劉紅，葉榮

復(fù)旦大學(xué)基礎(chǔ)醫(yī)學(xué)院，上海 200032

脂酰化(fatty acylation)是蛋白修飾的重要方式之一，這種修飾可以是靜態(tài)的，也可以是動態(tài)的，修飾后蛋白的功能呈現(xiàn)多樣性。近年來隨著方法學(xué)的進(jìn)步，人們對脂?；肿訖C(jī)制的認(rèn)識也逐漸清晰[1,2]。蛋白的脂?；揎椫饕凶貦磅；?palmitoylation)、豆蔻酰化(myristoylation)、異戊烯化(prenylation)和糖基化磷脂酰肌醇(glycosylphosphatidylinositol, GPI)共價結(jié)合4種方式。脂?；砂l(fā)生在蛋白合成過程中或合成完成后，修飾位點可在蛋白的N端、C端或中間[3]。脂?；牡鞍淄ǔ６ㄎ挥诩?xì)胞膜結(jié)構(gòu)附近，這是因為與脂類共價結(jié)合導(dǎo)致蛋白與膜的親和力增加，去除這些脂質(zhì)基團(tuán)會影響這些修飾后蛋白的生物學(xué)功能。脂質(zhì)基團(tuán)在蛋白上形成特定的脂質(zhì)錨，隨后插入膜結(jié)構(gòu)的磷脂雙分子層內(nèi)，從而穩(wěn)定蛋白與膜的結(jié)合，也有利于將蛋白導(dǎo)入一些特定亞細(xì)胞的膜結(jié)構(gòu)域如脂筏等發(fā)揮其功能[4]。病毒在宿主細(xì)胞內(nèi)的復(fù)制過程中，一些病毒蛋白發(fā)生脂酰化，這些修飾促進(jìn)病毒識別及進(jìn)入細(xì)胞，幫助病毒蛋白在細(xì)胞內(nèi)運輸及定位，從而有利于病毒復(fù)制和裝配。一些重要病毒蛋白的脂?；绞郊捌渖飳W(xué)功能的變化見圖1。

圖1 重要病毒蛋白的脂?；问紽ig.1 Diagram summary of the fatty acylation of viral proteins

1 Ⅰ型病毒膜蛋白的棕櫚?；?/h2>

棕櫚?；ǔ０l(fā)生在含天冬氨酸-組氨酸-組氨酸-半胱氨酸(Asp-His-His-Cys，DHHC)保守序列的Cys富集區(qū)(cysteine-rich domain, CRD)。含DHHC-CRD結(jié)構(gòu)的蛋白廣泛存在于從低等真核生物如酵母到包括人在內(nèi)的哺乳動物細(xì)胞中，目前已從人細(xì)胞中分離鑒定出23種含類似結(jié)構(gòu)的蛋白[5]。蛋白的棕櫚?；哂锌赡嫘?，蛋白在棕櫚酰基轉(zhuǎn)移酶(palmitoylacyl transferase, PAT)作用下發(fā)生棕櫚?；?，轉(zhuǎn)運至不同的膜結(jié)構(gòu)，然后在棕櫚酰蛋白硫酯酶(palmitoyl protein thioesterase, PPT)作用下去棕櫚?；ㄟ^這種循環(huán)實現(xiàn)對蛋白功能的不同調(diào)控[3,6]。大部分含DHHC-CRD結(jié)構(gòu)的蛋白具有PAT活性，兼具酶和底物的特性[6,7]?？拷缒^(qū)的Cys棕櫚?；稍鰪?qiáng)跨膜區(qū)的疏水性，使跨膜蛋白容易定位至脂筏[8]。最常發(fā)生棕櫚酰化的Ⅰ型病毒膜蛋白包括流感病毒(influenza virus, FluV)的血凝素(hemagglutinin, HA)、人類免疫缺陷病毒(human immunodeficiency virus, HIV)的包膜蛋白(envelope, Env)、冠狀病毒(coronavirus, CoV)的棘突蛋白(spike, S)等[9]。此外，副黏病毒F蛋白、披膜病毒包膜糖蛋白(envelope glycoprotein, gp)、桿狀病毒gp64等也可發(fā)生棕櫚?；痆10-12]。

流感病毒HA的棕櫚酰化研究開展得最早、最廣泛。其前體分子HA0由細(xì)胞蛋白酶裂解為N端HA1和C端HA2[13]。HA的棕櫚酰化位點為Cys殘基。甲型流感病毒有2個Cys殘基位于胞質(zhì)尾區(qū)(cytoplasmic tail, CT)、1個位于跨膜區(qū)(transmembrane domain, TMD)；乙型流感病毒HA 的2個Cys殘基均位于胞質(zhì)尾區(qū)；丙型流感病毒HA-酯酶融合糖蛋白(HA-esterase fusion glycoprotein, HEF)只有1個Cys殘基，位于TMD與胞質(zhì)尾區(qū)之間。這些位點突變導(dǎo)致感染性病毒顆粒產(chǎn)生明顯減少[14-16]。 HA棕櫚?；兄诙ㄎ恢?，一些亞型的HA如H1、H2和H7等出現(xiàn)在富含鞘磷脂和膽固醇的特異性膜結(jié)構(gòu)域[17,18]。脂筏是病毒成熟相關(guān)蛋白的集聚位點，也是病毒蛋白激活細(xì)胞信號轉(zhuǎn)導(dǎo)的平臺，因此脂筏中除了HA，其他流感病毒蛋白也常富集于此[19,20]。HA通常在內(nèi)質(zhì)網(wǎng)(endoplasmic reticulum, ER)發(fā)生棕櫚?；?，然后裝配入病毒顆粒，與基質(zhì)蛋白1相互作用參與病毒的出芽過程[21]。流感病毒不能形成合胞體，表明HA介導(dǎo)的細(xì)胞-細(xì)胞融合受到影響，可能與HA的去脂酰化有關(guān)[22]。研究還發(fā)現(xiàn)，甲型流感病毒H1、H7和乙型流感病毒HA的棕櫚?；?，對病毒感染過程中膜融合孔的形成和增大及水性融合通道的形成有明顯促進(jìn)作用[23-26]。

HIV Env被細(xì)胞蛋白酶裂解為2個亞單位：gp120和gp41。gp120與受體結(jié)合，gp41具有包括膜融合在內(nèi)的多種功能[27]。gp41胞質(zhì)尾區(qū)含有2個棕櫚?；稽c：Cys-764和Cys-837[28,29]。其中1個位點突變成Ser，HIV-1感染性降低2倍；2個位點同時突變成Ser，HIV-1感染性則降低60倍[30,31]。病毒裝配出芽過程中，要求gp160胞質(zhì)區(qū)與Gag相互作用，單個棕櫚?；稽c突變對Env-Gag的相互作用影響很小，雙位點同時突變導(dǎo)致Env-Gag相互作用減弱，從而影響Env特異性裝配至病毒顆粒[28,32]。然而，棕櫚?；趃p160表達(dá)、細(xì)胞內(nèi)運輸及細(xì)胞-細(xì)胞膜融合過程中的作用仍未完全闡明，有待進(jìn)一步研究[29]。用β-甲基環(huán)糊精除去細(xì)胞的膽固醇后，HIV-1不但感染性降低，而且合胞體的形成減少[33-35]。HIV病毒顆粒的組裝及出芽依賴脂筏結(jié)構(gòu)，棕櫚?；腅nv在細(xì)胞膜內(nèi)側(cè)錨定，但2個棕櫚酰化位點缺失后，Env失去了脂筏定位能力，不能抵抗非離子去垢劑的作用[29]。

冠狀病毒棘突蛋白為典型的Ⅰ型跨膜糖蛋白，以三聚體的形式發(fā)揮生物學(xué)功能，不僅決定冠狀病毒的宿主特異性，還介導(dǎo)病毒與細(xì)胞膜融合[36]。棘突蛋白膜內(nèi)區(qū)有1個含8～10個Cys的保守區(qū)域，容易發(fā)生棕櫚?；揎梉37]。嚴(yán)重急性呼吸綜合征(severe acute respiratory syndrome，SARS)冠狀病毒和鼠冠狀病毒的棘突蛋白發(fā)生棕櫚?；螅坏鞍捉閷?dǎo)的細(xì)胞-細(xì)胞融合、病毒感染性及病毒顆粒裝配受影響[38-40]。重組鼠冠狀病毒表達(dá)和羥胺處理實驗發(fā)現(xiàn)，棘突蛋白的棕櫚?；竽?nèi)區(qū)3個以上Cys參與，棕櫚?；欣谄涠ㄎ恢林41]。棕櫚?；赡転榧坏鞍滋峁┦荏w結(jié)合和膜融合所必要的膜錨定力，從而增強(qiáng)病毒的感染性。

2 丙型肝炎病毒NS4B與核心蛋白的棕櫚?；?/h2>

丙型肝炎病毒(hepatitis C virus, HCV)非結(jié)構(gòu)蛋白NS4B含有261個氨基酸，是高度疏水的跨膜蛋白，參與基因組復(fù)制及病毒顆粒的組裝和釋放[42]。NS4B在HCV RNA復(fù)制復(fù)合體的形成過程中，C端膜內(nèi)區(qū)發(fā)生棕櫚?；米貦磅；种苿?-溴代棕櫚酸鹽處理后RNA復(fù)制明顯降低[43]。用蛋白轉(zhuǎn)運抑制劑布雷菲德菌素A(brefeldin A)破壞高爾基復(fù)合體，NS4B棕櫚?；⒉皇苡绊?，表明修飾發(fā)生在蛋白進(jìn)入高爾基復(fù)合體之前[43]。NS4B 的Cys-257是主要棕櫚?；稽c，但HCV 基因型3、5和6的NS4B第257位不是Cys，而是Thr或Tyr[43]。所有HCV基因型NS4B末端的Cys-261均高度保守，其突變可明顯減弱病毒RNA復(fù)制。但由于Cys-261也是NS3蛋白酶的識別位點，這些功能減低與棕櫚?；年P(guān)系仍無法確定。另外，Cys-257和Cys-261同時突變時，NS4B本身聚合作用及其與NS5A或其他蛋白的相互作用明顯減弱[44]。

HCV核心蛋白由191個氨基酸殘基組成，在含脂滴的內(nèi)質(zhì)網(wǎng)上與病毒RNA結(jié)合形成核衣殼顆粒[45]?？拷诵牡鞍證端疏水部位的Cys-172是主要棕櫚酰化位點，該位點突變后引起核心蛋白與脂滴相互作用減弱，導(dǎo)致內(nèi)質(zhì)網(wǎng)上核心蛋白減少，形成的病毒顆粒也減少[46]。用信號肽酶裂解核心蛋白使疏水尾部減少至4個氨基酸殘基，可導(dǎo)致其與內(nèi)質(zhì)網(wǎng)結(jié)合不穩(wěn)定，而棕櫚?；欣诓《竞诵呐c內(nèi)質(zhì)網(wǎng)的穩(wěn)定結(jié)合[47]。

3 HIV Nef蛋白和乙型肝炎病毒L蛋白的豆蔻?；?/h2>

發(fā)生豆蔻?；牡鞍淄ǔ：刑禺愋员Ｊ匦蛄蠫ly-X-X-X-Ser/Thr，且Gly后通常為1個Cys殘基，修飾的豆蔻?；鶊F(tuán)從豆蔻酰CoA獲得，由N-豆蔻酰轉(zhuǎn)移酶(N-myristoyltransferase, NMT)催化[48]。NMT分為NMT1和NMT2兩種，包括1個催化結(jié)構(gòu)域和1個N端結(jié)構(gòu)域，后者可特異性結(jié)合至核糖體[49]。蛋白的N端如果集中出現(xiàn)堿性氨基酸殘基，可作為蛋白豆蔻酰化的附加信號。這些堿性氨基酸殘基與磷脂頭部帶負(fù)電荷的基團(tuán)發(fā)生靜電作用，豆蔻酰基作為“靜電開關(guān)”調(diào)控該蛋白表面的正電荷，從而改變蛋白與膜的親和力[2]。發(fā)生豆蔻?；牟《镜鞍子卸喾N，其中HIV-1的Nef蛋白和乙型肝炎病毒(hepatitis B virus，HBV)的L蛋白豆蔻酰化對病毒的復(fù)制和組裝是必需的。

HIV-1 Nef是相對分子質(zhì)量為27 000的磷酸化蛋白，早期被認(rèn)為是負(fù)調(diào)控因子，與細(xì)胞的膜結(jié)構(gòu)結(jié)合從而抑制病毒基因組表達(dá)[50]。后期研究發(fā)現(xiàn)Nef具有增強(qiáng)病毒復(fù)制的能力，用特異性抑制劑阻斷其N端結(jié)構(gòu)域與核糖體的結(jié)合可抑制HIV-1顆粒的產(chǎn)生[51]。Nef豆蔻酰化由NMT2催化，其N端Thr通過靜電作用首先結(jié)合至雙層脂質(zhì)膜一側(cè)表面，再與肉豆蔻酸一起進(jìn)入膜內(nèi)。此時Nef的構(gòu)象并沒有變化，而NMT的構(gòu)象發(fā)生明顯變化[51]。Nef發(fā)生豆蔻?；瘯r，其N端區(qū)域也需Arg和Lys等堿性氨基酸殘基及至少1個豆蔻?；禺愋曰?，其中Lys被認(rèn)為是Nef進(jìn)入脂筏的關(guān)鍵因素[52]。然而，相對于細(xì)胞蛋白，Nef豆蔻?；淖饔幂^弱，表明豆蔻?；皇荖ef與細(xì)胞膜結(jié)合的主要因素[53]。Nef豆蔻?；部捎绊懠?xì)胞內(nèi)蛋白功能來增強(qiáng)病毒復(fù)制(如下調(diào)細(xì)胞表面CD4，病毒出芽時與CD4結(jié)合受阻)，從而影響感染性病毒顆粒的形成[54]。Nef還可下調(diào)細(xì)胞表面主要組織相容性復(fù)合體(major histocompatibility complex，MHC)Ⅰ類分子，促進(jìn)HIV-1免疫逃避，介導(dǎo)細(xì)胞內(nèi)信號轉(zhuǎn)導(dǎo)及T細(xì)胞活化，增加病毒顆粒產(chǎn)生[55]。

另外，豆蔻?；腘ef可優(yōu)先激活巨噬細(xì)胞。與腫瘤壞死因子α(tumor necrosis factor α，TNF-α)相似，可溶性Nef激活絲裂原活化蛋白激酶(mitogen-activated protein kinase，MAPK)/核因子κB(nuclear factor κB，NF-κB)信號途徑，刺激產(chǎn)生炎性細(xì)胞因子和趨化因子，如白細(xì)胞介素1β(interleukin 1β，IL-1β)、IL-6、TNF-α、巨噬細(xì)胞炎性蛋白1α(macrophage inflammatory protein 1α，MIP-1α)、MIP-1β等，激活巨噬細(xì)胞[56]。G2A突變后，Nef無豆蔻?；荒芗せ頜APK/NF-κB信號途徑，導(dǎo)致CD163表達(dá)及巨噬細(xì)胞活化降低[56,57]。Nef激活巨噬細(xì)胞后形成納米管，并與MHCⅡ、TGN蛋白及SH3蛋白等結(jié)合，誘導(dǎo)抗炎因子產(chǎn)生。Nef進(jìn)入B細(xì)胞，使其分化因子增加，從而干擾生發(fā)中心因子的產(chǎn)生，與HIV-1導(dǎo)致的淋巴瘤發(fā)生有關(guān)[58]。

HBV外膜蛋白由S、M和L組成。L比M多了preS1序列，含21～47個氨基酸殘基。HBV外膜蛋白也參與包裝丁型肝炎病毒(hepatitis D virus, HDV)，preS1序列是與肝細(xì)胞受體結(jié)合位點的可能區(qū)域[59,60]。最近發(fā)現(xiàn)牛磺膽酸鈉共轉(zhuǎn)運肽(sodium taurocholate cotransporting polypeptide, NTCP)作為HBV和HDV的功能性受體，能特異性結(jié)合該區(qū)域[61]。早期研究發(fā)現(xiàn)，preS1豆蔻?；瘜Σ《境鲅繜o明顯影響，將豆蔻?；盘柸斯と笔?，仍能產(chǎn)生與野毒株形狀無明顯差別的病毒顆粒[62]。后來才發(fā)現(xiàn)，preS1 N端的Gly突變使結(jié)合至宿主細(xì)胞膜上的L蛋白減少，導(dǎo)致病毒喪失感染性，但Gly豆蔻?；⒉皇遣《狙b配所必需的，提示preS1的豆蔻?；钱a(chǎn)生有感染性HBV的決定性因素[63]。HBV與細(xì)胞膜的結(jié)合主要位于preS1的第9～18位氨基酸殘基，第28～48位氨基酸殘基具有輔助病毒結(jié)合的功能，豆蔻?；l(fā)生在N端Gly殘基[63,64]。preS1豆蔻?；髽?gòu)象發(fā)生改變，蛋白結(jié)合至細(xì)胞膜，從而影響宿主細(xì)胞的脂質(zhì)雙分子層或表面成分，促使病毒結(jié)合和定位至宿主細(xì)胞上，病毒顆粒與HBV受體的親和性明顯增加[64]。最近有研究發(fā)現(xiàn)，豆蔻?；盘柸笔У牡鞍滓材艽龠M(jìn)中和抗體產(chǎn)生，為HBV疫苗研制提供了新思路[65]。

此外，乳頭瘤病毒VP2、小RNA病毒VP4、呼腸孤病毒μ1蛋白也常發(fā)生豆蔻酰化[66-68]。豬繁殖與呼吸綜合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV)、沙粒病毒及蛙病毒的一些結(jié)構(gòu)和非結(jié)構(gòu)蛋白也具有不同程度的豆蔻?；痆69-71]。這些蛋白豆蔻?；饕绊懖《具M(jìn)入細(xì)胞的過程和病毒顆粒的裝配。

4 HDV L蛋白的異戊烯化

1978年，異戊烯化作為參與酵母雜交的一個主要因素被首次證明，隨后發(fā)現(xiàn)哺乳動物細(xì)胞蛋白和HDV L蛋白也存在這種修飾[72-74]。異戊烯化包括15-C的法尼酰基化(farnesylation)和20-C的雙牻牛兒?；?geranylgeranylation)，通常發(fā)生在蛋白C端特異性序列CAAX(C，半胱氨酸；A，脂肪族氨基酸；X，任意氨基酸)的Cys殘基，而X殘基的性質(zhì)決定發(fā)生法尼?；€是雙牻牛兒酰基化[75]。異戊烯基通過硫酯鍵連接至Cys殘基的過程由異戊烯基轉(zhuǎn)移酶催化，通常不可逆。蛋白的異戊烯化對其膜結(jié)合功能至關(guān)重要，如阻斷Ras蛋白法尼?；赡孓D(zhuǎn)Ras轉(zhuǎn)化細(xì)胞的功能[76]。此外，異戊烯化也可介導(dǎo)蛋白-蛋白間相互作用，并在蛋白運輸中發(fā)揮作用[77]。

HDV包膜與HBV相同，核心部分由HDV RNA和HDV抗原(HDV antigen，HDAg)組成，其中大HDAg(Large HDAg, L-HDAg)C端包含異戊烯化位點CXXX(C，半胱氨酸；X，任意氨基酸)，由法尼酰基轉(zhuǎn)移酶和雙牻牛兒?；D(zhuǎn)移酶Ⅰ催化，另一些能被雙牻牛兒?；D(zhuǎn)移酶Ⅱ催化，其識別的序列不同[78]。HDV顆粒的裝配需HBV表面抗原(HBV surface antigen，HBsAg)與L-HDAg相互作用，異戊烯修飾的L-HDAg易運輸至內(nèi)質(zhì)網(wǎng)膜上，同時L-HDAg的異戊烯化也反饋抑制HDV RNA的合成，促使HDV RNA與L-HDAg一起裝配成HDV顆粒[79]。根據(jù)HDAg異戊烯化設(shè)計抑制劑，可干擾HDV組裝，目前已用于臨床治療慢性丁型肝炎[80,81]。

5 朊蛋白與GPI共價結(jié)合

GPI與蛋白質(zhì)共價結(jié)合是一種間接脂酰化修飾，修飾的蛋白通常附著于細(xì)胞膜的外側(cè)，參與膜蛋白轉(zhuǎn)運、信息傳遞、細(xì)胞黏附和補(bǔ)體調(diào)節(jié)等作用。哺乳動物有150多種蛋白以此種方式與膜結(jié)合，包括水解酶、受體、黏附分子、補(bǔ)體抑制因子和功能不詳?shù)谋砻婵乖萚82]。GPI由糖基化的磷脂和乙醇胺殘基組成，排列復(fù)雜，被其修飾的蛋白以糖基-磷脂酰-肌醇的順序錨定于細(xì)胞膜外側(cè)。如果用特異性磷脂酶C處理細(xì)胞表面，GPI錨定的蛋白被釋放出來[83]。GPI錨定的蛋白通常在N端有信號序列，在C端有疏水序列[84]。最早證明發(fā)生GPI修飾的病毒蛋白為登革病毒NS1[85]。最近發(fā)現(xiàn)，PRRSV GP4 C端的GPI結(jié)合位點突變后，其不能進(jìn)入脂筏，從而不能與受體CD163結(jié)合，導(dǎo)致病毒喪失感染性[86]。

朊病毒(prion)感染導(dǎo)致腦神經(jīng)元退化，其主要成分是朊蛋白(prion protein，PrP)，由正常細(xì)胞編碼的PrP前體蛋白稱為細(xì)胞型朊蛋白(cellular prion protein，PrPc)，不致病，PrPc的異構(gòu)體羊癢疫朊蛋白(scrapie prion protein，PrPsc)為其致病形式[87]。PrPc是一種糖基化膜蛋白，包括N端信號肽序列、中間高度保守疏水區(qū)和C端GPI錨定區(qū)。PrPc在核糖體合成后被轉(zhuǎn)運至粗面內(nèi)質(zhì)網(wǎng)和高爾基復(fù)合體內(nèi)進(jìn)行糖基化，然后在高爾基復(fù)合體上與GPI飽和酰基及脂筏內(nèi)飽和鞘脂類結(jié)合，最后轉(zhuǎn)運定位至細(xì)胞膜[88]。成熟的朊蛋白通常以與脂筏結(jié)合的形式存在，但未糖基化的PrPc前體在內(nèi)質(zhì)網(wǎng)也能與脂筏結(jié)合，這種結(jié)合能促進(jìn)蛋白的折疊[89]。PrPsc中GPI錨的生物學(xué)作用很難用實驗證實，因為PrPsc的聚合形式保護(hù)GPI不被磷脂酶C消化。人們首次證明GPI錨定與PrPsc致病性的關(guān)系，是用缺失特定GPI錨定元件的轉(zhuǎn)基因小鼠產(chǎn)生大量PrPsc蛋白，但發(fā)現(xiàn)這些PrPsc蛋白并未引起動物產(chǎn)生與羊癢疫相似的臨床癥狀[90]。GPI錨通過改變膜結(jié)構(gòu)及胞質(zhì)內(nèi)磷脂變化，致使PrPc交聯(lián)或PrPsc聚集，引起突觸發(fā)生變性，導(dǎo)致海綿樣病變。磷脂酶的消化會導(dǎo)致PrPc的交聯(lián)形式消失[91]。因為朊病毒引起突觸變性取決于其GPI錨組成，提示可針對GPI錨建立治療朊病毒感染疾病的新策略。

6 結(jié)語

脂?；鳛橐环N常見的蛋白修飾方式，參與細(xì)胞內(nèi)多種蛋白的膜相關(guān)功能的發(fā)揮；但長期以來由于方法上的限制，該研究進(jìn)展緩慢。近年來，新的標(biāo)記和標(biāo)記方法的出現(xiàn)，脂?；芯吭絹碓绞艿街匾?，其中脂?；鞍捉M學(xué)的開展為鑒定被修飾蛋白及參與修飾反應(yīng)的酶具有重要推進(jìn)作用。病毒感染過程中合成蛋白的膜結(jié)合能力對病毒的復(fù)制與致病性至關(guān)重要，尤其是一些包膜病毒的裝配。近年來，PrPsc和HDAg脂?；揎椦芯繛橄嚓P(guān)藥物開發(fā)提供了新靶點和新思路。同樣，一些病毒蛋白有明確的結(jié)構(gòu)和功能特點，也是研究脂?；牧己媚Ｐ?，可加快脂酰化機(jī)制的闡明。

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