溶酶體酶PPT1介導(dǎo)的細(xì)胞穩(wěn)態(tài)調(diào)控及疾病發(fā)生的研究進(jìn)展

周欣怡，常棟，徐黃瑩，關(guān)瑞琪，嚴(yán)程浩，尹秋媛，孫建偉

綜 述

溶酶體酶PPT1介導(dǎo)的細(xì)胞穩(wěn)態(tài)調(diào)控及疾病發(fā)生的研究進(jìn)展

周欣怡，常棟，徐黃瑩，關(guān)瑞琪，嚴(yán)程浩，尹秋媛，孫建偉

云南大學(xué)生命科學(xué)中心、云南省細(xì)胞代謝與疾病重點(diǎn)實(shí)驗(yàn)室、省部共建云南生物資源保護(hù)與利用國家重點(diǎn)實(shí)驗(yàn)室，生命科學(xué)學(xué)院，昆明 650500

棕櫚酰蛋白質(zhì)硫酯酶1 (palmitoyl protein thioesterase 1，PPT1)是一種溶酶體酶，具有對(duì)蛋白質(zhì)行使去棕櫚?；揎椀墓δ埽谡{(diào)控細(xì)胞器(如溶酶體、線粒體)功能、脂質(zhì)代謝和Ca2+轉(zhuǎn)運(yùn)等方面具有重要作用。PPT1在神經(jīng)系統(tǒng)疾病和癌癥的發(fā)生發(fā)展中發(fā)揮了重要的作用，然而其調(diào)控作用機(jī)制仍未被完全闡明。本文綜述了目前PPT1在脂質(zhì)代謝調(diào)控、物質(zhì)轉(zhuǎn)運(yùn)調(diào)控、鈣穩(wěn)態(tài)調(diào)控過程中的作用及最新研究進(jìn)展，總結(jié)并探討了PPT1在神經(jīng)系統(tǒng)疾病及腫瘤發(fā)生發(fā)展中作用及調(diào)控機(jī)制，為將來更深層次研究PPT1的功能及調(diào)控機(jī)制并開發(fā)相關(guān)疾病治療的新藥物提供借鑒和參考。

棕櫚酰蛋白質(zhì)硫酯酶1，去棕櫚?；?，溶酶體，疾病發(fā)生

棕櫚酰蛋白質(zhì)硫酯酶1(palmitoyl protein thioesterase 1，PPT1)在牛腦細(xì)胞質(zhì)的研究中首次發(fā)現(xiàn)并被純化出來[1]，PPT1主要定位在溶酶體中，在細(xì)胞中行使去棕櫚?；墓δ躘2]。PPT1可以去棕櫚?；疓AP43 (growth associated protein 43)、PSD-95 (postsynaptic density protein 95)等多個(gè)蛋白，影響溶酶體的功能和穩(wěn)態(tài)[3]，表明PPT1在維持溶酶體正常生理功能中具有重要作用。

PPT1與疾病的發(fā)生發(fā)展有著密切的關(guān)系。嬰兒神經(jīng)元蠟樣脂褐素沉積癥(infantile neuronal ceroid lipofuscinosis，INCL)，也被稱為嬰兒巴頓病，是一種罕見的隱性遺傳神經(jīng)退行性疾病，患有INCL的嬰兒一般在18個(gè)月左右出現(xiàn)視覺運(yùn)動(dòng)缺陷、認(rèn)知障礙、癲癇發(fā)作等癥狀[4]。研究人員發(fā)現(xiàn)該疾病是由于神經(jīng)元細(xì)胞內(nèi)基因突變導(dǎo)致PPT1功能受損，細(xì)胞內(nèi)類脂質(zhì)不能被及時(shí)分解，在神經(jīng)元細(xì)胞內(nèi)沉積，最終導(dǎo)致神經(jīng)元功能障礙和死亡，造成INCL[5,6]。此外，PPT1與腫瘤的發(fā)生發(fā)展也密切相關(guān)，在多種腫瘤中都存在PPT1過表達(dá)的情況，且高表達(dá)的PPT1與腫瘤惡性程度呈正相關(guān)。研究表明抑制的表達(dá)或活性，可以抑制腫瘤細(xì)胞的增殖和侵襲，靶向PPT1對(duì)多種腫瘤有較強(qiáng)的治療效果[7]。以上研究表明，PPT1是治療INCL和癌癥的有效靶點(diǎn)，因此，研究PPT1在疾病中的作用及機(jī)制、研發(fā)靶向PPT1的藥物是未來神經(jīng)性疾病和腫瘤治療的重要方向之一。本文圍繞PPT1的結(jié)構(gòu)和功能及其在疾病發(fā)生發(fā)展中作用及分子調(diào)控機(jī)制展開綜述，最后對(duì)PPT1在臨床運(yùn)用方面的價(jià)值進(jìn)行了探討和展望。

1 PPT1的結(jié)構(gòu)

基因定位于人類1號(hào)染色體短臂3區(qū)4帶2亞帶(1p34.2)，全長25,751 bp。有19個(gè)轉(zhuǎn)錄本，其中最常見的轉(zhuǎn)錄本由9個(gè)外顯子組成，外顯子1～8長約70～140 bp，被長短不一的內(nèi)含子間隔開來，最大的外顯子9位于靠近3'端的位置。自然界中存在220個(gè)PPT1的直系同源物，在不同的模式生物(秀麗隱桿線蟲、擬南芥等)中都能找到的同源基因。已有的研究表明，第4外顯子活性位點(diǎn)附近的突變可導(dǎo)致神經(jīng)退行性疾病INCL[5,8]，這提示PPT1與INCL具有潛在聯(lián)系。在正常人體中，PPT1一般在腦、眼和脾臟等器官中特異性表達(dá)[9]，然而在一些類型(乳腺癌、食管腺癌等)腫瘤患者的原發(fā)灶中，PPT1的表達(dá)則明顯高于癌旁和正常組織，與腫瘤患者的不良預(yù)后密切相關(guān)[10]。

PPT1蛋白由306個(gè)氨基酸組成，分子量約35 kDa[11]，PPT1二聚體采用α/β折疊，包含N端α螺旋、β折疊區(qū)、C端α螺旋等多個(gè)重要的結(jié)構(gòu)(圖1)[12,13]。N端α螺旋位于PPT1分子的一側(cè)，與另一個(gè)亞基的β折疊區(qū)域相互作用，負(fù)責(zé)PPT1的組裝；β折疊區(qū)包含多個(gè)β片層，形成PPT1的主要結(jié)構(gòu)框架；C端α螺旋與連接區(qū)域一起形成了PPT1的另一側(cè)，該側(cè)上的活性位點(diǎn)負(fù)責(zé)棕櫚酰輔酶A的水解[13]。通過多波長反常衍射相位法(multiwavelength anomalous diffraction，MAD)測定PPT1的晶體結(jié)構(gòu)，發(fā)現(xiàn)PPT1晶體結(jié)構(gòu)中的活性位點(diǎn)是由Ser115- His289-Asp233組成的催化三聯(lián)體，Ser115通過其羥基與脂質(zhì)酰基結(jié)合，形成酯鍵從而水解?；?，Asp233的負(fù)電荷與Ser115的羥基形成氫鍵，同時(shí)能在Ser115和His289之間提供催化作用；His289的環(huán)氨基和Ser115的羥基能夠在催化反應(yīng)中形成過渡態(tài)，穩(wěn)定其催化結(jié)構(gòu)[13]。糖基化是PPT1翻譯后修飾的形式之一，此前在非神經(jīng)元細(xì)胞中的研究已證明，PPT1的3個(gè)N-糖基化位點(diǎn)的突變會(huì)顯著降低其酶活性[14]；研究發(fā)現(xiàn)，PPT1特定的氨基酸殘基位點(diǎn)(天冬酰胺197和232位點(diǎn))N-糖基化增強(qiáng)了其形成復(fù)合物的能力[15]。蛋白印跡分析表明，PPT1蛋白主要以二聚體或三聚體的形式存在，在去糖基化過程中演變成單個(gè)大小29 kDa的蛋白質(zhì)[16]。

圖1 PPT1的三級(jí)結(jié)構(gòu)

黃色標(biāo)記為糖基化位點(diǎn)：Asn197、Asn212、Asn232，淡藍(lán)色標(biāo)記為催化三聯(lián)體：Ser115、Asp233、His289。數(shù)據(jù)來自于https://www.rcsb.org/3d-view/1EI9/1。

2 PPT1的功能

2.1 PPT1調(diào)控溶酶體功能和穩(wěn)態(tài)

溶酶體是細(xì)胞中的降解中心和信號(hào)樞紐，在細(xì)胞穩(wěn)態(tài)、發(fā)育和衰老中發(fā)揮重要作用，溶酶體的生物發(fā)生和活性受到多種細(xì)胞內(nèi)和細(xì)胞外信號(hào)的調(diào)節(jié)[17]。PPT1的去棕櫚酰化參與了包括自噬-溶酶體途徑等多個(gè)細(xì)胞反應(yīng)過程：通過水解脂酰化的蛋白質(zhì)，在溶酶體內(nèi)調(diào)節(jié)脂類代謝和蛋白質(zhì)降解，從而維持溶酶體的正常功能[18]。PPT1主要定位于溶酶體，其功能的缺失可導(dǎo)致溶酶體pH值升高，溶酶體酶活降低，調(diào)節(jié)溶酶體的功能和穩(wěn)態(tài)，進(jìn)而影響溶酶體與其他細(xì)胞器之間的通訊和交流[19]。

已有研究表明在敲除小鼠的大腦中高度棕櫚?；牡鞍踪|(zhì)GAP43(介導(dǎo)神經(jīng)元軸突延伸、改變神經(jīng)細(xì)胞形態(tài)的重要蛋白)異常地積聚在內(nèi)質(zhì)網(wǎng)(endoplasmic reticulum，ER)中，在培養(yǎng)的INCL細(xì)胞中過表達(dá)GAP43發(fā)現(xiàn)其與ER標(biāo)記蛋白Grp-78/Bip (glucose-regulated protein 78/binding immunoglobulin protein)有明顯的共定位[20,21]，GAP43在內(nèi)質(zhì)網(wǎng)的異常積累導(dǎo)致內(nèi)質(zhì)網(wǎng)應(yīng)激，進(jìn)而激活未折疊蛋白反應(yīng)(unfolded protein response, UPR)，最終激活凋亡因子caspase 4、caspase 12導(dǎo)致神經(jīng)細(xì)胞凋亡[22]。這表明敲除或去棕櫚?；δ軉适Ш骃-酰化蛋白從溶酶體逆向運(yùn)輸?shù)紼R，激活UPR促進(jìn)神經(jīng)細(xì)胞的凋亡，具體機(jī)制還需要進(jìn)一步的研究。在內(nèi)質(zhì)網(wǎng)應(yīng)激條件下，定位于溶酶體的PPT1部分異位到質(zhì)膜的微結(jié)構(gòu)域DRM (detergent- resistant microdomain，DRM)上，棕櫚?；?去棕櫚?；Ш?，導(dǎo)致細(xì)胞死亡，這從另一個(gè)角度說明了PPT1具有維持細(xì)胞穩(wěn)態(tài)的重要作用[23]。另有報(bào)道稱，在PPT1缺陷的成纖維細(xì)胞中，溶酶體相關(guān)膜蛋白1-陽性(lysosomal associated membrane protein 1，LAMP1-positive)空泡形成顯著增多，這進(jìn)一步說明了PPT1缺失導(dǎo)致了溶酶體功能紊亂[24,25]。溶酶體的運(yùn)動(dòng)、融合和分泌等過程受細(xì)胞骨架變化的影響，α微管蛋白1a (α-1a-tubulin)、β肌動(dòng)蛋白(β-actin)和α4肌動(dòng)蛋白 (α4-actin)等細(xì)胞骨架蛋白能被PPT1去棕櫚酰化，導(dǎo)致細(xì)胞骨架結(jié)構(gòu)的變化，這提示PPT1也通過行使其去棕櫚?；墓δ軐?dǎo)致細(xì)胞骨架的變化，進(jìn)而影響溶酶體功能[26,27]。也有研究表明敲除通過減少內(nèi)吞體運(yùn)輸復(fù)合物Rab7-RILP (Rab- interacting lysosomal protein)的相互作用，阻止自噬小體-溶酶體融合，進(jìn)而間接影響溶酶體功能[28]。以上說明PPT1通過行使其去棕櫚?；墓δ?，對(duì)維持溶酶體功能和穩(wěn)態(tài)具有十分重要的作用。

2.2 PPT1調(diào)控鈣穩(wěn)態(tài)

鈣離子是細(xì)胞內(nèi)重要的信號(hào)分子，在細(xì)胞內(nèi)廣泛參與多種生理和代謝過程，細(xì)胞內(nèi)鈣離子水平的動(dòng)態(tài)平衡對(duì)于維持正常細(xì)胞功能和生理過程至關(guān)重要[29]。研究表明，PPT1在腫瘤細(xì)胞中參與調(diào)節(jié)細(xì)胞內(nèi)的鈣穩(wěn)態(tài)，從而促進(jìn)腫瘤細(xì)胞的增殖和侵襲，PPT1缺陷導(dǎo)致了神經(jīng)元內(nèi)ER鈣平衡紊亂，進(jìn)而引發(fā)細(xì)胞凋亡和神經(jīng)炎癥反應(yīng)[30]。研究人員發(fā)現(xiàn)PPT1小鼠(該模型是指通過Cre/LoxP技術(shù)敲除的外顯子4，PPT1小鼠不能合成功能完整的PPT1蛋白，以此模擬INCL患者中PPT1蛋白質(zhì)的缺失)鈣穩(wěn)態(tài)與WT小鼠細(xì)胞相比存在明顯差異，敲除的神經(jīng)細(xì)胞能夠從谷氨酸誘導(dǎo)的鈣瞬變中恢復(fù)，并將其細(xì)胞內(nèi)鈣調(diào)節(jié)回靜止水平，這表明PPT1參與激活神經(jīng)保護(hù)機(jī)制[31]。這一結(jié)果表明PPT1在細(xì)胞和組織水平的鈣穩(wěn)態(tài)調(diào)控中發(fā)揮了重要作用，并影響細(xì)胞的生理和代謝過程。研究還發(fā)現(xiàn)PPT1缺陷小鼠的肌醇3-磷酸受體-1 (inositol 1,4,5-trisphosphate receptor type1，IP3R1)的表達(dá)水平降低，導(dǎo)致溶酶體鈣離子穩(wěn)態(tài)失衡，這加劇了溶酶體膜的損傷，進(jìn)一步引發(fā)了氧化應(yīng)激反應(yīng)[19,33]。此外，PPT1缺陷導(dǎo)致了溶酶體轉(zhuǎn)運(yùn)蛋白類別的毒蕈堿受體(transient receptor potential mucolipin 1，TRPML1)功能異常，導(dǎo)致鈣離子平衡紊亂[19]。這些研究提示PPT1主要通過調(diào)節(jié)鈣離子轉(zhuǎn)運(yùn)以調(diào)節(jié)鈣穩(wěn)態(tài)，其缺陷會(huì)導(dǎo)致鈣穩(wěn)態(tài)紊亂，導(dǎo)致細(xì)胞功能異常。

2.3 PPT1調(diào)控脂質(zhì)穩(wěn)態(tài)

正常的脂質(zhì)穩(wěn)態(tài)對(duì)于中樞神經(jīng)系統(tǒng)功能的正常發(fā)揮和維持至關(guān)重要[34]。有研究表明PPT1缺失導(dǎo)致脂質(zhì)的異常積累，當(dāng)PPT1缺乏時(shí)，皮質(zhì)神經(jīng)元大量死亡，大多數(shù)組織中表現(xiàn)出鞘脂激活蛋白(sphingolipid activator proteins，也稱作saposins) A和D的積累[35]，同時(shí)影響了它們的亞細(xì)胞定位。在相關(guān)模式生物研究中發(fā)現(xiàn)，PPT1缺陷小鼠體內(nèi)的脂質(zhì)穩(wěn)態(tài)在早期發(fā)生了一系列的變化，且PPT1在脂蛋白代謝調(diào)節(jié)中能夠起到新的作用[36]。PPT1缺乏與能量平衡和脂質(zhì)代謝的改變密切相關(guān)[37,38]，尤其在低溫條件刺激下，敲除小鼠無法滿足能量及脂質(zhì)代謝的需求，這是因?yàn)榈蜏貤l件下，棕色脂肪組織中缺乏脂解，無法維持體溫[39]。此外，PPT1能顯著調(diào)控膽固醇的水平，PPT1缺失導(dǎo)致神經(jīng)元膽固醇生物合成受損[31]。有研究表明，膽固醇生物合成受限可能是PPT1缺乏導(dǎo)致的間接結(jié)果，PPT1的表達(dá)水平與脂筏組分改變有關(guān)，PPT1的活性受到脂質(zhì)筏的調(diào)節(jié)，它與脂質(zhì)筏中的特定膜蛋白相互作用，并定位到這些區(qū)域，這種相互作用有助于PPT1在脂質(zhì)筏中的定位和功能，反映了維持脂筏的完整性和功能性的代償機(jī)制，研究人員認(rèn)為與PPT1潛在底物棕櫚酰化蛋白集中在脂筏的胞質(zhì)側(cè)有關(guān)[23,40, 41]。在PPT1缺失的神經(jīng)元中，發(fā)現(xiàn)結(jié)合和運(yùn)輸脂質(zhì)的載脂蛋白A-1 (apolipoprotein A-1，apoA-1)攝取量的增加[42]；支持細(xì)胞中(sertoli cells) 是一種具有護(hù)理功能的細(xì)胞，通過向發(fā)育中的生殖細(xì)胞提供營養(yǎng)和能量支持，對(duì)維持精子發(fā)生至關(guān)重要，研究發(fā)現(xiàn)在原代培養(yǎng)的支持細(xì)胞中，PPT1能響應(yīng)膽固醇的處理并上調(diào)其表達(dá)水平；同時(shí)，支持細(xì)胞中PPT1的特異性敲除會(huì)導(dǎo)致精子質(zhì)量差和精子畸形率高，從而造成雄性小鼠的生育能力下降，生殖細(xì)胞與精上皮中的支持細(xì)胞粘附不良，PPT1通過調(diào)節(jié)支持細(xì)胞的溶酶體功能和膽固醇代謝來影響精子質(zhì)量和雄性小鼠生育能力[43,44]。綜上所述，PPT1在調(diào)控脂質(zhì)穩(wěn)態(tài)中發(fā)揮著重要作用。

2.4 PPT1調(diào)控線粒體穩(wěn)態(tài)

線粒體是細(xì)胞內(nèi)能量代謝的重要細(xì)胞器，同時(shí)也是細(xì)胞內(nèi)鈣離子調(diào)控和凋亡信號(hào)傳遞的重要場所，線粒體穩(wěn)態(tài)的維持對(duì)于細(xì)胞生存和功能至關(guān)重要[45]。PPT1的缺陷會(huì)導(dǎo)致線粒體數(shù)量減少、形態(tài)異常和功能受損(線粒體膜磷脂代謝紊亂，線粒體的膜電位降低、鈣離子平衡紊亂、活性氧過度產(chǎn)生)[46, 47]。線粒體穩(wěn)態(tài)失衡最終會(huì)導(dǎo)致神經(jīng)細(xì)胞能量代謝紊亂、細(xì)胞凋亡數(shù)量的增加，從而引起神經(jīng)退行性疾病等。已有報(bào)道稱，在PPT1缺失患者細(xì)胞中觀察到球形斑點(diǎn)狀的異常線粒體形態(tài)，并且PPT1缺陷的人成纖維細(xì)胞對(duì)外源活性氧誘導(dǎo)的細(xì)胞死亡表現(xiàn)出更高的敏感性[46]。研究人員結(jié)合線粒體熒光染料(Mito Tracker)以及電鏡技術(shù)，發(fā)現(xiàn)線粒體富集在核周，并且線粒體總量減少，表明PPT1缺陷造成線粒體網(wǎng)絡(luò)形態(tài)及功能發(fā)生改變[48]。在體內(nèi)能量物質(zhì)循環(huán)和運(yùn)輸?shù)难芯堪l(fā)現(xiàn)，神經(jīng)元中PPT1的缺失導(dǎo)致線粒體電子傳遞鏈復(fù)合物I和IV的功能改變[49]。PPT1雖然是定位于溶酶體中的去棕櫚?；?，但在線粒體的功能及穩(wěn)態(tài)的調(diào)控中也發(fā)揮著重要的作用。

3 PPT1與疾病的發(fā)生發(fā)展

3.1 PPT1與神經(jīng)系統(tǒng)疾病

嬰兒神經(jīng)元蠟樣脂褐素沉著癥(INCL)是一種嚴(yán)重的神經(jīng)退行性疾病，INCL通常在嬰兒期開始發(fā)作，表現(xiàn)為進(jìn)行性的神經(jīng)系統(tǒng)退化，這種疾病的典型癥狀是智力和運(yùn)動(dòng)能力發(fā)育遲緩、癲癇發(fā)作、視力喪失、肌張力增高、肌肉萎縮以及認(rèn)知和行為問題等[50,51]，INCL病理的一個(gè)標(biāo)志是脂褐素的進(jìn)行性積累，最顯著地存在于神經(jīng)系統(tǒng)中，也存在于其他一些組織中。引起INCL的突變分布在整個(gè)編碼區(qū)，主要是第4外顯子活性位點(diǎn)附近R122W(Arg122Trp)突變和第5外顯子R151*(Arg151STOP)突變，導(dǎo)致PPT1結(jié)構(gòu)的明顯改變，從而影響PPT1的功能，導(dǎo)致溶酶體無法有效降解儲(chǔ)存在神經(jīng)細(xì)胞中的脂褐素，最終導(dǎo)致這些儲(chǔ)存物在腦中沉積引發(fā)INCL[13,52]。目前關(guān)于該疾病的發(fā)病機(jī)制尚不明確，結(jié)合病理水平的相關(guān)數(shù)據(jù)以及PPT1在分子水平的已有研究，提出了PPT1引起INCL致病機(jī)制的猜想：在癥狀前的最早階段(即在子宮和新生兒中)，神經(jīng)元軸突和軸突的連接中斷，導(dǎo)致回路形成功能障礙[53,54]；1個(gè)月齡的PPT1小鼠表現(xiàn)出突觸前蛋白的表達(dá)減少，沃勒變性(wallerian degeneration)異常，小鼠視皮層檢測到突觸后結(jié)構(gòu)和分子缺陷，導(dǎo)致了興奮毒性[55,56]；5個(gè)月后，GABA神經(jīng)元群(GABAergic neuron populations)在丘腦和皮質(zhì)開始退化[56]。INCL的病理性級(jí)聯(lián)可能在子宮內(nèi)就已出現(xiàn)，異常的神經(jīng)元回路形成導(dǎo)致隨后的功能障礙，缺乏PPT1會(huì)導(dǎo)致軸突和樹突發(fā)生缺陷[54]，突觸前蛋白(如synaptosome associated protein 25，SNAP25)水平降低，減緩沃勒變性的相關(guān)蛋白質(zhì)的表達(dá)[57]。此外，在PPT1小鼠神經(jīng)元中，突觸前小泡釋放受到抑制[58]，PPT1的缺乏導(dǎo)致VAMP2 (vesicle associated membrane protein 2)和SNAP25的持續(xù)膜結(jié)合，從而損害小鼠的囊泡循環(huán)，大腦皮質(zhì)中突觸前囊泡池的減小，失調(diào)的囊泡回收以及突觸傳遞的減少導(dǎo)致軸突“垂死”病理。在分離的神經(jīng)元培養(yǎng)和幼年鼠視皮質(zhì)中，PPT1缺失的神經(jīng)元顯示出不成熟的樹突形態(tài)，對(duì)于各種形式的突觸可塑性至關(guān)重要的突觸后N-甲基-D-天冬氨酸受體(N-methyl-D-aspartate receptor，NMDAR)的表達(dá)異常，導(dǎo)致神經(jīng)元突觸鈣內(nèi)流增加，損傷神經(jīng)元[27,55]。PPT1參與軸突生長、延伸和樹突棘形態(tài)發(fā)生途徑，PPT1缺失導(dǎo)致膠質(zhì)細(xì)胞激活、突觸前小泡功能障礙和突觸后功能障礙。在小鼠模型中，V型ATP酶(V-ATPase)的V0a1 (v-ATPase subunit a1 of the v0 sector)亞基被誤送到質(zhì)膜上，而非溶酶體膜上，V0a1向溶酶體膜的運(yùn)輸被改變，V-ATPase的活性下降，溶酶體的pH升高，溶酶體中酶的活性降低，降解物質(zhì)的能力下降，從而導(dǎo)致蠟樣物質(zhì)的積累[19,61～63]。在PPT1功能缺失的小鼠模型中，膠質(zhì)纖維酸性蛋白(glial fibrillary acidic protein，GFAP) Cys 291位的棕櫚酰化修飾水平升高，導(dǎo)致星形膠質(zhì)細(xì)胞的過度活化和增殖，加重了INCL的病理進(jìn)程[64,65]。PPT1催化S-棕櫚?；鞍?蠟樣成分)的去棕櫚酰化，這是溶酶體水解酶降解過程所必需的，PPT1缺乏導(dǎo)致脂質(zhì)修飾蛋白在溶酶體中儲(chǔ)存積累，從而導(dǎo)致INCL[28]。儲(chǔ)存物的積累導(dǎo)致不同形式的細(xì)胞應(yīng)激，還導(dǎo)致MHC (major histocompatibility complex)I類分子在預(yù)期靶細(xì)胞上的表達(dá)和促炎細(xì)胞因子的上調(diào)[30,66]。此外，在PPT1的小鼠研究中已有報(bào)道證明神經(jīng)炎癥是INCL重要發(fā)病機(jī)制之一，小鼠體內(nèi)觀察到神經(jīng)炎癥變化顯著，包括前細(xì)胞因子的產(chǎn)生增加，炎癥細(xì)胞的募集，小膠質(zhì)細(xì)胞活化[67]。PPT1缺失還導(dǎo)致ER應(yīng)激激活，從而介導(dǎo)了敲除小鼠腦組織的細(xì)胞凋亡增加[68]。綜上，導(dǎo)致INCL的原因包含以下三方面：（1）蛋白質(zhì)降解通路受損：PPT1作為一個(gè)參與蛋白質(zhì)的降解和代謝的溶酶體酶，其缺失導(dǎo)致溶酶體功能受損，影響蛋白質(zhì)的正常降解，導(dǎo)致異常蛋白質(zhì)的積累；（2）自噬通路異常：自噬是一種細(xì)胞內(nèi)的降解過程，通過溶酶體將細(xì)胞內(nèi)的廢棄物和異常蛋白質(zhì)降解，PPT1缺失影響自噬通路的正常功能，導(dǎo)致細(xì)胞無法有效清除異常蛋白質(zhì)和廢物，從而導(dǎo)致它們在細(xì)胞中的積累；（3）氧化應(yīng)激和炎癥反應(yīng)：PPT1缺失導(dǎo)致氧化應(yīng)激的增加和炎癥反應(yīng)的激活。這些異常生理過程進(jìn)一步加速神經(jīng)細(xì)胞的退化和死亡，促進(jìn)INCL疾病的發(fā)展。

在臨床研究方面，INCL的另一個(gè)主要臨床特征是產(chǎn)生進(jìn)行性的、逐漸惡化的癲癇病。在PPT1小鼠疾病進(jìn)展的最后階段，每個(gè)皮層區(qū)域間神經(jīng)元群所出現(xiàn)的分級(jí)影響導(dǎo)致了IV層顆粒神經(jīng)元的缺失，并最終引起癲癇發(fā)作。自發(fā)性癲癇的發(fā)作主要發(fā)生在皮層中間神經(jīng)元數(shù)量減少后，且會(huì)受到多種NCL (neuronal ceroid lipofuscinosis，NCL)形式的影響[69～71]，同時(shí)PPT1小鼠體內(nèi)的早期星形細(xì)胞和進(jìn)展性星形細(xì)胞增多促進(jìn)癲癇發(fā)作[72～74]。

3.2 PPT1調(diào)控腫瘤發(fā)生與進(jìn)展

已有的研究表明，PPT1在腫瘤發(fā)生及進(jìn)展中發(fā)揮重要作用[21]。目前關(guān)于PPT1的研究主要集中在PPT1在不同腫瘤中的表達(dá)與腫瘤生長、進(jìn)展以及預(yù)后之間的關(guān)系，根據(jù)TCGA數(shù)據(jù)庫分析，與配對(duì)的正常組織相比，在11種腫瘤中表達(dá)水平顯著升高(圖2)，并與腫瘤的病理分型、惡性程度、侵襲性和轉(zhuǎn)移等都有相關(guān)性。PPT1異常表達(dá)導(dǎo)致mTOR信號(hào)通路的過度激活，這種過度激活通過增加蛋白合成、抑制自噬和加快細(xì)胞周期進(jìn)程等方式來促進(jìn)腫瘤細(xì)胞的生長和增殖[75]。研究表明，在口腔鱗狀細(xì)胞癌(OSCC)使用erianin (一種天然的聯(lián)芐基化合物，具有抗癌作用)處理之后，觀察到PPT1的表達(dá)降低，mTOR與RHEB (ras homolog enriched in brain)的相互作用減少，mTOR的磷酸化減弱，這些結(jié)果提示PPT1可能通過調(diào)節(jié)mTOR與RHEB的相互作用從而調(diào)控mTOR通路[76]。研究人員利用CRISPR/Cas9技術(shù)敲除，與WT細(xì)胞相比，敲除細(xì)胞中磷酸化S6核糖體蛋白(mTOR信號(hào)通路下游效應(yīng)分子之一)降低，說明敲除抑制mTORC1活性，而且敲除后LC3B-II (light chain 3B isoform II)/LC3B-I (light chain 3B isoform I) (細(xì)胞自噬的標(biāo)志)水平增加，這表明PPT1可以通過mTOR信號(hào)通路調(diào)控細(xì)胞的生長和增殖[21]。另外，通過肝癌(HCC)患者的組織芯片分析，發(fā)現(xiàn)PPT1的表達(dá)水平與肝細(xì)胞癌的不良預(yù)后呈正相關(guān)，PPT1在HCC中的高表達(dá)促進(jìn)了細(xì)胞周期和上皮-間充質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition，EMT)途徑，這表明PPT1通過EMT途徑促進(jìn)肝癌細(xì)胞的增殖、遷移、侵襲[77]。此外，HCC單細(xì)胞RNA測序分析顯示，主要在HCC的巨噬細(xì)胞中表達(dá)，的豐度與患者的生存期有關(guān)，KEGG通路和GO生物過程富集進(jìn)一步分析表明與腫瘤代謝、免疫、腫瘤微環(huán)境和細(xì)胞外基質(zhì)密切相關(guān)，這說明PPT1也能通過腫瘤微環(huán)境影響腫瘤的發(fā)生與進(jìn)展[78～80]。研究表明，PPT1的過表達(dá)可以減少人神經(jīng)母細(xì)胞瘤細(xì)胞免受短鏈神經(jīng)酰胺或磷脂酰肌醇3-激酶抑制劑誘導(dǎo)的細(xì)胞凋亡，PPT1的過表達(dá)導(dǎo)致Ras的膜結(jié)合減少以及抗凋亡蛋白激酶Akt (protein kinase B，PK-B)的磷酸化(活性增加)，增強(qiáng)了細(xì)胞抗凋亡的能力，而敲降則增強(qiáng)了細(xì)胞對(duì)死亡的敏感性[81,82]。綜上，PPT1導(dǎo)致癌癥發(fā)生可能的原因如下：PPT1的缺失或功能異常導(dǎo)致自噬的紊亂，細(xì)胞內(nèi)有害物質(zhì)的積累，從而促進(jìn)癌癥的發(fā)生；PPT1的異常表達(dá)或功能失調(diào)導(dǎo)致mTORC1信號(hào)通路的異常激活，促進(jìn)癌細(xì)胞的增殖和轉(zhuǎn)移；在腫瘤微環(huán)境中發(fā)揮作用，PPT1通過影響腫瘤細(xì)胞與周圍環(huán)境的相互作用，促進(jìn)腫瘤免疫逃逸，進(jìn)一步影響腫瘤的發(fā)生和進(jìn)展；此外，PPT1還能通過調(diào)控細(xì)胞內(nèi)的氧化應(yīng)激反應(yīng)，維持細(xì)胞的氧化還原平衡進(jìn)而調(diào)控腫瘤的發(fā)生發(fā)展。另外，其他的一些研究也表明PPT1在腫瘤的發(fā)生發(fā)展中起著重要的作用[65,83,84]，根據(jù)TCGA癌癥患者的生存分析表明，的高表達(dá)與食管癌、頭頸癌腎細(xì)胞癌和肝細(xì)胞癌等多種癌癥的總生存期縮短有關(guān)，這表明PPT1在癌癥的發(fā)生和進(jìn)展中起著重要的調(diào)控作用，然而對(duì)于PPT1在癌癥中的確切作用機(jī)制和其作為治療靶點(diǎn)的潛力[85～87]，仍然需要更多的研究來加以確認(rèn)和深入探究。

圖2 PPT1在不同腫瘤組織與配對(duì)正常組織中的mRNA表達(dá)水平

紅色為腫瘤組織，黑色為正常組織，*表示<0.05。數(shù)據(jù)來自于http://gepia2.cancer-pku.cn。

總之，PPT1在疾病發(fā)生發(fā)展中發(fā)揮重要作用，PPT1的突變或者缺失會(huì)影響溶酶體的功能并造成脂褐素等物質(zhì)在神經(jīng)細(xì)胞中積累，最終導(dǎo)致神經(jīng)系統(tǒng)的退化和損傷，引發(fā)神經(jīng)退行性疾病，如INCL等。PPT1過表達(dá)會(huì)保護(hù)細(xì)胞免于細(xì)胞死亡，促進(jìn)腫瘤發(fā)生，而反義PPT1增加細(xì)胞死亡抑制腫瘤的發(fā)生(圖3)。

圖3 PPT1功能缺失導(dǎo)致多種疾病發(fā)生

4 靶向PPT1的臨床治療手段

基因的突變已經(jīng)被發(fā)現(xiàn)與INCL的發(fā)生有關(guān)，此外，PPT1還被發(fā)現(xiàn)在腫瘤等其他一些疾病的發(fā)生和發(fā)展中扮演重要的角色，因此PPT1作為潛在的治療靶點(diǎn)也受到了越來越多的關(guān)注。目前的研究已發(fā)現(xiàn)了一些可以影響PPT1的功能的臨床藥物和抑制劑(圖3)，如：Didemnin B (從加勒比被囊動(dòng)物中分離的環(huán)狀抗增殖肽)、白藜蘆醇(resveratrol，RSV)等。下表總結(jié)了一些臨床上靶向PPT1的藥物及小分子，用來治療PPT1調(diào)控失調(diào)導(dǎo)致的疾病(表1)。

此外，酶替代治療、干細(xì)胞治療等方法也被用于恢復(fù)PPT1的功能[93, 94]。酶替代療法利用溶酶體酶可以通過甘露糖-6-磷酸或甘露糖受體系統(tǒng)介導(dǎo)轉(zhuǎn)運(yùn)這一特性，由細(xì)胞分泌并被鄰近細(xì)胞吸收[95]，在PPT1小鼠靜脈注射重組人源PPT1 (recombinant human PPT1，rhPPT1)，可以顯著降低內(nèi)臟組織中脂褐素的積累，且通過植入式泵或定期鞘內(nèi)注射將重組酶長期輸送到中樞神經(jīng)系統(tǒng)對(duì)中樞神經(jīng)系統(tǒng)有顯著益處[96]。近年來干細(xì)胞技術(shù)的進(jìn)步，提高了神經(jīng)元干細(xì)胞移植治療INCL的可能性，當(dāng)正常(PPT1陽性)人神經(jīng)元干細(xì)胞移植到PPT1缺陷小鼠中時(shí)，細(xì)胞能在大腦中廣泛移植和遷移，并提供足夠水平的PPT1活性來減少脂褐素的積累，延緩宿主神經(jīng)元的喪失并改善運(yùn)動(dòng)功能[97]。然而這些治療方法在實(shí)驗(yàn)室和動(dòng)物模型中顯示出一定的潛力，但仍需要進(jìn)一步的研究和臨床試驗(yàn)來評(píng)估其安全性和療效。

5 結(jié)語與展望

棕櫚酰化是一種可逆的翻譯后修飾，其中16-C棕櫚酸通過硫酯鍵共價(jià)連接到靶蛋白內(nèi)的一個(gè)或多個(gè)半胱氨酸殘基上[98]。棕櫚?；诙喾N生物學(xué)功能中起著關(guān)鍵作用，包括膜錨定，囊泡運(yùn)輸，信號(hào)轉(zhuǎn)導(dǎo)和細(xì)胞結(jié)構(gòu)的維持[99,100]。PPT1作為一種溶酶體酶，它在棕櫚酰化蛋白的降解過程中起著重要作用。此外，PPT1還參與了生物體內(nèi)線粒體、溶酶體的功能及穩(wěn)態(tài)的調(diào)控，調(diào)節(jié)體內(nèi)蛋白質(zhì)、脂質(zhì)、鈣離子的運(yùn)輸。PPT1的功能缺陷會(huì)導(dǎo)致溶酶體功能障礙，進(jìn)而引發(fā)一系列的神經(jīng)退行性疾病，如嬰兒神經(jīng)元蠟樣脂褐素沉積癥、癲癇等。本文詳細(xì)討論了PPT1在溶酶體功能中的作用，以及其在疾病發(fā)生中的重要性，探討了PPT1在不同疾病發(fā)生中的作用及分子機(jī)制的最新的研究進(jìn)展，還討論了靶向PPT1的潛在治療策略，包括藥物干預(yù)、基因療法等。

迄今為止，對(duì)于PPT1在胞內(nèi)的轉(zhuǎn)運(yùn)、代謝、相互作用等一系列生理生化過程的研究仍有限，從已有的研究來看，PPT1不僅在維持細(xì)胞內(nèi)環(huán)境穩(wěn)態(tài)方面發(fā)揮重要作用，且在神經(jīng)退行性疾病以及腫瘤發(fā)生發(fā)展過程中也起到了重要作用。但對(duì)于PPT1的具體調(diào)控機(jī)制以及靶向PPT1的酶激活劑、抑制劑等方面的研究還有待進(jìn)一步的加強(qiáng)。PPT1介導(dǎo)的去棕櫚酸化及溶酶體功能調(diào)控在疾病，尤其是腫瘤發(fā)生發(fā)展中的功能及機(jī)制還需進(jìn)一步研究，利用小鼠等模型動(dòng)物探討PPT1突變介導(dǎo)的溶酶體功能失調(diào)在不同腫瘤發(fā)生中的作用及機(jī)制是重要的研究方向之一?；赑PT1在腫瘤中高表達(dá)并促進(jìn)其發(fā)生發(fā)展的重要作用，因此未來開發(fā)靶向PPT1的干預(yù)措施，能為疾病的治療提供可靠的策略。

表1 靶向PPT1的臨床藥物及抑制劑

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Progress on lysosomal PPT1-mediated regulation of cellular homeostasis and pathogenesis

Xinyi Zhou, Dong Chang, Huangying Xu, Ruiqi Guan, Chenghao Yan, Qiuyuan Yin, Jianwei Sun

Palmitoyl protein thioesterase 1(PPT1) is a lysosomal enzyme that catalyzes the protein depalmitoylation. It is considered to play a crucial role in regulating lysosomes, mitochondria and lipid metabolism. PPT1 has been reported to play an important role in the occurrence and progression of diseases, such as neurological diseases and cancers. However, the regulatory mechanisms remain unknown. In this review, we summarize the progress of PPT1 function and mechanisms in neurological disorders and cancers, which will provide as reference and guidance for exploring the regulatory mechanisms of PPT1 and developing new drugs for treating related diseases in the future.

Palmitoyl protein thioesterase 1; depalmitoylation; lysosome; disease

2023-07-07;

2023-09-19;

2023-09-28

國家自然科學(xué)基金(編號(hào): 82273460，32260167)和云南大學(xué)研究生科研創(chuàng)新項(xiàng)目(編號(hào): ZC-22223017, ZC-22223104)資助[Supported by the National Natural Science Foundation of China (Nos. 82273460，32260167) and Yunnan University Graduate Research Innovation Project (Nos. ZC-22223017, ZC-22223104).

周欣怡，碩士研究生，專業(yè)方向：腫瘤轉(zhuǎn)移與發(fā)生機(jī)制。E-mail: 1295351648@qq.com

常棟，碩士研究生，專業(yè)方向：腫瘤轉(zhuǎn)移與發(fā)生機(jī)制。E-mail: 13648831440@139.com

周欣怡和常棟并列第一作者。

孫建偉，博士，教授，研究方向：腫瘤發(fā)生與轉(zhuǎn)移機(jī)制研究。E-mail: jwsun@ynu.edu.cn

尹秋媛，博士，副研究員，研究方向：溶酶體的發(fā)生機(jī)制。E-mail: qiuyuan-_yin@ynu.edu.cn

10.16288/j.yczz.23-186

(責(zé)任編委: 史岸冰)