息學(xué)分析基因本體論(gene ontology,GO)注釋應(yīng)用于富集的蛋白質(zhì)種類分析。主要從細(xì)胞組分、分子功能和生物學(xué)過程3個(gè)方面進(jìn)行分類注釋。對于已經(jīng)確定而未被UniProt-GOA 數(shù)據(jù)庫注釋的蛋白質(zhì)則應(yīng)用Inter ProScan軟件,基于蛋白質(zhì)的氨基酸序列對齊方法進(jìn)行GO注釋。Kac蛋白質(zhì)的京都基因和基因組百科全書(Kyoto Encyclopedia of Genes and Genomes,KEGG)通路繪制應(yīng)用KEGG線上服務(wù)工具KAAS進(jìn)行。Fisher′s 修正P<0.05為有意義富集。采用亞細(xì)胞定位預(yù)測軟件wolfpsort預(yù)測亞細(xì)胞結(jié)構(gòu)定位。除此之外,在GO注釋、KEGG通路與蛋白質(zhì)結(jié)構(gòu)域分析的基礎(chǔ)上各自進(jìn)行再次功能富集,最后在富集基礎(chǔ)上進(jìn)行聚類分析。

結(jié)　　　果

HSCs中Kac蛋白質(zhì)的確定和分析質(zhì)譜分析可快速確定并精確量化上千種蛋白質(zhì),并可分析HSCs激活過程中蛋白質(zhì)修飾的動(dòng)態(tài)變化。聚苯乙烯皿中培養(yǎng)的原代HSCs可自行活化,并出現(xiàn)纖維母細(xì)胞的一些生物學(xué)特征,可作為一種體外HSCs活化模型[14]。由于HSCs分離比較困難,且需要大量的蛋白質(zhì)用以蛋白質(zhì)組學(xué)對比分析,因此對于HSCs活化細(xì)胞蛋白質(zhì)組學(xué)的研究還非常有限。

現(xiàn)有的靜息與活化的HSCs對比性蛋白質(zhì)質(zhì)譜分析于2000年完成。Kristensen等[15]于活化的HSCs中確定了43種表達(dá)改變的蛋白質(zhì),并首次提供了HSCs蛋白質(zhì)組學(xué)分析,包含150種蛋白質(zhì)。隨著穩(wěn)定同位素標(biāo)記和蛋白質(zhì)鑒定技術(shù)的發(fā)展,JI等[16]于2014年對HSCs活化過程中蛋白質(zhì)組學(xué)進(jìn)行了研究,共測定2 417種蛋白質(zhì),并對其中2 322種蛋白質(zhì)進(jìn)行了量化。但至今仍缺乏HSCs活化過程中乙?；鞍踪|(zhì)修飾研究。本研究通過乙酰化肽段的選擇性富集、超高分辨質(zhì)譜(Orbitrap)和生物信息學(xué)分析鑒定乙酰化蛋白質(zhì)。蛋白質(zhì)乙酰化分析的主要環(huán)節(jié)為(圖1):(1) 由大鼠肝臟提取的原代HSCs制備一組活化與未活化HSCs對照細(xì)胞樣本,并用胰蛋白酶進(jìn)行消化,經(jīng)iTTRAQ/TMT標(biāo)記,應(yīng)用抗乙?；贵w進(jìn)行肽段分離;(2) 將分離后的乙酰化肽段經(jīng)HPLC-MS/MS進(jìn)行質(zhì)譜分析;(3) 乙?；稽c(diǎn)先通過序列對比,再根據(jù)蛋白質(zhì)功能、所關(guān)聯(lián)的通路及相互作用網(wǎng)絡(luò)提示的蛋白質(zhì)乙酰化特質(zhì)來確定。

Whole-cell extracts were prepared from primary and activated HSCs.Then immune-affinity enrichment and high-sensitivity mass spectrometry were followed by bioinformatical analysis.

圖1HSCs中Kac蛋白質(zhì)肽段鑒定流程圖

Fig 1Process chart of identifying Kac peptides in HSCs

本研究共確定了470個(gè)乙酰化位點(diǎn),對應(yīng)于309個(gè)不同蛋白質(zhì)。并對其中的150種蛋白質(zhì)進(jìn)行了量化分析,最終確定109種蛋白質(zhì)乙?；亩螖?shù)目增多(活化/靜息>1.3),18種蛋白質(zhì)乙酰化肽段數(shù)目減少(活化/靜息<0.77)。平均每個(gè)HSCs乙?；鞍踪|(zhì)包含約1.5個(gè)乙酰化位點(diǎn)。乙酰化位點(diǎn)的數(shù)量分布顯示(圖2A):超過70%的蛋白質(zhì)包含1個(gè)乙?；稽c(diǎn),約30%的蛋白質(zhì)在多個(gè)賴氨酸位點(diǎn)出現(xiàn)乙?；揎?。另有34個(gè)存在高度乙?；牡鞍踪|(zhì)擁有3個(gè)以上的乙?；稽c(diǎn),包括組蛋白 H2A,其乙?；稽c(diǎn)位于第4、7、11個(gè)賴氨酸位點(diǎn)(圖2 B),且在兩個(gè)結(jié)構(gòu)域范圍內(nèi)。

圖3 A選取了蛋白質(zhì)乙?；亩螖?shù)目顯著增多或減少的部分乙酰化蛋白質(zhì),可見每個(gè)蛋白質(zhì)的乙?；稽c(diǎn)個(gè)數(shù)及乙?；亩涡蛄小D3B為HSCs蛋白質(zhì)中新確定的具有代表性的乙?；?xì)絲蛋白(filamin-α)的MS/MS質(zhì)譜圖,包含2個(gè)乙酰化位點(diǎn),分別為K299和K700。

我們同樣對所確定的乙?；鞍踪|(zhì)亞細(xì)胞結(jié)構(gòu)定位進(jìn)行了分析,結(jié)果顯示,乙?；亩卧龆嗟牡鞍踪|(zhì)(圖4 A)、乙?；亩螠p少的蛋白質(zhì)(圖4B)及乙酰化總蛋白質(zhì)(圖4C)都與胞質(zhì)的聯(lián)系最為密切,而且50%乙?；亩螠p少的蛋白質(zhì)都位于胞質(zhì)中。與乙?；亩螠p少蛋白質(zhì)相比,乙?；亩卧龆嗟牡鞍踪|(zhì)更多集中于線粒體(33% vs.11%)。與對照組相比,部分乙?；亩卧龆嗟牡鞍踪|(zhì)位于質(zhì)膜上,如Q5BJZ3、D3ZXD2和Q62638等;而乙?；亩螠p少的蛋白質(zhì)則未出現(xiàn)在質(zhì)膜上。

A:Number of Kac sites identified per protein;B:Annotation of Kac sites in Histone H2A.Z protein.

圖2HSCs中蛋白質(zhì)的乙?；稽c(diǎn)分析

Fig 2Acetylation sites analysis of the proteins in HSCs

乙酰化蛋白質(zhì)的功能富集分析基于GO注釋,我們從細(xì)胞組分、分子功能和生物學(xué)過程對309種乙?；鞍踪|(zhì)進(jìn)行了分類分析。結(jié)果顯示,乙酰化蛋白質(zhì)主要分布于細(xì)胞亞級(16.2%)、細(xì)胞部分亞級(16.2%)及細(xì)胞器亞級(15.2%)(圖5)。分子功能注釋顯示,38.5%的蛋白質(zhì)與綁定功能相關(guān),26.9%的蛋白質(zhì)與催化活性相關(guān),還有15.4%的蛋白質(zhì)與結(jié)構(gòu)分子活性相關(guān)。生物學(xué)過程分析顯示,18.3%的乙?；鞍踪|(zhì)參與細(xì)胞學(xué)過程,16.7%的蛋白質(zhì)與單個(gè)細(xì)胞器過程相關(guān),13.3%的蛋白質(zhì)與代謝過程相關(guān)。GO富集分析表明HSCs中乙?；鞍踪|(zhì)分布廣泛。

根據(jù)乙?；亩螖?shù)目變化高低對量化的乙酰化蛋白進(jìn)行分類:Q1～Q4。GO富集基礎(chǔ)上的聚類包括之前進(jìn)行的細(xì)胞組分、生物學(xué)過程和分子功能分析(圖6)。我們發(fā)現(xiàn)大部分乙?；鞍踪|(zhì)定位于線粒體,如GO富集的線粒體,線粒體膜、線粒體內(nèi)膜,線粒體基質(zhì)和線粒體被膜,而這些都是高度富集于Q4的乙?；亩螖?shù)目顯著增高的蛋白。線粒體是細(xì)胞代謝和能量產(chǎn)生過程中具有中心作用的一個(gè)細(xì)胞器[17],具有氧化糖類、脂肪酸和氨基酸的能力,并可以通過內(nèi)膜形成H+梯度來產(chǎn)生ATP。線粒體同時(shí)在許多重要的細(xì)胞學(xué)過程中具有中心作用,包括合成代謝過程中前體的提供及協(xié)調(diào)核-線粒體交流。此外,乙?；禺愋晕稽c(diǎn)足以改變依賴ATP的核小體的動(dòng)力學(xué)以及染色體重組[18-19]?，F(xiàn)今許多對于線粒體的研究已經(jīng)將蛋白質(zhì)賴氨酸N-端的乙酰化作為其修飾蛋白質(zhì)功能的主要調(diào)節(jié)機(jī)制[20]。

A:Identification of the modification sites of HSCs;B:MS/MS spectra of acetylpeptides from filamin-α (C0JPT7).Acetylpeptide AYGPGIEPTGNMVK(ac)K with a Kac site at K299.Acetylpeptide TGVAINKPAEFTVDAK(ac)HAGK with a Kac site at K700.

圖3HSCs中蛋白質(zhì)的乙?；畔⒓癕S/MS質(zhì)譜分析

Fig 3Acetylation information and MS/MS spectra of proteins in HSCs

細(xì)胞中蛋白質(zhì)以代謝蛋白質(zhì)為主,而這些蛋白質(zhì)多屬于線粒體蛋白質(zhì)[21-22]。這一結(jié)果提示線粒體中的乙酰化蛋白質(zhì)在HSCs活化過程中可能具有相關(guān)作用。與乙?；亩卧龆嗟牡鞍踪|(zhì)相反,乙?；亩螠p少的蛋白質(zhì)集中定位于細(xì)胞核。乙?；鞍踪|(zhì)所涉及的生物學(xué)過程如圖6所示。乙?；亩卧龆嗟牡鞍踪|(zhì)(Q3)高度富集于各種代謝生物學(xué)過程,包括二羧酸代謝、單個(gè)有機(jī)體代謝、酮酸代謝、三羧酸代謝及有機(jī)酸代謝等,這或許可以揭示激活和代謝調(diào)節(jié)之間的相互作用。另一方面,乙?；亩螠p少的蛋白質(zhì)也涉及多種調(diào)節(jié)過程,如細(xì)胞增殖的正向調(diào)節(jié)、蛋白質(zhì)磷酸化調(diào)節(jié)及磷酸化代謝調(diào)節(jié)過程等。乙?；亩卧龆嗟牡鞍踪|(zhì)(Q4)分子功能GO富集聚類比較集中,主要是肽酶活性。

代謝信號通路中的乙?；饔肒EGG通路富集顯示乙?；鞍踪|(zhì)涉及多種信號通路(圖7)。許多參與中心性能量代謝通路和氨基酸代謝通路的蛋白質(zhì)存在乙?；揎?。具體來說,使用KEGG通路將這些蛋白質(zhì)富集于相關(guān)的信號通路(圖7),可見乙?；鞍踪|(zhì)存在于多種蛋白質(zhì)和氨基酸的生物合成、三羧酸循環(huán)、碳代謝、亨廷頓病、酒精性中毒及黏著斑信號通路。

A:Proteins whose acetylpeptides were increased;B:Proteins whose acetylpeptides were reduced;C:All acetylated proteins.

圖4乙?；鞍踪|(zhì)的亞細(xì)胞結(jié)構(gòu)定位

Fig 4Subcellular location classification of acetylated proteins

The cellular components,molecular functions and biological processes of the acetylated proteins in HSCs were analyzed by gene ontology analysis.The column number means acetylated proteins (the ratio of acetylated proteins).

圖5乙?；鞍椎腉O富集分析

Fig 5GO enrichment analysis of acetylated proteins

HSCs激活時(shí)需要大量的能量來維持其細(xì)胞增殖、細(xì)胞外基質(zhì)分泌和細(xì)胞收縮等生物學(xué)行為[23]。三羧酸循環(huán)途徑是發(fā)生在線粒體中的細(xì)胞氧化供能的最終途徑,也是糖、脂肪和氨基酸代謝的重要途徑[24]。包括三羧酸循環(huán)在內(nèi)的新陳代謝途徑的改變是許多疾病發(fā)展的重要特點(diǎn)?；蚪M學(xué)研究表明:三羧酸循環(huán)中的重要酶——延胡索酸水化酶[25]和異檸檬酸脫氫酶(isocitrate dehydrogenase,IDH)[26]及其代謝產(chǎn)物與腫瘤發(fā)生相關(guān)。

乙?；亩嗡缴险{(diào)的多種酶參與了三羧酸循環(huán)(圖8)。包括IDH、丙酮酸脫氫酶、烏頭酸水合酶、檸檬酸合酶(citrate synthase,CS)等,共13個(gè)。IDH是TCA循環(huán)中第2個(gè)調(diào)節(jié)酶,IDH1在線粒體和胞質(zhì)中都存在,本研究發(fā)現(xiàn)其發(fā)生了乙?；⑶乙阴；亩螖?shù)目明顯升高(FDR:2.63735E-8,Fold:4.40),而IDH2只存在于線粒體中,乙?；亩螖?shù)目增高(FDR:2.65831E-20,Fold:2.15)。IDH1和IDH2發(fā)生乙?；乙阴；亩螖?shù)目增加,說明乙?；腎DH可能參與了HSCs的激活。

有研究表明CS在卵巢癌細(xì)胞中高度表達(dá),而CS基因沉默能夠抑制細(xì)胞增殖并降低細(xì)胞侵襲和遷移能力,CS可能是卵巢癌發(fā)生發(fā)展的潛在調(diào)控因素[27]。CS發(fā)生了顯著的乙?；亩螖?shù)目增加(圖8),進(jìn)而可能影響下一步的脂肪酸代謝及氨基酸代謝。

Kac sites belong to Q1 (0

圖6量化的Kac位點(diǎn)的GO富集聚類分析

Fig 6GO enrichment-based clustering analysis for quantified Kac sites

圖7KEGG通路富集注釋

Fig 7KEGG pathway analysis

經(jīng)KEGG通路富集結(jié)果可見,在發(fā)生乙?；牡鞍字?乙?；亩卧龆嗟亩喾N蛋白質(zhì),如超氧化物歧化酶、連接蛋白3、連接蛋白5和線粒體通透性轉(zhuǎn)換孔,同樣參與了多種代謝信號通路,包括氧自由基信號通路、P53信號通路及細(xì)胞凋亡等。

討　　　　論

本研究首次報(bào)道了HSCs活化過程中乙?；鞍椎馁|(zhì)信息數(shù)據(jù)。Kac是一種重要的PTM,可以影響多種代謝通路。本研究結(jié)果采用高靈敏度蛋白組學(xué)方法確定了HSCs激活過程中發(fā)生乙?；牡鞍踪|(zhì)Kac位點(diǎn),最終于309種蛋白質(zhì)中確定了470個(gè)Kac位點(diǎn),每種蛋白質(zhì)所對應(yīng)的Kac位點(diǎn)數(shù)目不同;并對其中的150種蛋白質(zhì)進(jìn)行了量化分析。結(jié)果顯示有109種蛋白質(zhì)的乙酰化肽段數(shù)目顯著增多,18種蛋白質(zhì)的乙?；亩螖?shù)目減少;進(jìn)一步的功能分析顯示乙酰化蛋白質(zhì)涉及多種特定的功能和生物學(xué)過程。在細(xì)胞組分富集方面,多種乙酰化蛋白質(zhì)與線粒體相關(guān);結(jié)合生物學(xué)過程分析,乙酰化蛋白質(zhì)涉及大部分能量代謝,提示乙?；揎椩谄渌鞍踪|(zhì)生成和氨基酸生成過程中可能也有調(diào)節(jié)作用。研究顯示,主要代謝通路中的蛋白質(zhì)都存在乙?；揎?這些通路包括糖酵解、脂肪酸氧化、三羧酸循環(huán)和氨基酸生成,都是HSCs能量代謝網(wǎng)絡(luò)中非常重要的組分。

本研究是在前期CCAAT增強(qiáng)子結(jié)合蛋白(CCAAT enhancer binding protein,C/EBP)-α乙?；饔玫难芯炕A(chǔ)上開展的。C/EBP-α對脂肪細(xì)胞的分化具有重要的調(diào)節(jié)作用,經(jīng)乙?；种苿┨幚砗蟮腍SCs中C/EBP-α蛋白質(zhì)水平升高,HSCs凋亡增加,平滑肌肌動(dòng)蛋白與Ⅰ 型膠原蛋白水平降低。干擾C/EBP-α乙?；稽c(diǎn),經(jīng)TSA處理后,C/EBP-α蛋白質(zhì)水平增加現(xiàn)象消失。提示蛋白質(zhì)乙?；贖SCs中具有一定的調(diào)節(jié)作用,進(jìn)而對HSCs激活過程中的乙酰化作用進(jìn)行了質(zhì)譜鑒定分析。

The identified lysine-acetylated proteins that acetyl-peptides increased significantly were shown in red,and the proteins in light turquoise were background.

圖8三羧酸循環(huán)中涉及的乙?；割惣跋嚓P(guān)通路

Fig 8Acetylate metabolicenzymes and related pathways in TCA cycle

Kac是一種保守的PTM,可以連接Ac-CoA代謝和細(xì)胞信號傳導(dǎo)[28-30]。線粒體中的乙酰輔酶A合成酶2(Acs2)是一種催化乙酸鹽轉(zhuǎn)化成為Ac-CoA的酶類,參與調(diào)節(jié)脂肪酸代謝過程,Ac-CoA水平能夠根據(jù)代謝狀態(tài)的變化而波動(dòng)[31]。本研究證實(shí)了激活的HSCs中Acs2存在乙?；揎棽橛幸阴；亩嗡降纳?而同時(shí)Acs2的酶活性也受到賴氨酸琥珀?；恼{(diào)節(jié)[32]。乙酰化修飾是與賴氨酸琥珀?；揎椣嗷ジ偁幍?這說明在蛋白質(zhì)的乙酰化與琥珀?；g存在著串?dāng)_現(xiàn)象,兩者相互作用對細(xì)胞的蛋白質(zhì)活性與代謝通路進(jìn)行調(diào)節(jié)

在本研究中,黏著斑的幾個(gè)組成分子(filamin-α、ILK和actin)同樣發(fā)生了乙?；?這可能與活化HSCs產(chǎn)生ECM增多并沉積相關(guān)。多個(gè)研究表明抑制某些蛋白質(zhì)的去乙?；饔每梢砸种苹罨疕SCs的增殖,并促進(jìn)其凋亡[11,13,33]。本研究只是對乙?；稽c(diǎn)和乙酰化肽段數(shù)目變化進(jìn)行了測定,而乙?；降淖兓€需要參考蛋白質(zhì)水平的變化。本研究首次在HSCs中探測了蛋白質(zhì)組學(xué)的乙?；稽c(diǎn)修飾,結(jié)果提示Kac修飾或許在HSCs活化過程中具有重要的調(diào)節(jié)作用。

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E-mail:xpliu1228@fudan.edu.cn

Proteomics analysis on lysine acetylation of proteins in actived hepatic stellate cells of rats

ZHAI Ying-zhen1, DING Di2, HOU Chen-jian1, TAO Li-li4, LIU Xiu-ping1,3△

(1Department of Pathology,School of Basic Medical Sciences,Fudan University,Shanghai 200032,China;2Department of Pathology,Zhongshan Hospital,Fudan University,Shanghai 200032,China;3Department of Pathology,The Fifth People's Hospital of Shanghai,Fudan University,Shanghai 200240,China;4Department of Pathology,Perking University Shenzhen Hospital,Shenzhen 518000,Guangdong Province,China)

ObjectiveTo investigate the lysine acetylation (Kac) of proteins in activated hepatic stellate cells (HSCs) of rats.MethodsPathogen-free male SD rats were used for the isolation of HSCs,and the primary cells were divided into control group (< 2 days of culture) and active group (≥10 days of culture).In the two groups,iTRAQ/TMT labeling were performed on the samples,and then the labelled proteins were followed by immune-affinity isolation of acetyl-peptides through anti-acetyl lysine antibodies.2D nano-LC-MS/MS was used to identify acetyl-proteome of HSCs.Finally,the quantitative analysis of acetylation sites and bioinformatics analysis were carried out.Results A total of 309 acetylated proteins with 470 Kac sites were identified in HSCs,of which 150 proteins with 231 Kac sites were quantified.Compared with quiescent HSCs,acetyl-peptides of 109 proteins showed significant increase in activated HSCs,and acetyl-peptides of 18 proteins showed significant reduce in activated HSCs.Bioinformatics and physiological analyses of detected proteins suggested that these acetylated proteins were widely located in cytosol and mitochondria,which contain 38.1% and 30%,respectively.GO enrichment-based clustering analysis indicated that Kac of these proteins may strictly regulate energy metabolism.In addition,KEGG pathway enrichment-based clustering analysis suggested that these protein significantly up-regulated in TCA cycle,which have intense relationship with mitochondria.ConclusionsKac of proteins in HSCs were investigated for the first time,and quantitative comparison of acetyl-proteome before and after HSCs activation were extensively studied in order to reveal the relationship between acetylation and HSCs activation,and to provide the potential target spots to inhibit the fibrosis of liver.

hepatic stellate cell;lysine acetylation;LC-MS/MS;post-translational modifications;rat

R364.2,R34

Adoi: 10.3969/j.issn.1672-8467.2016.05.002

2016-02-22;編輯:段佳)

*This work was supported by the National Natural Science Foundation of China (81272387,81470857),the Natural Science Foundation of Shanghai,China (134119b1100),the PhD Start-up Fund of Natural Science Foundation of Guangdong Province,China (2015A030310031) and the Research Project of Health and Family Planning Commission of Shenzhen Municipality (201401048).

國家自然科學(xué)基金(81272387,81470857);上海市自然科學(xué)基金(134119b1100);廣東省自然科學(xué)基金博士科研啟動(dòng)項(xiàng)目(2015A030310031);深圳市衛(wèi)計(jì)委課題(201401048)