王欣悅，石田培，趙志達(dá)，胡文萍，尚明玉，張莉

基于綿羊胚胎骨骼肌蛋白質(zhì)組學(xué)的PI3K-AKT信號(hào)通路分析

王欣悅，石田培，趙志達(dá)，胡文萍，尚明玉，張莉

（中國(guó)農(nóng)業(yè)科學(xué)院北京畜牧獸醫(yī)研究所，北京 100193）

【】綿羊是重要的經(jīng)濟(jì)動(dòng)物，其骨骼肌生長(zhǎng)發(fā)育與產(chǎn)肉性能密切相關(guān)。胚胎期是綿羊骨骼肌生長(zhǎng)發(fā)育的關(guān)鍵階段，挖掘分析綿羊胚胎骨骼肌蛋白質(zhì)組數(shù)據(jù)，為揭示綿羊肌肉發(fā)育重要時(shí)間節(jié)點(diǎn)、篩選綿羊胚胎骨骼肌生長(zhǎng)發(fā)育調(diào)控蛋白質(zhì)提供依據(jù)。本團(tuán)隊(duì)已對(duì)妊娠第85天、第105天和第135天的中國(guó)美利奴綿羊胚胎背最長(zhǎng)肌進(jìn)行串聯(lián)質(zhì)譜(tandem mass tag, TMT)蛋白質(zhì)定量，鑒定到1316種差異豐度蛋白質(zhì)?，F(xiàn)利用GO、KEGG和R等方法對(duì)這些差異豐度蛋白質(zhì)開(kāi)展聚類、功能注釋和通路分析等生物信息學(xué)分析?；谇捌谘芯拷Y(jié)果對(duì)差異豐度蛋白質(zhì)進(jìn)行R語(yǔ)言聚類，分析結(jié)果顯示，cluster 5類蛋白在胚胎骨骼肌第105天具有較高豐度。對(duì)cluster 5 蛋白進(jìn)行GO和KEGG富集分析發(fā)現(xiàn)，該類蛋白質(zhì)參與胞內(nèi)蛋白質(zhì)代謝過(guò)程，顯著富集于PI3K-AKT信號(hào)通路中，而在該信號(hào)通路中RAC-β絲氨酸/蘇氨酸蛋白激酶X1(AKT2)具有較高表達(dá)豐度。蛋白質(zhì)生物信息學(xué)結(jié)果表明，AKT2蛋白由481個(gè)氨基酸構(gòu)成，AKT2蛋白理論分子量為55.58kD，由66個(gè)帶正電荷的氨基酸殘基和72個(gè)帶負(fù)電荷的氨基酸殘基組成，理論等電點(diǎn)為6.08，親水性平均系數(shù)-0.454，屬于親水性蛋白。預(yù)測(cè)AKT2蛋白的481個(gè)氨基酸全部位于膜外，屬于膜受體蛋白。AKT2蛋白有12個(gè)N-端糖基化位點(diǎn)，71個(gè)磷酸化位點(diǎn)，與蛋白酶K相似度為99%，屬于蛋白酶催化亞基家族。綿羊胚胎骨骼肌蛋白質(zhì)組數(shù)據(jù)發(fā)現(xiàn)，第105天是綿羊胚胎骨骼肌纖維由增殖分化到增大增粗的轉(zhuǎn)折點(diǎn)，具有調(diào)控綿羊胚胎骨骼肌纖維生長(zhǎng)發(fā)育作用的PI3K-AKT信號(hào)通路在該節(jié)點(diǎn)顯著富集，AKT2是調(diào)控該信號(hào)通路的重要候選蛋白。綜上，本研究結(jié)果對(duì)揭示胚胎骨骼肌生長(zhǎng)發(fā)育及其調(diào)控分子機(jī)制具有重要理論指導(dǎo)意義。

綿羊()； 胚胎背最長(zhǎng)??； 蛋白質(zhì)組學(xué)；生物信息學(xué)分析

0 引言

【研究意義】胚胎期是綿羊骨骼肌生長(zhǎng)發(fā)育的重要時(shí)期。胚胎骨骼肌纖維在該時(shí)期發(fā)生增殖、分化、融合、增粗及成熟等生物過(guò)程，直接影響出生后骨骼肌的生長(zhǎng)[1]。因此，分析綿羊胚胎骨骼肌蛋白質(zhì)組學(xué)數(shù)據(jù)對(duì)闡明其生長(zhǎng)發(fā)育機(jī)制、篩選重要調(diào)控蛋白具有重要意義。【前人研究進(jìn)展】骨骼肌生長(zhǎng)發(fā)育研究一直備受關(guān)注，早期研究較多的，又稱，是一種肌肉生長(zhǎng)抑制素，對(duì)家畜肌肉生長(zhǎng)發(fā)育具有重要作用，其活性的喪失或降低會(huì)促進(jìn)動(dòng)物肌肉的發(fā)育。隨后，發(fā)現(xiàn)、、和肌源性調(diào)節(jié)因子（myogenic regulatory factors,）調(diào)控肌源性祖細(xì)胞、成肌細(xì)胞和肌纖維的生長(zhǎng)[2-4]。肌源性調(diào)節(jié)因子4（myogenic regulatory factor 4,）、肌源因子5（myogenic factor 5,）、肌源性分化因子（myogenic differentiation 1,）和肌細(xì)胞生成素（）是決定肌纖維最終分化的調(diào)控因子，Six家族蛋白質(zhì)是參與肌肉早期發(fā)育的轉(zhuǎn)錄因子，并在胚胎骨骼肌發(fā)育過(guò)程中發(fā)揮重要作用[5-6]。研究發(fā)現(xiàn)PI3K-AKT等信號(hào)通路與骨骼肌生長(zhǎng)發(fā)育密切相關(guān)，可以誘導(dǎo)肌肉的生成、調(diào)控基因的表達(dá)和成肌分化[7-8]。綿羊骨骼肌結(jié)構(gòu)特征研究表明，綿羊胚胎期第50天至第100天是肌纖維生長(zhǎng)發(fā)育的關(guān)鍵階段，此階段以后肌纖維的種類、數(shù)量和狀態(tài)不再發(fā)生變化[9-10]。【本研究切入點(diǎn)】蛋白組學(xué)研究技術(shù)為揭示家畜骨骼肌生長(zhǎng)發(fā)育提供了有效的技術(shù)手段。目前，蛋白質(zhì)組學(xué)技術(shù)廣泛應(yīng)用于豬、雞、牛和羊等動(dòng)物的骨骼肌生長(zhǎng)發(fā)育研究。通過(guò)該技術(shù)，研究人員已挖掘出一批調(diào)控骨骼肌生長(zhǎng)發(fā)育的關(guān)鍵蛋白[11-14]。但現(xiàn)階段，對(duì)綿羊胚胎骨骼肌蛋白質(zhì)組學(xué)的研究非常少。本團(tuán)隊(duì)前期利用TMT技術(shù)[15]對(duì)胚胎期第85天(D85N)、第105天(D105N)和第135天(D135N)的綿羊胚胎背最長(zhǎng)肌進(jìn)行蛋白質(zhì)定量研究，并鑒定到1316種差異豐度蛋白。本研究在此基礎(chǔ)上利用生物信息學(xué)技術(shù)對(duì)差異豐度蛋白質(zhì)進(jìn)行分析與篩選[16]?！緮M解決的關(guān)鍵問(wèn)題】通過(guò)進(jìn)一步分析差異豐度蛋白，揭示綿羊胚胎骨骼肌重要發(fā)育時(shí)間節(jié)點(diǎn)、挖掘發(fā)育相關(guān)調(diào)控蛋白，分析預(yù)測(cè)候選調(diào)控蛋白功能與結(jié)構(gòu)，為提高綿羊產(chǎn)肉性能、闡明綿羊胚胎骨骼肌生長(zhǎng)發(fā)育蛋白質(zhì)調(diào)控機(jī)制提供新思路。

1 材料與方法

試驗(yàn)于2018年7月在中國(guó)農(nóng)業(yè)科學(xué)院北京畜牧獸醫(yī)研究所完成。

1.1 前期綿羊胚胎骨骼肌蛋白質(zhì)組學(xué)分析

選擇體況良好、體重相近的中國(guó)美利奴綿羊成年母羊進(jìn)行同期發(fā)情與人工輸精。通過(guò)手術(shù)法采集妊娠D85N、D105N和D135N母羊的胚胎相同部位的背最長(zhǎng)肌(每階段3個(gè)生物學(xué)重復(fù))為樣品進(jìn)行TMT蛋白質(zhì)組學(xué)定量。通過(guò)對(duì)二級(jí)質(zhì)譜數(shù)據(jù)進(jìn)行Maxquant （v1.5.2.8）檢索（數(shù)據(jù)庫(kù)為NCBI Ovis aries Oar_v4.0 https://www.ncbi.nlm.nih.gov/genome/?term=Ovis+aries），設(shè)置D105N vs D85N、D135N vs D105N和D135N vs D85N 3個(gè)比較組進(jìn)行分析，共鑒定到1316種差異豐度蛋白質(zhì)。本試驗(yàn)將利用GO、KEGG和R等生物信息學(xué)數(shù)據(jù)分析軟件和平臺(tái)對(duì)這些差異豐度蛋白質(zhì)進(jìn)行分析和篩選。

1.2 差異豐度蛋白質(zhì)聚類分析

為進(jìn)一步分析差異豐度蛋白質(zhì)功能，篩選調(diào)控綿羊胚胎骨骼肌生長(zhǎng)發(fā)育候選蛋白，利用R中Mfuzz算法對(duì)前期定量到的1316種差異豐度蛋白進(jìn)行表達(dá)模式聚類分析[17-18]。

1.3 cluster 5蛋白GO和KEGG分析

利用InterProScan v.5.14-53.0（http://www.ebi.ac. uk/interpro/）、KAAS v.2.0（http://www. genome.jp/ kaas-bin/kaas_main）、KEGG mapper V2.5（http://www. kegg.jp/kegg/mapper.html）和Perl module（v.1.31 https://metacpan.org/pod/Text::NSP::Measures::2D::Fisher）等軟件對(duì)cluster 5蛋白進(jìn)行功能注釋及富集分析。

1.4 AKT2蛋白生物信息學(xué)分析

使用ExPASy網(wǎng)站的ProtParam（http://web.expasy. org/protparam/）預(yù)測(cè)和分析蛋白質(zhì)的分子量、等電點(diǎn)等物理參數(shù)[19]； TMHMM軟件（http://www.cbs.dtu.dk/ services/TMHMM-2.0/）對(duì)蛋白進(jìn)行跨膜區(qū)域預(yù)測(cè)[20]；使用Expasy（http://www.expasy.org/proteomics）軟件分析蛋白質(zhì)潛在的磷酸化和糖基化等位點(diǎn)[21-23]；利用Protein Homology/analogY Recognition Engine V 2.0（Phyre2，http://www.sbg.bio.ic.ac.uk/phyre2/html/page. cgi?id=index）預(yù)測(cè)蛋白質(zhì)的三級(jí)結(jié)構(gòu)。

2 結(jié)果

2.1 差異豐度蛋白質(zhì)表達(dá)模式聚類分析

R語(yǔ)言表達(dá)模式聚類分析表明，cluster 5蛋白在D105N時(shí)具有較高表達(dá)趨勢(shì)（圖1）。通過(guò)GO和KEGG分析發(fā)現(xiàn)，cluster 5蛋白質(zhì)參與胞內(nèi)蛋白質(zhì)代謝過(guò)程，并顯著富集于PI3K-AKT信號(hào)通路。同時(shí)，ATK2蛋白在PI3K-AKT信號(hào)通路中顯著上調(diào)（圖2-4）。

圖1 差異豐度蛋白質(zhì)表達(dá)模式聚類分析

Y軸：生物學(xué)過(guò)程Y: Enrichment index; X軸：富集指數(shù)X: Biology process

Y軸: 通路名稱Y: Pathway name; X軸: Fisher精確測(cè)試P值X: Fisher’s exact test p-value

紅色：顯著富集的上調(diào)基因 Red:Significant enrichment up-regulation gene

2.2 AKT2蛋白生物信息學(xué)分析

2.2.1 AKT2蛋白的理化性質(zhì) AKT2蛋白由481個(gè)氨基酸構(gòu)成。使用ProtParam在線軟件分析AKT2蛋白的理化性質(zhì)，推測(cè)其分子式為C2490H3865N673O724S24，分子量為55.58kD，理論等電點(diǎn)（pI）為6.08，半衰期均是30 h，不穩(wěn)定系數(shù)32.36，屬于穩(wěn)定蛋白。脂肪系數(shù)為76.61，親水性平均系數(shù)（GRAVY）是-0.454，屬于親水性蛋白。負(fù)電荷（Asp + Glu）氨基酸殘基72個(gè)，正電荷（Arg + Lys）氨基酸殘基66個(gè)。

2.2.2 AKT2蛋白跨膜結(jié)構(gòu)分析及其潛在N-糖基化、磷酸化位點(diǎn)預(yù)測(cè) TMHMM在線預(yù)測(cè)表明，AKT2蛋白的481個(gè)氨基酸沒(méi)有位于細(xì)胞膜上和膜內(nèi)，全部位于膜外，屬膜受體蛋白（圖5）。PSORT II Prediction分析結(jié)果表明AKT2蛋白主要在65.2% 胞質(zhì)、4.3% 線粒體、17.4%細(xì)胞核、4.3%分泌包囊、4.3%細(xì)胞支架。使用NetNGlyc 1.0 Server和NetPhos 3.1 Server分別預(yù)測(cè)AKT2蛋白N-端糖基化和磷酸化情況，結(jié)果顯示：AKT2蛋白有12個(gè)N-糖基化位點(diǎn)，71個(gè)磷酸化位點(diǎn)，其中26個(gè)絲氨酸（Ser）磷酸化位點(diǎn)、26個(gè)蘇氨酸（Thr）磷酸化位點(diǎn)、19個(gè)酪氨酸（Tyr）磷酸化位點(diǎn)（圖6）。

圖5 AKT2蛋白跨膜結(jié)構(gòu)分析

（a）AKT2 12個(gè)N-糖基化位點(diǎn)(a)12 N-glycosylation sites in AKT2;（b）AKT2 71個(gè)磷酸化位點(diǎn)(b)71 phosphorylation sites in AKT2

Fig .6 Prediction on glycosylation and phosphorylation sites of AKT2 protein

2.2.3 AKT2蛋白三級(jí)結(jié)構(gòu)預(yù)測(cè) PHYER2預(yù)測(cè)結(jié)果顯示，AKT2蛋白具有α-螺旋及無(wú)規(guī)則卷曲等結(jié)構(gòu)，三級(jí)結(jié)構(gòu)整體呈晶體結(jié)構(gòu)，與蛋白酶K相似度為99%，屬于蛋白酶催化亞基（圖7）。

圖7 AKT2蛋白3D結(jié)構(gòu)預(yù)測(cè)

3 討論

本文利用GO、KEGG和R語(yǔ)言等方法對(duì)差異豐度蛋白質(zhì)進(jìn)行聚類、功能注釋和通路富集等生物信息學(xué)分析，分析結(jié)果對(duì)揭示綿羊胚胎骨骼肌生長(zhǎng)發(fā)育關(guān)鍵窗口期、篩選調(diào)控蛋白具有重要意義。胚胎時(shí)期骨骼肌大部分由生肌節(jié)中的肌肉前體細(xì)胞發(fā)育而來(lái)，這些肌肉前體細(xì)胞會(huì)在初級(jí)生肌節(jié)中分化成單核肌肉細(xì)胞，初級(jí)生肌節(jié)最終生成脊椎動(dòng)物早期的肌肉組織[24-25]。前期研究表明，綿羊胚胎骨骼肌纖維在胚胎期第85天至第105天增殖分化，在第105天至第135天增大增粗，而這些差異豐度蛋白質(zhì)主要富集于能夠調(diào)控肌纖維發(fā)生生長(zhǎng)的代謝及氧化磷酸化等信號(hào)通路[8, 26-30]。本研究中，綿羊胚胎骨骼肌蛋白質(zhì)組學(xué)數(shù)據(jù)R語(yǔ)言分析發(fā)現(xiàn)，cluster 5蛋白在胚胎發(fā)育第105天具有較高表達(dá)豐度（圖1）。相關(guān)文獻(xiàn)報(bào)道綿羊胚胎骨骼肌纖維在胚胎期第50天至第100天基本發(fā)育完成，并在第100天左右開(kāi)始分化[31]。因此，初步判斷D105N是調(diào)控綿羊胚胎骨骼肌發(fā)育轉(zhuǎn)折點(diǎn)。

KEGG分析發(fā)現(xiàn)，cluster 5蛋白質(zhì)在PI3K-AKT信號(hào)通路中顯著富集。由此推斷，PI3K-AKT信號(hào)通路可能對(duì)胚胎時(shí)期骨骼肌發(fā)育轉(zhuǎn)折及調(diào)控具有重要的作用。PI3K-AKT信號(hào)通路參與骨骼肌生長(zhǎng)發(fā)育，能夠調(diào)控細(xì)胞周期、細(xì)胞凋亡和蛋白質(zhì)合成等生物過(guò)程[32]。研究表明，PI3K-AKT信號(hào)通路能夠調(diào)控肌漿蛋白形成，促進(jìn)肌肉分化和肥大[33]。本研究發(fā)現(xiàn)PI3K-AKT信號(hào)通路在綿羊胚胎骨骼肌發(fā)育轉(zhuǎn)折過(guò)程中具有重要作用，AKT作為第二信使在該通路中扮演重要的角色。PI3K-AKT信號(hào)通路下游的轉(zhuǎn)導(dǎo)因子AKT/PKB在調(diào)節(jié)個(gè)體發(fā)育、生長(zhǎng)和細(xì)胞存活過(guò)程中發(fā)揮著重要作用[34]。ATK是一種保守的絲/蘇氨酸蛋白激酶，可以調(diào)控動(dòng)物胚胎發(fā)育及幼體生長(zhǎng)，而ATK2是ATK的不同亞基也具有相同作用[35]。在正常生理?xiàng)l件下，PI3K-AKT信號(hào)通路由受體酪氨酸激酶（RTK）激活，并通過(guò)活化PI3K誘導(dǎo)PIP3激活A(yù)KT，上調(diào)下游靶基因從而調(diào)節(jié)細(xì)胞周期及分化。本研究中，RAC-β絲/蘇氨酸蛋白激酶X1（ATK2）顯著富集于PI3K-AKT信號(hào)通路，成肌調(diào)控因子Myostatin作為AKT的活化因子之一，也可以通過(guò)激活PI3K-AKT信號(hào)通路來(lái)調(diào)控肌肉生長(zhǎng)[36-38]。因此，PI3K-AKT信號(hào)通路可以通過(guò)調(diào)控和（肌酸激酶）骨骼肌發(fā)育分化標(biāo)志分子表達(dá)來(lái)調(diào)控骨骼肌纖維發(fā)育及分化[39-40]。

AKT2蛋白在胞質(zhì)比例較高，屬于膜受體蛋白。由此可以推斷，該蛋白可能在核膜上大量分布，并在蛋白質(zhì)翻譯時(shí)具有重要作用。而AKT2蛋白與蛋白酶催化亞基的三級(jí)結(jié)構(gòu)具有較高的同源性，表明該蛋白可能是蛋白質(zhì)翻譯時(shí)重要的催化激活因子。同時(shí)，該蛋白的三級(jí)結(jié)構(gòu)整體較為復(fù)雜，存在α-螺旋及無(wú)規(guī)則卷曲等結(jié)構(gòu)，可能對(duì)配體或受體蛋白的識(shí)別和結(jié)合具有重要作用。該蛋白大量磷酸化修飾位點(diǎn)的發(fā)現(xiàn)表明，可逆磷酸化調(diào)控可能在實(shí)現(xiàn)AKT2蛋白質(zhì)功能中起到重要作用。綜上，AKT2蛋白不僅在PI3K-AKT信號(hào)通路中具有重要的信號(hào)傳導(dǎo)及調(diào)控功能，還在綿羊胚胎骨骼肌發(fā)育分化時(shí)具有關(guān)鍵的調(diào)控作用，但AKT2蛋白調(diào)控肌纖維發(fā)育分化的分子機(jī)制還有待進(jìn)一步驗(yàn)證和研究。

4 結(jié)論

通過(guò)GO二級(jí)注釋、KEGG富集及R語(yǔ)言表達(dá)模式聚類等分析發(fā)現(xiàn)，蛋白質(zhì)功能和富集通路均與個(gè)體發(fā)育和骨骼肌生長(zhǎng)發(fā)育相關(guān)，第105天是綿羊胚胎骨骼肌纖維由增殖分化到增大增粗的轉(zhuǎn)折點(diǎn)，PI3K-AKT信號(hào)通路對(duì)骨骼肌纖維生長(zhǎng)發(fā)育轉(zhuǎn)換具有調(diào)控作用。候選蛋白質(zhì)生物信息學(xué)分析表明，ATK2具有重要催化調(diào)控功能，是調(diào)控PI3K-AKT信號(hào)通路信號(hào)傳導(dǎo)的重要候選蛋白。

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The Analysis of PI3K-AKT Signal Pathway Based on the Proteomic Results of Sheep Embryonic Skeletal Muscle

WANG XinYue, Shi TianPei, ZHAO ZhiDa, HU WenPing, Shang MingYu, Zhang Li

（Institute of Animal Sciences, Chinese Academy of Agriculture Sciences, Beijing 100193）

【】Sheep is an important economic livestock and its skeletal muscle growth and development have a deep bond with meat production traits. The sheep embryonic period is an essential stage for skeletal muscle growth, analyzing and mining the proteome data of sheep embryonic skeletal muscle in this period has a great significance to reveal the muscle development process and screen their key regulation proteins.【】The longissimus dorsi of Chinese merino sheep at embryonic age of 85 days, 105days and 135days were selected for protein qualification by using tandem mass tag (TMT) and 1316 differential abundance proteins were obtained finally. GO, KEGG and R bioinformatic methods were used to cluster, annotate and analyze the differential abundance proteins. And the candidate proteins were testified by using bioinformatic methods.【】Based on the previous results, the cluster analysis on differential abundance proteins illustrated that the cluster 5 proteins were significantly expressed on embryonic age of 105 dayswith high abundance.GO and KEGG analysis on cluster 5 proteins showed these proteins were significantly involved in protein metabolism biology process and notably enriched in PI3K-AKT signal pathway in which RAC-beta serine/threonine-protein kinase isoform X1(ATK2) has a high abundance. Meanwhile, the results of bioinformatics showed that the AKT2 was composed of 481 amino acids and the theoretical molecular weight was 55.58kD. It consists of 66 positively charged amino acid residues and 72 negatively charged amino acid residues, the theoretical isoelectric point was 6.08, the hydrophilic average coefficient was -0.454, 12 N-terminal glycosylation sites and 71 phosphorylation sites were found in AKT2. The homology of AKT2 and protein kinase-like (PK-like) was 99% and it belongs to the family of protein kinases catalytic subunit.【】The proteome data analysis of sheep embryonic skeletal muscle showed that embryonic age of 105 daysis a key point of sheep embryonic skeletal fiber cell from proliferation and differentiation to hypertrophy. The PI3K-AKT signaling pathway which has function of regulating growth and development of embryonic skeletal muscle fibers was significantly enriched, and ATK2 is a keycandidateregulation protein in this pathway. To summarize, the study has a theoretical guiding significance to reveal the growth and development and its molecular regulation mechanism of embryonic skeletal muscle.

sheep (); embryonic longissimus dorsi; proteomic; bioinformatics analysis

10.3864/j.issn.0578-1752.2020.14.018

2019-08-29；

2020-03-30

國(guó)家自然基金聯(lián)合基金重點(diǎn)支持項(xiàng)目（U1503285）、中國(guó)農(nóng)業(yè)科學(xué)院基本科研業(yè)務(wù)費(fèi)重大項(xiàng)目?jī)?chǔ)備計(jì)劃 (Y2017XM02)

王欣悅，E-mail：wxyanimalgenetic@163.com。通信作者張莉，E-mail：zhangli07@caas.cn

（責(zé)任編輯 林鑒非）