Induction of autophagy and inhibition of tumorigenesis by beclin 1

Liang, XH; Jackson, S; Seaman, M; et al.

Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor

Yue, ZY; Jin, SK; Yang, CW; et al.

Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease

Ravikumar, B; Vacher, C; Berger, Z; et al.

Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice

Komatsu, M; Waguri, S; Ueno, T; et al.

細(xì)胞自噬調(diào)控的分子機(jī)制研究進(jìn)展

李樂興，戴漢川

細(xì)胞自噬的研究方法

馬泰，孫國平，李家斌

細(xì)胞自噬

·編者按·

自噬（Autophagy，或稱自體吞噬）是細(xì)胞將自身一些蛋白質(zhì)和細(xì)胞器包裹在特定的膜結(jié)構(gòu)中，送入溶酶體（酵母的液泡）降解，產(chǎn)生能量和小分子，如氨基酸等供細(xì)胞再次利用的過程。這是一個(gè)受到緊密調(diào)控的步驟，此步驟是細(xì)胞生長、發(fā)育與穩(wěn)態(tài)中的常規(guī)步驟，它幫助細(xì)胞產(chǎn)物在合成、降解以及接下來的循環(huán)中保持一個(gè)平衡狀態(tài)。細(xì)胞自噬的研究是目前生物醫(yī)學(xué)領(lǐng)域熱點(diǎn)之一，廣泛參與各種生理和病理過程。日本科學(xué)家大隅良典因“對細(xì)胞自噬機(jī)制的發(fā)現(xiàn)”獲得2016年度的諾貝爾生理學(xué)或醫(yī)學(xué)獎。

1963年“自噬”的概念由比利時(shí)科學(xué)家Christian de Duve 在溶酶體國際會議上首先提出，Christian De Duve本人也因?yàn)殚_發(fā)了新的細(xì)胞研究手段，發(fā)現(xiàn)了溶酶體這一新的細(xì)胞器而獲得1974年諾貝爾生理學(xué)或醫(yī)學(xué)獎。當(dāng)代的自噬研究是1990年代酵母的研究人員通過識別的自噬相關(guān)基因而被推動。

細(xì)胞自噬主要有3種形式：微自噬（microautophagy)、巨自噬（macroautophagy）和分子伴侶介導(dǎo)的自噬（Chaperone-mediated autophagy，CMA）。通常所說的自噬即指巨自噬，也是目前研究最多的?，F(xiàn)在研究人員普遍采用電鏡、免疫熒光、蛋白質(zhì)印跡等方法檢測自噬體及其標(biāo)志蛋白。

本專題得到專家丁樹哲教授（華東師范大學(xué)）的大力支持。

·熱點(diǎn)數(shù)據(jù)排行·

截至 2016年10月19日，中國知網(wǎng)（CNKI）和Web of Science（WOS）的數(shù)據(jù)報(bào)告顯示，以“細(xì)胞自噬”為詞條可以檢索到的期刊文獻(xiàn)分別為 1035、 8569條，本專題將相關(guān)數(shù)據(jù)按照：研究機(jī)構(gòu)發(fā)文數(shù)、作者發(fā)文數(shù)、期刊發(fā)文數(shù)、被引用頻次進(jìn)行排行，結(jié)果如下。

研究機(jī)構(gòu)發(fā)文數(shù)量排名（CNKI）

研究機(jī)構(gòu)發(fā)文數(shù)量排名（WOS）

作者發(fā)文數(shù)量排名（WOS）

（數(shù)據(jù)來源：中國知網(wǎng)、Web of Science，檢索時(shí)間：2016-10-19）

期刊發(fā)文數(shù)量排名（CNKI）

期刊發(fā)文數(shù)量排名（WOS）

根據(jù)中國知網(wǎng)（CNKI）數(shù)據(jù)報(bào)告，以“細(xì)胞自噬”等為詞條可以檢索到的高被引論文排行結(jié)果如下。

國內(nèi)數(shù)據(jù)庫高被引論文排行

根據(jù)Web of Science統(tǒng)計(jì)數(shù)據(jù)，以“細(xì)胞自噬”為詞條可以檢索到的高被引論文排行結(jié)果如下。

國外數(shù)據(jù)庫高被引論文排行

·經(jīng)典文獻(xiàn)推薦·

基于Web of Science檢索結(jié)果，利用Histcite軟件選取LCS（Local Citation Score，本地引用次數(shù)）TOP 30文獻(xiàn)作為節(jié)點(diǎn)進(jìn)行分析，得到本領(lǐng)域推薦的經(jīng)典文獻(xiàn)如下。

本領(lǐng)域經(jīng)典文獻(xiàn)

來源出版物：FEBS Letters, 1993, 333(1-2): 169-174

Induction of autophagy and inhibition of tumorigenesis by beclin 1

Liang, XH; Jackson, S; Seaman, M; et al.

Abstract: The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumour cells. However, little is known about the genetic mediators of autophagy in mammalian cells or their role in tumour development. The mammalian gene encoding Beclin 1, a novel Bcl-2-interacting, coiled-coil protein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40%-75% of sporadic human breast cancers and ovarian cancers. Here we show, using gene-transfer techniques, that beclin 1 promotes autophagy in autophagy-defective yeast with a targeted disruption of agp6/vps30, and in human MCF7 breast carcinoma cells. The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice. Furthermore, endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia. Thus, beclin 1 is a mammalian autophagy gene that can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma. These findings

suggest that decreased expression of autophagy proteins may contribute to the development or progression of breast and other human malignancies.

來源出版物：Nature, 1999, 402(6762): 672-676

Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor

Yue, ZY; Jin, SK; Yang, CW; et al.

Abstract: The biochemical properties of beclin 1 suggest a role in two fundamentally important cell biological pathways: autophagy and apoptosis. We show here that beclin 1-/-mutant mice die early in embryogenesis and beclin 1+/-mutant mice suffer from a high incidence of spontaneous tumors. These tumors continue to express wild-type beclin 1 mRNA and protein, establishing that beclin 1 is a haploinsufficient tumor suppressor gene. Beclin 1-/-embryonic stem cells have a severely altered autophagic response, whereas their apoptotic response to serum withdrawal or UV light is normal. These results demonstrate that beclin 1 is a critical component of mammalian autophagy and establish a role for autophagy in tumor suppression. They both provide a biological explanation for recent evidence implicating beclin 1 in human cancer and suggest that mutations in other genes operating in this pathway may contribute to tumor formation through deregulation of autophagy.

來源出版物：Proceedings of the National Academy of Sciences, 2003, 100(25): 15077-15082

Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease

Ravikumar, B; Vacher, C; Berger, Z; et al.

Abstract: Huntington disease is one of nine inherited neurodegenerative disorders caused by a polyglutamine tract expansion. Expanded polyglutamine proteins accumulate abnormally in intracellular aggregates. Here we show that mammalian target of rapamycin (mTOR) is sequestered in polyglutamine aggregates in cell models, transgenic mice and human brains. Sequestration of mTOR impairs its kinase activity and induces autophagy, a key clearance pathway for mutant huntingtin fragments. This protects against polyglutamine toxicity, as the specific mTOR inhibitor rapamycin attenuates huntingtin accumulation and cell death in cell models of Huntington disease, and inhibition of autophagy has the converse effects. Furthermore, rapamycin protects against neurodegeneration in a fly model of Huntington disease, and the rapamycin analog CCI-779 improved performance on four different behavioral tasks and decreased aggregate formation in a mouse model of Huntington disease. Our data provide proof-of-principle for the potential of inducing autophagy to treat Huntington disease.

來源出版物：Nature Genetics, 2004, 36(6): 585-595

Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice

Komatsu, M; Waguri, S; Ueno, T; et al.

Abstract: Autophagy is a membrane-trafficking mechanism that delivers cytoplasmic constituents into the lysosome/vacuole for bulk protein degradation. This mechanism is involved in the preservation of nutrients under starvation condition as well as the normal turnover of cytoplasmic component. Aberrant autophagy has been reported in several neurodegenerative disorders, hepatitis, and myopathies. Here, we generated conditional knockout mice of Atg7, an essential gene for autophagy in yeast. Atg7 was essential for ATG conjugation systems and autophagosome formation, amino acid supply in neonates, and starvation-induced bulk degradation of proteins and organelles in mice. Furthermore, Atg7 deficiency led to multiple cellular abnormalities, such as appearance of concentric membranous structure and deformed mitochondria, and accumulation of ubiquitin-positive aggregates. Our results indicate the important role of autophagy in starvation response and the quality control of proteins and organelles in quiescent cells.

來源出版物：The Journal of Cell Biology, 2005, 169(3): 425-434

·推薦綜述·

細(xì)胞自噬調(diào)控的分子機(jī)制研究進(jìn)展

李樂興，戴漢川

細(xì)胞自噬是廣泛存在于真核細(xì)胞內(nèi)的一種溶酶體依賴性的降解途徑。細(xì)胞自噬導(dǎo)致細(xì)胞內(nèi)長壽命蛋白和受損傷細(xì)胞器的降解，使細(xì)胞在應(yīng)激條件下循環(huán)利用營養(yǎng)物質(zhì)和三羧酸循環(huán)產(chǎn)生的ATP繼續(xù)生存。根據(jù)細(xì)胞內(nèi)底物運(yùn)送到溶酶體腔方式的不同，哺乳動物細(xì)胞自噬可分為巨自噬（macroautophagy）、微自噬（microautophagy）和分子伴侶介導(dǎo)的自噬（chaperone-mediated autophagy，CMA）三種主要方式，但目前研究最為廣泛的是巨自噬。研究表明，細(xì)胞自噬在細(xì)胞內(nèi)穩(wěn)態(tài)、癌癥、心力衰竭、神經(jīng)退行性疾病、傳染病、衰老相關(guān)性疾病等生命過程中發(fā)揮著重要作用。近年來，隨著自噬基因和及其功能被相繼發(fā)現(xiàn)，自噬成為繼凋亡（apoptosis）之后生命科學(xué)最熱的研究領(lǐng)域之一。本文綜述了近年來細(xì)胞自噬的研究進(jìn)展，以期有助于細(xì)胞自噬調(diào)控的深入研究，并為治療心臟疾病（如動脈粥樣硬化）、癌癥（如乳腺癌）等提供理論基礎(chǔ)。

1 細(xì)胞自噬的過程

自噬包括生理?xiàng)l件的基礎(chǔ)型自噬和應(yīng)激條件下的誘導(dǎo)型自噬。細(xì)胞自噬主要包括以下過程：（1）自噬誘導(dǎo)，諸如輻射、饑餓、缺氧、細(xì)菌入侵、生長因子匱乏等多種因素均可誘導(dǎo)自噬發(fā)生；（2）囊泡（吞噬泡）成核，細(xì)胞內(nèi)質(zhì)網(wǎng)膜、線粒體外膜、高爾基復(fù)合體膜或質(zhì)膜等構(gòu)成囊泡的雙層或多層膜結(jié)構(gòu)，30多種 Atg（autophagy-related）蛋白在囊泡膜上順序耦聯(lián)組裝成PAS（pre-autophagosomal structures）。其中，Beclin1/ BECN1（Atg6）作為構(gòu)成III類PI3K（phosphoinositide 3-kinase）復(fù)合體的支架蛋白，通過Vps34-p150復(fù)合體與 Atg9、Atg14L 和 UVRAG（ultraviolet radiation resistance-associatedgene protein）等蛋白結(jié)合，形成PI3K核心復(fù)合體啟動自噬；（3）自噬體的成熟，PI3K核心復(fù)合體轉(zhuǎn)變成 PtdIns（3）P（phosphatidylinositol 3-phosphate），PtdIns（3）P作為“著陸臺”，募集自噬蛋白進(jìn)入到吞噬泡（phagophore）成核中心，在泛素化耦聯(lián)系統(tǒng)的輔助下，Atg10、Atg7、Atg3、Atg8/LC3、 Atg4和Atg12-Atg5-Atg16L1等最終被募集到PAS，參與囊泡膜延伸和自噬體（autophagosome）的成熟；（4）自噬溶酶體的融合，自噬體通過酸化與溶酶體融合形成自噬溶酶體（autophagolysosome）。自噬體膜的受體蛋白SQSTM1/p62接受酶作用的底物（錯(cuò)誤折疊蛋白或蛋白聚合體），降解 p62及其靶蛋白，清除線粒體、內(nèi)質(zhì)網(wǎng)等受損細(xì)胞器，釋放出營養(yǎng)物質(zhì)和ATP供細(xì)胞再利用。此外，p62還與自噬活性呈負(fù)相關(guān)性，反映自噬溶酶體溶解酶活性和自噬潮（autophagic flux）的強(qiáng)弱，因此可作為自噬的分子標(biāo)記。

2 細(xì)胞自噬的功能

生理性自噬是細(xì)胞的自我保護(hù)機(jī)制，有益于細(xì)胞的生長發(fā)育，保護(hù)細(xì)胞防止代謝應(yīng)激和氧化損傷，對維持細(xì)胞內(nèi)穩(wěn)態(tài)以及細(xì)胞產(chǎn)物的合成、降解和循環(huán)再利用具有重要作用；但自噬過度可能導(dǎo)致代謝應(yīng)激、降解細(xì)胞成分、細(xì)胞死亡等，打破細(xì)胞生長和死亡（細(xì)胞死亡至少分為三種形態(tài)學(xué)上不同的進(jìn)程，即細(xì)胞凋亡、自噬性細(xì)胞死亡和壞死，此處所指的死亡可能伴隨著細(xì)胞自噬，過度自噬以一種不同于凋亡和壞死的方式使細(xì)胞死亡，但自噬與二者還有一定的關(guān)聯(lián)性，比如 Bcl-2和Beclin1之間的互作）間的平衡。自噬在多種生理病理過程中發(fā)揮重要作用。缺血再灌注顯著上調(diào)Beclin1，激發(fā)心肌細(xì)胞自噬；Beclin1表達(dá)下調(diào)抑制自噬，減弱心肌損傷。研究認(rèn)為，自噬是動脈粥樣硬化發(fā)展過程中的一種保護(hù)機(jī)制，因?yàn)樽允赏ㄟ^加工氧化修飾蛋白使斑塊固化，而自噬缺陷加劇動脈粥樣硬化。在腫瘤細(xì)胞生物學(xué)上，根據(jù)細(xì)胞基因組成和細(xì)胞所處環(huán)境的變化，自噬既能抑制腫瘤抑制因素發(fā)揮作用，自噬基因的缺失又可促進(jìn)腫瘤的生成。研究發(fā)現(xiàn)，一方面，自噬可使乳腺癌細(xì)胞繼續(xù)生存；而另一方面，自噬又可導(dǎo)致結(jié)腸癌細(xì)胞HCT116的死亡。目前普遍認(rèn)為，在乏氧、營養(yǎng)缺乏、代謝應(yīng)激等條件以及抗癌治療（如化學(xué)療法、放射療法）等環(huán)境下，癌細(xì)胞通過自噬可以繼續(xù)生存。此外，自噬還可能在衰老、炎癥、細(xì)胞凋亡、胞內(nèi)病原體入侵、神經(jīng)退行性疾病等方面發(fā)揮著重要作用。

3 細(xì)胞自噬調(diào)控的分子機(jī)制

3.1 泛素樣蛋白系統(tǒng)對細(xì)胞自噬的調(diào)控

泛素化是在翻譯后水平上進(jìn)行蛋白修飾的一種方式，參與蛋白酶體依賴性蛋白水解、蛋白功能調(diào)控、亞細(xì)胞分布和/或蛋白質(zhì)互作。在泛素激活酶（ubiquitin-activating enzyme，El）、泛素接合酶（ubiquitin-conjugating enzyme，E2）以及泛素蛋白連接酶（ubiquitin-protein ligase，E3）的連續(xù)作用下，泛素與底物蛋白特定的Lys殘基共價(jià)結(jié)合完成泛素化。同時(shí)，泛素化也是一種可逆性的過程，可由去泛素化酶將泛素從蛋白質(zhì)上除去。泛素化主要包括以下3步酶促反應(yīng)過程：（1）在ATP作用下，E1可在其Cys和泛素的C-端的Gly之間形成巰酯鍵，即E1-SH～Ub，從而激活泛素；（2）在ATP和E2酶作用下，泛素從E1轉(zhuǎn)移到E2上，同樣以巰酯鍵的方式結(jié)合（E2-SH～Ub）；（3）E3酶可以特異性識別底物蛋白并與之結(jié)合，與此同時(shí) E2將激活的泛素直接轉(zhuǎn)移到某些 E3結(jié)合的底物上，經(jīng)過多個(gè)重復(fù)，多個(gè)泛素之間通過Lys相互連接，在底物上形成多泛素鏈。E1-樣酶Atg7和E2-樣酶Atg10泛素樣反應(yīng)后，泛素樣蛋白Atg12與Atg5 Lys130共價(jià)耦聯(lián)，Atg16L1作為連接蛋白，增強(qiáng)Atg12和E3泛素連接酶樣蛋白Atg5間的互作，而后Atg12-Atg5與Atg16L1形成E3連接酶樣復(fù)合體并定位于PAS。半胱氨酸酶Atg4酶切LC3并暴露C-端最后5個(gè)Gly殘基，在E2-樣酶Atg3輔助下，與磷脂酰乙醇胺（phosphatidylethanolamine，PE）發(fā)生E3-樣共軛形成脂化的LC3（LC3-II）并定位于PAS，吞噬泡加工成為成熟自噬體。

3.2 mTOR信號通路對細(xì)胞自噬的調(diào)控

mTOR（mammalian target of rapamycin）屬于Ser/Thr激酶，參與細(xì)胞發(fā)育、核糖體生成和代謝調(diào)控等生物學(xué)過程。mTOR包括雷帕霉素敏感型mTORC1和雷帕霉素非敏感型 mTORC2。mTORC1通過磷酸化ULK1-Atg13-RB1CC1-C12orf44/Atg101復(fù)合體使其失活，從而負(fù)調(diào)控細(xì)胞自噬體的形成，其活化程度可反映自噬水平，如果阻斷mTORC1的功能，Ser/Thr激酶可磷酸化Atg1復(fù)合體并激活自噬。mTORC2的磷酸化能激活 Akt（PKB）和 Atg1抑制自噬，也可上調(diào) HIF1A（hypoxia-inducible factor 1A）的表達(dá)。mTOR調(diào)控細(xì)胞自噬主要包括mTOR非依賴性和mTOR依賴性兩條信號通路。

3.2.1 mTOR非依賴性信號通路

有實(shí)驗(yàn)發(fā)現(xiàn)，Mst1（mammalian Ste20-like kinase 1）可使 Beclin1BH3結(jié)構(gòu)域 N-端的 Thr108磷酸化，增強(qiáng)Beclin1與Bcl-2和/或Bcl-xL疏水溝α3螺旋間的互作，使Beclin1同源二聚體穩(wěn)定，減弱Atg14L與Beclin1的結(jié)合，降低 Beclin1-PI3K-Atg14L復(fù)合體脂激酶 Vps34的活性以抑制自噬。Molejon等認(rèn)為，VMP1（vacuole membraneprotein 1）20位氨基酸殘基C-端親水性結(jié)構(gòu)域（VMP1-AtgD）與Beclin1 BH3結(jié)構(gòu)域結(jié)合致使Bcl-2與Beclin1解離，最終形成VMP1-Beclin1-hVps34-Atg14L復(fù)合體共同定位于PAS，啟動PI3P生成、泛素樣級聯(lián)反應(yīng)和囊泡的形成。有趣的是，棉酚衍生物ApoG2與Mst1作用相反，ApoG2破壞Beclin1和Bcl-2/xL的互作，釋放出 Beclin1 BH3結(jié)構(gòu)域，從而誘導(dǎo)自噬，但氯喹（chloroquine，CQ）與 ApoG2結(jié)合可阻斷自噬體與溶酶體的融合。而EGFR（epidermal growthfactor receptor）通過磷酸化Beclin1多個(gè)位點(diǎn)的酪氨酸，增強(qiáng)Beclin1與抑制劑的結(jié)合能力，降低Vps34脂激酶活性以抑制自噬。

3.2.2 mTOR依賴性信號通路

Qased等發(fā)現(xiàn)，Ser/Thr蛋白激酶 ATM（ataxia telangiectasia mutated）屬 PIKK（PI3K-related protein kinase）家族，ATM C-端序列與PI3K催化區(qū)同源，其能夠刺激 LBK/AMPK/TSC2通路的下游信號，抑制mTORC1。mTORC1被抑制后可激活 ULK1（unc-51 like autophagy activatingkinase 1），ULK1通過與UVRAG結(jié)合再使 Beclin1Ser14磷酸化，從而增強(qiáng) Beclin1-Vps34-Atg14L復(fù)合體的活性，啟動自噬。此外，作為ULK復(fù)合體的重要組成成分，F(xiàn)IP200的缺失會造成MEF（mouse embryonicfibroblast）的Atg14-Atg1-WIPI誘導(dǎo)缺陷。然而，Efeyan等指出，Rag GTPases活化后能夠募集mTORC1進(jìn)入到溶酶體表面導(dǎo)致自噬缺陷。除前述作用外，ApoG2亦可抑制線粒體電子傳遞以產(chǎn)生ROS（reactive oxygen species），ROS通過提高 ERK（extracellular regulated protein kinases）、JNK（c-Jun N-terminal kinases）（靶作用于Bim和Atg5）的磷酸化水平，加快HMGB1（high-mobility group box 1）從細(xì)胞核到細(xì)胞質(zhì)的轉(zhuǎn)運(yùn)，以及抑制mTOR信號，啟動細(xì)胞自噬。但NAC（N-acetyl-cysteine）可減弱HMGB1從細(xì)胞核到細(xì)胞質(zhì)的轉(zhuǎn)運(yùn)，誘導(dǎo)細(xì)胞凋亡和殺傷。同樣，EGFR也可使PI3K、Akt和mTOR的酪氨酸磷酸化，負(fù)調(diào)控細(xì)胞自噬。

3.2.3 其他信號對細(xì)胞自噬的調(diào)控

研究表明，在細(xì)胞核中，p53可通過sestrin1/2蛋白激活A(yù)MPKmTORC1信號通路，從而抑制mTORC1以

上調(diào)自噬水平；也可通過激活DAPK1（death-associated proteinkinase 1），磷酸化Beclin1，促進(jìn)細(xì)胞自噬；還能通過激活抗凋亡蛋白 Bcl-2家族，解除 Bcl-2/xL與Beclin1之間的抑制作用而上調(diào)細(xì)胞自噬。而在細(xì)胞質(zhì)中，p53缺失的癌細(xì)胞的自噬水平上調(diào)，重新載入 p53后可下調(diào)細(xì)胞自噬水平。還有研究表明，脂多糖（lipopolysaccharide，LPS）可通過 TLR（Toll like receptor）調(diào)節(jié)細(xì)胞自噬的水平。在天然免疫研究中發(fā)現(xiàn)，LPS能誘導(dǎo)小鼠單核巨噬細(xì)胞和人巨噬細(xì)胞自噬體形成，抑制TLR4后自噬體形成明顯減少。LPS/TLR4信號通路介導(dǎo)的自噬可加強(qiáng)TLR4信號通路中髓樣分化蛋白（myeloid differentiation factor 88，MyD88）或IFN誘導(dǎo)接頭蛋白[Toll/interleukin （IL）-1receptor homology domain （TIR）-containing adaptorinducing interferon（IFN）-β，TRIF]與自噬蛋白 Beclin1的相互作用，抑制Beclin1和自噬信號通路中Bcl-2的結(jié)合，增強(qiáng)NF-κB核轉(zhuǎn)錄因子的活性。

此外，PI3K-Akt-FoxO信號通路可介導(dǎo)谷氨酰胺合成酶的活化，參與募集Atg蛋白，提高LC3和ULK2的共定位水平。ox-LDL（oxidized low-densitylipoprotein）極大地促進(jìn)動脈粥樣硬化的發(fā)生、發(fā)展，適當(dāng)濃度（10～40 μg/mL）的ox-LDL可激活保護(hù)性細(xì)胞自噬，致使內(nèi)皮細(xì)胞、血管細(xì)胞和巨噬細(xì)胞的溶酶體降解ox-LDL。

3.3 miRNA對細(xì)胞自噬的調(diào)控

microRNA（miRNA）是一類長約22 nt的內(nèi)源性非編碼小 RNA分子，在轉(zhuǎn)錄后水平調(diào)控基因的表達(dá)。研究表明，miRNA參與細(xì)胞生長發(fā)育、炎癥、腫瘤、衰老、凋亡等多種生理病理過程。近年來，還發(fā)現(xiàn)miRNA參與了細(xì)胞自噬調(diào)控，在自噬的發(fā)生和形成中發(fā)揮重要作用。miRNA與其靶mRNA 3′-UTR部分互補(bǔ)序列配對，通過降解mRNA和/或抑制蛋白翻譯來調(diào)控基因表達(dá)，并且miRNA與其靶mRNA的序列同源性決定了是降解mRNA還是抑制翻譯。營養(yǎng)饑餓、缺氧、雷帕霉素等可誘導(dǎo)細(xì)胞自噬，但多數(shù) miRNA在自噬過程的不同階段可通過作用于Atg蛋白以拮抗這種誘導(dǎo)作用，抑制細(xì)胞自噬，對細(xì)胞造成傷害，且無細(xì)胞特異性。

3.3.1 囊泡（吞噬泡）成核階段

在正常生長條件下，抗凋亡蛋白家族 Bcl-2（包括Bcl-2、Bcl-xL、Mcl-1、A1、Bcl-W和Rubicon）與Beclin1結(jié)合能力最強(qiáng)，Beclin1BH3結(jié)構(gòu)域與Bcl-2和/或Bcl-xL的疏水溝互作，負(fù)調(diào)控Beclin1-Vps34 PI3K-p150核心復(fù)合體的形成和活性，形成Beclin1同源二聚體抑制自噬；當(dāng)自噬被誘導(dǎo)時(shí)，Beclin1與Bcl-2解離啟動自噬。此外，Beclin1 CCD（coiled-coil domain）結(jié)構(gòu)域也可與Bcl-2和/或Bcl-xL的BH4結(jié)構(gòu)域互作，但miR-376b、miR-216a、miR-30a、miR-30d都可靶向 Beclin1，抑制其表達(dá)，減弱Beclin1與Bcl-2的結(jié)合和解離能力，從而調(diào)控自噬。Atg9作為唯一的跨膜蛋白，定位于PAS、線粒體和高爾基復(fù)合體，啟動脂質(zhì)從生物膜轉(zhuǎn)運(yùn)到PAS，介導(dǎo)組裝完整的囊泡膜，但miR-34a抑制Atg9A表達(dá)，中斷囊泡成核。Atg14L可以調(diào)節(jié)脂激酶 Vps34活性，并可募集 ULK1以使 Beclin1磷酸化，但 miR-195靶抑制Atg14，以抑制細(xì)胞自噬。PI3KC3是PI3K復(fù)合體的核心蛋白，miR-338-5p通過抑制PI3KC3的表達(dá)，阻斷囊泡成核，從而負(fù)調(diào)控細(xì)胞自噬。

3.3.2 自噬體的成熟階段

miR-216a、miR-181-a、miR-30a和miR-30d靶作用于Atg5，miR-375、miR-199a-5p靶作用于Atg7，miR-23b、miR-30d靶作用于 Atg12，抑制泛素化蛋白表達(dá)，負(fù)調(diào)控囊泡膜延伸。除可脂化LC3-I外，Atg4酶（Atg4B）同樣可以介導(dǎo)自噬體膜表面的LC3蛋白發(fā)生去脂化作用，從而使細(xì)胞自噬循環(huán)利用LC3蛋白。研究發(fā)現(xiàn)，miR-376b靶作用于Atg4C、miR-101靶作用于Atg4D以抑制細(xì)胞自噬。Suzuki等認(rèn)為，LC3側(cè)鏈lys49參與調(diào)控LC3和Atg13 C-端LIR（LC3 interactingregion，氨基酸序列H441D442D443F444V445M446I447）間的互作。miR-204通過靶向抑制LC3B的表達(dá)，阻斷LC3B的脂化，從而抑制自噬體的成熟。LC3-II與自噬體形成有關(guān)，而未成熟的LC3-I是可溶的。因此，LC3-II或LC3-II/LC3-I比值常用作自噬體形成的分子標(biāo)記，用于自噬現(xiàn)象的觀察和分析。

3.3.3 自噬溶酶體的融合階段

研究認(rèn)為，Atg8不僅參與膜延伸，且可經(jīng)接頭蛋白LIR結(jié)構(gòu)將錯(cuò)誤折疊或聚合蛋白質(zhì)、受損細(xì)胞器、病原體募集到自噬體膜進(jìn)行降解。酵母 Atg8的哺乳動物同源蛋白有7種，即LC3A、LC3B、LC3C、GABARAP、GABARAPL-1、GABARAPL-3、GABARAPL-2，因此LC3亞型與Atg13 LIR親和力的特異性也是影響自噬體形成的關(guān)鍵因素。但miR-204通過靶抑制LC3B表達(dá)，抑制自噬，從而降低自噬溶酶體的分解能力。

此外，miR-22、miR-138、miR-302b、miR-210分別靶作用于HMGB1、Mst1、EGFR、VMP1，且miR-22與HMGB1、Mst1與miR-138、miR-302b與EGFR、miR-

210與VMP1均呈負(fù)相關(guān)，這些miRNA通過抑制蛋白表達(dá)，從而負(fù)調(diào)控前述細(xì)胞自噬信號通路。Ucar等認(rèn)為，IGF-1（insulinlike growth factor-1）可上調(diào)miR- 212/132的表達(dá)，并且miR-212/132靶抑制FoxO3表達(dá)以致弱細(xì)胞自噬。miR-132還可激活PI3K-Akt-mTOR信號通路，抑制細(xì)胞自噬。hsa-let-7g可能通過下調(diào) LOX-1、ROS的表達(dá)，以抑制細(xì)胞自噬。miR-101還可靶作用于STMN1（在一定程度上拮抗 miR-101的抑制作用）、RAB5A，miR-30d靶作用于Atg2，miR-130a靶作用于Atg2B、DICER1，miR-224（可被自噬降解）可能靶作用于Smad4，這些均能夠抑制細(xì)胞自噬。

研究還發(fā)現(xiàn)，除多數(shù)miRNA對細(xì)胞自噬有抑制作用外，少數(shù)miRNA也可增強(qiáng)細(xì)胞自噬水平（表2）。Wan等認(rèn)為，miR-155、miR-7可靶作用于mTOR信號多種分子，如RHEB、RICTOR和RPS6KB2等，負(fù)調(diào)控PI3KAkt-mTOR信號通路，誘導(dǎo)細(xì)胞自噬。此外，miR-99a也可通過抑制mTOR信號、miR-18a通過上調(diào)ATM的表達(dá)以增強(qiáng)細(xì)胞自噬水平。

目前，細(xì)胞自噬與miRNA關(guān)系的研究還處于初級階段，關(guān)于miRNA如何參與調(diào)控自噬的研究有待進(jìn)一步闡明。隨著研究的深入，不同的miRNA在細(xì)胞自噬中的作用與機(jī)制將會更加清楚，對理解機(jī)體病理生理過程、抗感染以及天然免疫機(jī)制等方面具有重要意義。在自噬過程涉及的復(fù)雜的分子調(diào)節(jié)網(wǎng)絡(luò)中，解析 miRNA與細(xì)胞自噬關(guān)系，有助于尋找新的自噬調(diào)控靶點(diǎn)，也為揭示自噬分子調(diào)控機(jī)制提供新的思路和策略。

3.4 細(xì)胞自噬調(diào)控的其他機(jī)制

caspase屬于半胱天冬氨酸蛋白酶（cysteinyl containing aspartate-specific protease），在正常細(xì)胞中，caspase以無活性的酶原形式存在。MOMP（mitochondrialouter membrane permeabilisation）是激活caspase的主要機(jī)制之一，細(xì)胞色素C進(jìn)入到細(xì)胞質(zhì)啟動凋亡復(fù)合體的形成，激活 caspase-9，caspase-9被裂解后激活 caspase-3和caspase-7。細(xì)胞應(yīng)激激活caspase，活化的caspase能夠啟動細(xì)胞降解，調(diào)控細(xì)胞凋亡。研究發(fā)現(xiàn)，ATP合成增加能夠抑制細(xì)胞自噬，而凋亡蛋白caspase Dcp-1調(diào)控自噬潮（autophagicflux）的發(fā)生。pro-Dcp-1在線粒體聚集，Dcp-1與核苷酸轉(zhuǎn)移酶SesB互作，降低SesB的穩(wěn)定性并下調(diào)ATP的合成，從而啟動細(xì)胞自噬。Atg16L1蛋白發(fā)生Thr300Ala/Thr316Ala突變，能夠致使caspase-3對Atg16L1的降解敏感性增強(qiáng)，進(jìn)一步影響自噬體的形成。但此時(shí)，36 kDa和34 kDa裂解產(chǎn)物的出現(xiàn)，并不是由于 caspase-3活性增強(qiáng)所造成的。calpain是一種鈣依賴性的半胱氨酸蛋白酶，可在 Atg5蛋白的氨基末端進(jìn)行裂解，產(chǎn)生一個(gè)相對分子質(zhì)量為24000的產(chǎn)物，從而驅(qū)使細(xì)胞自噬向細(xì)胞凋亡轉(zhuǎn)變，將自噬與凋亡聯(lián)系起來。除Atg5蛋白外，Beclin1、Bax也是calpain的作用靶點(diǎn)，更說明自噬與凋亡有一定的關(guān)聯(lián)性。

AMPK（AMP-activated protein kinase）是一種異源三聚體，包括α催化亞基1個(gè)和調(diào)控型β、γ亞基各1個(gè)。多種應(yīng)激因素可激活A(yù)MPK，AMPK活化后既可抑制自噬，又可激活自噬。細(xì)胞滲透性核苷酸類似物AICAR（AICA riboside）可以激活肝細(xì)胞AMPK，AMPK磷酸化水平的提高能夠抑制細(xì)胞自噬，致使神經(jīng)元胞內(nèi)泛素化蛋白的累積；而在酵母和多種哺乳動物細(xì)胞內(nèi)，AMPK的活化可以激活自噬。Yu等認(rèn)為，蛋白磷酸酶PP2A（protein phosphatase 2A）調(diào)控AMPK α和γ亞基間的互作，使AMPK α亞基去磷酸化，從而使AMPK抑制mTOR磷酸化，啟動細(xì)胞自噬。

研究還認(rèn)為，蛋白乙?；嵌喾N細(xì)胞進(jìn)程的關(guān)鍵調(diào)控機(jī)制。Yi等對釀酒酵母進(jìn)行遺傳學(xué)分析證實(shí)，自噬過程必需組蛋白乙酰轉(zhuǎn)移酶Esa1的參與，且Atg3是Esa1的作用底物。Atg3的 K19和 K48發(fā)生乙?；刂艫tg3-Atg8互作和Atg8的脂化，進(jìn)而調(diào)控細(xì)胞自噬。去除脫乙酰酶Rpd3后，提高K19-K48的乙?；?，可以增強(qiáng)細(xì)胞自噬。

4 結(jié)語與展望

在植物、酵母、蠕蟲、鼠和人等真核生物中都發(fā)現(xiàn)細(xì)胞自噬現(xiàn)象，其與多種生理病理過程密切相關(guān)，成為生命科學(xué)領(lǐng)域研究的熱點(diǎn)之一。雖然，目前的研究在一定程度上闡明了細(xì)胞自噬調(diào)控的分子機(jī)制，但極其復(fù)雜的自噬過程因位置、狀態(tài)、環(huán)境、疾病的不同，信號通路、生物功能也會有所不同。因此，自噬體膜來源、自噬溶酶體融合機(jī)制、自噬相關(guān)蛋白功能、自噬調(diào)控網(wǎng)絡(luò)以及尚存爭議的自噬對細(xì)胞生存與死亡所發(fā)揮的雙重作用等，仍待進(jìn)一步研究，以期為人類預(yù)防和治療腫瘤、免疫性疾病、神經(jīng)退行性疾病和代謝性疾病等帶來新的希望。?

【作者單位：華中農(nóng)業(yè)大學(xué)動物醫(yī)學(xué)院】

（摘自《中國細(xì)胞生物學(xué)學(xué)報(bào)》2015年2期）

·高被引論文摘要·

被引頻次：52

細(xì)胞自噬的研究方法

馬泰，孫國平，李家斌

細(xì)胞自噬的研究是目前生物醫(yī)學(xué)領(lǐng)域熱點(diǎn)之一，廣泛參與各種生理和病理過程。目前普遍采用的自噬檢測方法包括電鏡、免疫熒光、蛋白質(zhì)印跡等方法檢測自噬體及其標(biāo)志蛋白。研究的深入對自噬的檢測方法也提出了更高的要求，自噬功能障礙包括自噬體形成和降解障礙，因此，準(zhǔn)確全面地評估自噬不僅包括自噬體的檢測，還包括動態(tài)觀察整個(gè)自噬性降解的過程是否順暢（即自噬潮分析）。另外，通過藥物或基因干預(yù)技術(shù)來人為地調(diào)控自噬以觀察其在體內(nèi)體外模型中的作用也是自噬分析的重要內(nèi)容。需要注意的是，任何一種方法單獨(dú)應(yīng)用均不能作為自噬的依據(jù)，對任何方法得到的結(jié)果進(jìn)行解釋時(shí)必須慎重，特別是不能將自噬體的增多減少或自噬相關(guān)蛋白表達(dá)的高低等同于自噬的增強(qiáng)或減弱。

細(xì)胞自噬；自噬體；微管相關(guān)蛋白1輕鏈3；自噬潮；檢測方法

來源出版物：生物化學(xué)與生物物理進(jìn)展, 2012, 39(3): 204-209

被引頻次：44

細(xì)胞自噬與腫瘤發(fā)生的關(guān)系

王寵，張萍，朱衛(wèi)國

摘要：細(xì)胞自噬（autophagy）是將細(xì)胞內(nèi)受損、變性或衰老的蛋白質(zhì)以及細(xì)胞器運(yùn)輸?shù)饺苊阁w進(jìn)行消化降解的過程。正常生理情況下，細(xì)胞自噬利于細(xì)胞保持自穩(wěn)狀態(tài)；在發(fā)生應(yīng)激時(shí)，細(xì)胞自噬防止有毒或致癌的損傷蛋白質(zhì)和細(xì)胞器的累積，抑制細(xì)胞癌變；然而腫瘤一旦形成，細(xì)胞自噬為癌細(xì)胞提供更豐富的營養(yǎng)，促進(jìn)腫瘤生長。因此，在腫瘤發(fā)生發(fā)展的過程中，細(xì)胞自噬的作用具有兩面性。盡管大多數(shù)抑癌蛋白可以激活細(xì)胞自噬這一結(jié)論被廣泛接受，但 p53作為重要的抑癌蛋白，在細(xì)胞核和細(xì)胞漿不同的亞細(xì)胞定位中對細(xì)胞自噬有著截然相反的調(diào)控。對于細(xì)胞自噬和癌癥發(fā)生之間關(guān)系亟待深入的研究，這將會有助于人類更好地認(rèn)識并最終攻克癌癥。本文將針對細(xì)胞自噬與腫瘤發(fā)生過程中主要的信號調(diào)節(jié)通路展開介紹。

關(guān)鍵詞：細(xì)胞自噬；癌癥；Beclin1；FoxO；mTOR；p53

來源出版物：中國生物化學(xué)與分子生物學(xué)報(bào), 2010, 26(11): 988-997

被引頻次：41

細(xì)胞自噬與腫瘤

杜海磊，邱偉華，楊衛(wèi)平

摘要：自噬又稱為 II型程序性細(xì)胞死亡（typeII programmed cell death）是以胞質(zhì)內(nèi)出現(xiàn)雙層膜結(jié)構(gòu)包裹長壽命蛋白和細(xì)胞器的自噬體為特征的細(xì)胞“自我消化”的一系列生化過程。自噬現(xiàn)象最早是 Ashford和Porter于1962年用電子顯微鏡在人的肝細(xì)胞中觀察到。近年來隨著酵母模型的建立，分子生物學(xué)及基因技術(shù)的發(fā)展，對自噬的研究有了很大的進(jìn)展。

關(guān)鍵詞：自吞噬作用；細(xì)胞凋亡；溶酶體；腫瘤；信號轉(zhuǎn)導(dǎo)

來源出版物：中國病理生理雜志, 2010, 26(2): 401-404

被引頻次：39

冬凌草甲素通過誘導(dǎo)人宮頸癌HeLa細(xì)胞自噬下調(diào)凋亡的機(jī)制

崔僑，田代真一，小野寺敏，等

摘要：研究冬凌草甲素通過誘導(dǎo)人宮頸癌 HeLa細(xì)胞自噬拮抗凋亡的機(jī)制。MTT法測定冬凌草甲素對HeLa細(xì)胞的細(xì)胞毒作用。通過相差顯微鏡觀察細(xì)胞形態(tài)學(xué)變化，用瓊脂糖凝膠電泳檢測DNA片段化，用流式細(xì)胞儀檢測細(xì)胞自噬和凋亡水平，用Westernblotting檢測分析藥物對蛋白質(zhì)表達(dá)的影響。冬凌草甲素明顯抑制HeLa細(xì)胞的增殖，誘導(dǎo)HeLa細(xì)胞凋亡，同時(shí)誘導(dǎo)HeLa細(xì)胞發(fā)生自噬。Westernblotting檢測結(jié)果表明，冬凌草甲素作用24 h后，促凋亡蛋白Bax、細(xì)胞色素c和控制Bax活力的去乙?；窼IRT-1的表達(dá)明顯改變。冬凌草甲素（64 μmol·L-1）誘導(dǎo)的自噬通過影響SIRT-1和線粒體途徑蛋白的表達(dá)下調(diào)凋亡。

關(guān)鍵詞：冬凌草甲素；HeLa細(xì)胞；自噬；細(xì)胞凋亡

來源出版物：藥學(xué)學(xué)報(bào), 2007, 42(1): 35-39

被引頻次：36

細(xì)胞自噬在腫瘤中作用的研究進(jìn)展

李國東，吳德全，李本義

摘要：自噬（autophagy）是廣泛存在于真核細(xì)胞中的基本生命現(xiàn)象，是程序性細(xì)胞死亡形式之一，近年來備受關(guān)注。自噬活性的變化與人類腫瘤的發(fā)生、發(fā)展密切相關(guān)，并可從多個(gè)層面影響腫瘤進(jìn)程，包括腫瘤細(xì)胞凋亡、血管生成及抗腫瘤治療等。本文就自噬及其在腫瘤中作用的研究進(jìn)展進(jìn)行簡要綜述。

關(guān)鍵詞：自噬；腫瘤；信號調(diào)控；腫瘤治療

來源出版物：癌癥, 2009, 28(4): 445-448

被引頻次：36

缺血/再灌注過程中心肌細(xì)胞自噬研究進(jìn)展

李欣志，劉建勛

摘要：自噬是一種廣泛存在于真核細(xì)胞中的生命現(xiàn)象，心肌細(xì)胞營養(yǎng)缺乏、缺血/再灌注損傷、心衰等均可誘發(fā)細(xì)胞自噬。缺血/再灌注過程中的心肌細(xì)胞自噬可以維持心肌細(xì)胞穩(wěn)態(tài)、減少細(xì)胞缺失，但是自噬作用也可導(dǎo)致心肌細(xì)胞死亡。

關(guān)鍵詞：自噬；缺血/再灌注；心肌細(xì)胞

來源出版物：中國藥理學(xué)通報(bào), 2008, 24(6): 704-707

被引頻次：32

自噬的抑制影響長春新堿誘導(dǎo)的肝癌細(xì)胞自噬性凋亡

彭心昭，陳英，樸英杰

摘要：目的：研究自噬特異性抑制劑3-甲基腺嘌呤（3-methyladenine，3MA）對長春新堿（vincristine，VCR）誘導(dǎo)的肝癌細(xì)胞系 HepG2細(xì)胞自噬性凋亡的影響。方法：利用已建立的VCR誘導(dǎo)HepG2細(xì)胞自噬性凋亡之模型，采用monodansylcadaverin（MDC）染色及流式細(xì)胞術(shù)熒光強(qiáng)度測定方法對自噬進(jìn)行定量研究，采用流式細(xì)胞術(shù)及DNA電泳檢測細(xì)胞凋亡。結(jié)果：3MA可以特異性地阻止自噬泡形成，還可以部分地抑制HepG2細(xì)胞自噬性凋亡。結(jié)論：自噬是VCR誘導(dǎo)的HepG2細(xì)胞自噬性凋亡所必需的。

關(guān)鍵詞：自吞噬作用/藥物作用；凋亡；長春新堿；3-甲基腺嘌呤

來源出版物：腫瘤, 2004, 24(1): 32-34

被引頻次：31

淋巴細(xì)胞自噬性凋亡的超微結(jié)構(gòu)細(xì)胞化學(xué)研究

樸英杰，黃行許，霍霞，等

摘要：本實(shí)驗(yàn)用透射電鏡和細(xì)胞化學(xué)染色法觀察放線菌酮處理和γ射線照射大鼠胸腺、脾臟和腸系膜淋巴結(jié)的超微結(jié)構(gòu)。結(jié)果顯示，放線菌酮注射后4 h，或γ射線照的2 h后引起大鼠胸腺、脾臟及腸系膜淋巴結(jié)中的淋巴細(xì)胞凋亡。凋亡淋巴細(xì)胞發(fā)生一系列的形態(tài)學(xué)變化，其胞核染色質(zhì)凝集、重排，使核呈不同的形狀；凋亡淋巴細(xì)胞的線粒體、內(nèi)質(zhì)網(wǎng)、溶酶體等細(xì)胞器大量增殖，我們稱之為細(xì)胞反跳現(xiàn)象；大部分凋亡淋巴細(xì)胞凋亡時(shí)呈現(xiàn)典型的自噬性凋亡特征，即內(nèi)質(zhì)網(wǎng)大量增殖、分割、包裹凋亡淋巴細(xì)胞內(nèi)變性的細(xì)胞成分形成大量自噬體，自噬體和溶酶體融合成為自噬佐調(diào)亡小體，和非自噬性凋亡形成的凋亡小體一起，由巨噬細(xì)胞消化分解。

關(guān)鍵詞：自噬性凋亡；淋巴細(xì)胞；放線菌酮；γ射線：超微細(xì)胞化學(xué)

來源出版物：第一軍醫(yī)大學(xué)學(xué)報(bào), 1996, 16(3): 165-171

被引頻次：25

自噬參與心臟疾病調(diào)控的研究進(jìn)展

謝鳳，柳威，陳臨溪

摘要：細(xì)胞自噬（autophagy）是將細(xì)胞內(nèi)受損、變性或衰老的蛋白質(zhì)以及細(xì)胞器運(yùn)輸?shù)饺苊阁w內(nèi)進(jìn)行消化降解的過程。細(xì)胞自噬既是一種廣泛存在的正常生理過程，又是細(xì)胞對不良環(huán)境的一種防御機(jī)制，參與多種疾病的病理過程。正常水平的自噬可以保護(hù)細(xì)胞免受環(huán)境刺激的影響，但自噬過度和自噬不足卻可能導(dǎo)致疾病的發(fā)生。在心臟中，心肌細(xì)胞自噬對維持心肌功能具有重要的作用，自噬的異?？赡軐?dǎo)致各種心肌疾病如溶酶體儲積癥（Danon disease）等。各種心血管刺激如心肌缺血（ischemia）、再灌注（reperfusion）損傷、慢性缺氧（chronic hypoxia）等均可誘導(dǎo)心肌細(xì)胞自噬增強(qiáng)。而這些情況下心肌細(xì)胞自噬的作用還不清楚：它是否是一種潛在的細(xì)胞存活機(jī)制還是導(dǎo)致細(xì)胞死亡或疾病發(fā)生的病理性機(jī)制，或者是同時(shí)具有兩種作用，目前還沒有定論。心臟疾病是心肌功能出現(xiàn)異常時(shí)產(chǎn)生的各種病理狀態(tài)的總稱。在多種心臟疾病中，均伴隨有心肌細(xì)胞自噬的改變，且影響著疾病的發(fā)生發(fā)展。在心肌肥厚

（hypertrophic cardiomyopathy）中，細(xì)胞自噬程度降低而加劇心肌肥厚；在心力衰竭（heart failure，HF）中，細(xì)胞自噬增強(qiáng)可導(dǎo)致心肌細(xì)胞自噬性死亡；而在心肌梗死（myocardial infarction，MI）中，細(xì)胞自噬增強(qiáng)可減小梗死面積。但是細(xì)胞自噬在心臟疾病中到底扮演著怎樣的角色，取決于細(xì)胞自噬發(fā)生的水平及病理狀態(tài)。目前越來越多的人開始關(guān)注藥物與細(xì)胞自噬調(diào)節(jié)之間的聯(lián)系，且主要集中于抗腫瘤藥物及心血管調(diào)節(jié)藥物的研究。另外，有報(bào)道維生素類以及雌激素受體拮抗劑他莫西芬對細(xì)胞自噬也具有調(diào)節(jié)作用。研究心肌細(xì)胞自噬與心臟疾病的關(guān)系，以及藥物對細(xì)胞自噬的調(diào)節(jié)，將有利于從自噬的角度探討心臟疾病的發(fā)生發(fā)展過程及機(jī)制，開發(fā)出治療心臟疾病的藥物。

關(guān)鍵詞：細(xì)胞自噬；心肌功能；心血管應(yīng)激；心臟疾?。凰幬?/p>

來源出版物：生物化學(xué)與生物物理進(jìn)展, 2012, 39(3): 224-233

被引頻次：23

PI3K/Akt/mTOR信號通路在巨噬細(xì)胞自噬及動脈粥樣硬化斑塊不穩(wěn)定中的作用

王和峰，翟純剛，龐文會，等

摘要：目的：探討磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）/哺乳動物雷帕霉素靶蛋白（mTOR）信號通路在巨噬細(xì)胞自體吞噬以及動脈粥樣硬化斑塊不穩(wěn)定中的作用。方法：利用Akt抑制劑康士得（20 μmol/L）、mTOR抑制劑雷帕霉素（10 nmol/L）及mTOR-siRNA（30 nmol/L）體外處理小鼠RAW 264.7巨噬細(xì)胞株48 h后，透射電鏡觀察巨噬細(xì)胞自噬體的變化，細(xì)胞免疫熒光法及Western blotting法檢測微管相關(guān)蛋白LC3-Ⅱ表達(dá)，實(shí)時(shí)熒光定量qRT-PCR和Western blotting法檢測Akt、mTOR及自噬相關(guān)蛋白Beclin 1的表達(dá)，ELISA檢測巨噬細(xì)胞分泌炎癥因子水平。體內(nèi)實(shí)驗(yàn)中，24只雄性新西蘭兔給予球囊損傷+1%膽固醇喂養(yǎng)8周，然后隨機(jī)分為對照組、康士得（1.0 mg·kg-1·d-1）組和雷帕霉素（0.5 mg·kg-1·d-1）組，每組8只，干預(yù)4周。血管內(nèi)超聲（IVUS）檢測斑塊的影像學(xué)特征，透射電鏡觀察斑塊中巨噬細(xì)胞超微結(jié)構(gòu)的改變，免疫熒光法檢測微管相關(guān)蛋白LC3-Ⅱ表達(dá)，免疫組織化學(xué)法檢測巨噬細(xì)胞Akt和mTOR的蛋白表達(dá)。結(jié)果：與對照組比較，康士得、雷帕霉素及mTOR-siRNA干預(yù)巨噬細(xì)胞后，透射電鏡下觀察到自噬體明顯增多，微管相關(guān)蛋白 LC3-Ⅱ和自噬相關(guān)蛋白Beclin 1的表達(dá)水平明顯上調(diào)，而Akt及mTOR的mRNA及蛋白表達(dá)水平明顯減少，巨噬細(xì)胞分泌的 IL-10明顯降低，而 IFN-γ的分泌顯著增加。體內(nèi)實(shí)驗(yàn)：IVUS顯示，與對照組比較，康士得組及雷帕霉素組的外彈性膜面積（EEMA）、斑塊面積（PA）及斑塊負(fù)荷（PB）明顯減少，透射電鏡下觀察到巨噬細(xì)胞中自噬體增加，組織免疫熒光法示 LC3-Ⅱ明顯增加，HE染色顯示斑塊纖維帽的厚度明顯增加，內(nèi)、中膜厚度顯著減低，組織免疫組化染色顯示巨噬細(xì)胞RAM-11及p-mTOR染色顯著減少。結(jié)論：選擇性抑制PI3K/Akt/mTOR信號通路能誘導(dǎo)巨噬細(xì)胞自噬，減少斑塊巨噬細(xì)胞的浸潤，抑制炎癥反應(yīng)進(jìn)而穩(wěn)定動脈粥樣硬化易損斑塊。

關(guān)鍵詞：PI3K/Akt/mTOR信號通路；巨噬細(xì)胞；自噬；易損斑塊

來源出版物：中國病理生理雜志, 2013, 29(3): 390-397

被引頻次：1702

Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice

Hara, T; Nakamura, K; Matsui, M; et al.

Abstract: Autophagy is an intracellular bulk degradation process through which a portion of the cytoplasm is delivered to lysosomes to be degraded. Although the primary role of autophagy in many organisms is in adaptation to starvation, autophagy is also thought to be important for normal turnover of cytoplasmic contents, particularly in quiescent cells such as neurons. Autophagy may have a protective role against the development of a number of neurodegenerative diseases. Here we report that loss of autophagy causes neurodegeneration even in the absence of any disease-associated mutant proteins. Mice deficient for Atg5 (autophagy-related 5) specifically in neural cells develop progressive deficits in motor function that are accompanied by the accumulation of cytoplasmic inclusion bodies in neurons. In Atg5-/-cells, diffuse, abnormal intracellular proteins accumulate, and then form aggregates and inclusions. These results suggest that the continuous clearance of diffuse cytosolic proteins through basal autophagy is important for preventing the accumulation of abnormal proteins, which can disrupt neural function and ultimately lead to neurodegeneration.

來源出版物：Nature, 2006, 441(7095): 885-889

被引頻次：1700

Bcl-2 antiapoptotic proteins inhibit Beclin 1-dependent autophagy

Pattingre, S; Tassa, A; Qu, XP; et al.

Abstract: Apoptosis and autophagy are both tightly regulated biological processes that play a central role in tissue homeostasis, development, and disease. The antiapoptotic protein, Bcl-2, interacts with the evolutionarily conserved autophagy protein, Beclin 1. However, little is known about the functional significance of this interaction. Here, we show that wild-type Bcl-2 antiapoptotic proteins, but not Beclin 1 binding defective mutants of Bcl-2, inhibit Beclin 1-dependent autophagy in yeast and mammalian cells and that cardiac Bcl-2 transgenic expression inhibits autophagy in mouse heart muscle. Furthermore, Beclin 1 mutants that cannot bind to Bcl-2 induce more autophagy than wild-type Beclin 1 and, unlike wild-type Beclin 1, promote cell death. Thus, Bcl-2 not only functions as an antiapoptotic protein, but also as an antiautophagy protein via its inhibitory interaction with Beclin 1. This antiautophagy function of Bcl-2 may help maintain autophagy at levels that are compatible with cell survival, rather than cell death.

來源出版物：Cell, 2005, 122(6): 927-939

被引頻次：1648

Induction of autophagy and inhibition of tumorigenesis by beclin 1

Liang, XH; Jackson, S; Seaman, M; et al.

Abstract: The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumour cells. However, little is known about the genetic mediators of autophagy in mammalian cells or their role in tumour development. The mammalian gene encoding Beclin 1, a novel Bcl-2-interacting, coiled-coil protein, has structural similarity to the yeast autophagy gene, apg6/vps30, and is mono-allelically deleted in 40%-75% of sporadic human breast cancers and ovarian cancers. Here we show using gene-transfer techniques, that beclin 1 promotes autophagy in autophagy-defective yeast with a targeted disruption of agp6/vps30, and in human MCF7 breast carcinoma cells. The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with inhibition of MCF7 cellular proliferation, in vitro clonigenicity and tumorigenesis in nude mice. Furthermore, endogenous Beclin 1 protein expression is frequently low in human breast epithelial carcinoma cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia. Thus, beclin 1 is a mammalian autophagy gene that can inhibit tumorigenesis and is expressed at decreased levels in human breast carcinoma. These findings suggest that decreased expression of autophagy proteins may contribute to the development or progression of breast and other human malignancies.

來源出版物：Nature, 1999, 402(6762): 672-676

被引頻次：1589

Loss of autophagy in the central nervous system causes neurodegeneration in mice

Komatsu, M; Waguri, S; Chiba, T; et al.

Abstract: Protein quality-control, especially the removal of proteins with aberrant structures, has an important role in maintaining the homeostasis of non-dividing neural cells. In addition to the ubiquitin-proteasome system, emerging evidence points to the importance of autophagy-the bulk protein degradation pathway involved in starvation-induced and constitutive protein turnover-in the protein qualitycontrol process. However, little is known about the precise roles of autophagy in neurons. Here we report that loss of Atg7 (autophagy-related 7), a gene essential for autophagy, leads to neurodegeneration. We found that mice lacking Atg7 specifically in the central nervous system showed behavioural defects, including abnormal limb-clasping reflexes and a reduction in coordinated movement, and died within 28 weeks of birth. Atg7 deficiency caused massive neuronal loss in the cerebral and cerebellar cortices. Notably, polyubiquitinated proteins accumulated in autophagydeficient neurons as inclusion bodies, which increased in size and number with ageing. There was, however, no obvious alteration in proteasome function. Our results indicate that autophagy is essential for the survival of neural cells, and that impairment of autophagy is implicated in the pathogenesis of neurodegenerative disorders involving ubiquitin-containing inclusion bodies.

來源出版物：Nature, 2006, 441(7095): 880-884

被引頻次：1454

p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy

Pankiv, S; Clausen, TH; Lamark, T ; et al.

Abstract: Protein degradation by basal constitutive

autophagy is important to avoid accumulation of polyubiquitinated protein aggregates and development of neurodegenerative diseases. The polyubiquitinbinding protein p62/SQSTM1 is degraded by autophagy. It is found in cellular inclusion bodies together with polyubiquitinated proteins and in cytosolic protein aggregates that accumulate in various chronic, toxic, and degenerative diseases. Here we show for the first time a direct interaction between p62 and the autophagic effector proteins LC3A and -B and the related γ-aminobutyrate receptor-associated protein and γ-aminobutyrate receptor-associated like proteins. The binding is mediated by a 22-residue sequence of p62 containing an evolutionarily conserved motif. To monitor the autophagic sequestration of p62- and LC3-positive bodies, we developed a novel pH-sensitive fluorescent tag consisting of a tandem fusion of the red, acid-insensitive mCherry and the acid-sensitive green fluorescent proteins. This approach revealed that p62- and LC3-positive bodies are degraded in autolysosomes. Strikingly, even rather large p62-positive inclusion bodies (2 μm diameter) become degraded by autophagy. The specific interaction between p62 and LC3, requiring the motif we have mapped, is instrumental in mediating autophagic degradation of the p62-positive bodies. We also demonstrate that the previously reported aggresome-like induced structures containing ubiquitinated proteins in cytosolic bodies are dependent on p62 for their formation. In fact, p62 bodies and these structures are indistinguishable. Taken together, our results clearly suggest that p62 is required both for the formation and the degradation of polyubiquitin-containing bodies by autophagy.

來源出版物： Journal of Biological Chemistry, 2007, 282(33): 24131-24145

被引頻次：1420

The role of autophagy during the early neonatal starvation period

Kuma, A; Hatano, M; Matsui, M ; et al.

Abstract: At birth the trans-placental nutrient supply is suddenly interrupted, and neonates face severe starvation until supply can be restored through milk nutrients. Here, we show that neonates adapt to this adverse circumstance by inducing autophagy. Autophagy is the primary means for the degradation of cytoplasmic constituents within lysosomes. The level of autophagy in mice remains low during embryogenesis; however, autophagy is immediately upregulated in various tissues after birth and is maintained at high levels for 3-12 h before returning to basal levels within 1-2 days. Mice deficient for Atg5, which is essential for autophagosome formation, appear almost normal at birth but die within 1 day of delivery. The survival time of starved Atg5-deficient neonates (approximately 12 h) is much shorter than that of wild-type mice (approximately 21 h) but can be prolonged by forced milk feeding. Atg5-deficient neonates exhibit reduced amino acid concentrations in plasma and tissues, and display signs of energy depletion. These results suggest that the production of amino acids by autophagic degradation of ‘self’ proteins, which allows for the maintenance of energy homeostasis, is important for survival during neonatal starvation.

來源出版物：Nature, 2004, 432(7020): 1032-1036

被引頻次：1395

Methods in mammalian autophagy research

Mizushima, N; Yoshimori, T; Levine, B

Abstract: Autophagy has been implicated in many physiological and pathological processes. Accordingly, there is a growing scientific need to accurately identify, quantify, and manipulate the process of autophagy. However, as autophagy involves dynamic and complicated processes, it is often analyzed incorrectly. In this Primer, we discuss methods to monitor autophagy and to modulate autophagic activity, with a primary focus on mammalian macroautophagy.

來源出版物：Cell, 2010, 140(3): 313-326

被引頻次：1290

Parkin is recruited selectively to impaired mitochondria and promotes their autophagy

Narendra, D; Tanaka, A; Suen, DF; et al.

Abstract: Loss-of-function mutations in Park2, the gene coding for the ubiquitin ligase Parkin, are a significant cause of early onset Parkinson’s disease. Although the role of Parkin in neuron maintenance is unknown, recent work has linked Parkin to the regulation of mitochondria. Its loss is associated with swollen mitochondria and muscle degeneration in Drosophila melanogaster, as well as mitochondrial dysfunction and increased susceptibility to mitochondrial toxins in other species. Here, we show that Parkin is selectively recruited to dysfunctional mitochondria with low membrane potential in mammalian cells. After recruitment, Parkin mediates the engulfment of mitochondria by autophagosomes and the selective elimination of

impaired mitochondria. These results show that Parkin promotes autophagy of damaged mitochondria and implicate a failure to eliminate dysfunctional mitochondria in the pathogenesis of Parkinson’s disease.

來源出版物：The Journal of Cell Biology, 2008, 183(5): 795-803

被引頻次：1280

p62/SQSTM1 forms protein aggregates degraded by autophagyand has a protective effect on huntingtin-induced cell death

Bjorkoy, G; Lamark, T; Brech, A ; et al.

Abstract: Autophagic degradation of ubiquitinated protein aggregates is important for cell survival, but it is not known how the autophagic machinery recognizes such aggregates. In this study, we report that polymerization of the polyubiquitin-binding protein p62/SQSTM1 yields protein bodies that either reside free in the cytosol and nucleus or occur within autophagosomes and lysosomal structures. Inhibition of autophagy led to an increase in the size and number of p62 bodies and p62 protein levels. The autophagic marker light chain 3 (LC3) colocalized with p62 bodies and coimmunoprecipitated with p62, suggesting that these two proteins participate in the same complexes. The depletion of p62 inhibited recruitment of LC3 to autophagosomes under starvation conditions. Strikingly, p62 and LC3 formed a shell surrounding aggregates of mutant huntingtin. Reduction of p62 protein levels or interference with p62 function significantly increased cell death that was induced by the expression of mutant huntingtin. We suggest that p62 may, via LC3, be involved in linking polyubiquitinated protein aggregates to the autophagy machinery.

來源出版物：The Journal of Cell Biology, 2005, 171(4): 603-614

被引頻次：1236

Inhibition of mTOR induces autophagy and reduces toxicity of polyglutamine expansions in fly and mouse models of Huntington disease

Ravikumar, B; Vacher, C; Berger, Z; et al.

Abstract: Huntington disease is one of nine inherited neurodegenerative disorders caused by a polyglutamine tract expansion. Expanded polyglutamine proteins accumulate abnormally in intracellular aggregates. Here we show that mammalian target of rapamycin (mTOR) is sequestered in polyglutamine aggregates in cell models, transgenic mice and human brains. Sequestration of mTOR impairs its kinase activity and induces autophagy, a key clearance pathway for mutant huntingtin fragments. This protects against polyglutamine toxicity, as the specific mTOR inhibitor rapamycin attenuates huntingtin accumulation and cell death in cell models of Huntington disease, and inhibition of autophagy has the converse effects. Furthermore, rapamycin protects against neurodegeneration in a fly model of Huntington disease, and the rapamycin analog CCI-779 improved performance on four different behavioral tasks and decreased aggregate formation in a mouse model of Huntington disease. Our data provide proof-of-principle for the potential of inducing autophagy to treat Huntington disease.

來源出版物：Nature Genetics, 2004, 36(6): 585-595

·推薦論文摘要·

巨噬細(xì)胞自噬在動脈粥樣硬化中的作用

許秋蓮，楊陽，田野

摘要：自噬通過溶酶體依賴的降解途徑維持細(xì)胞穩(wěn)態(tài)。最新研究顯示巨噬細(xì)胞自噬可以促進(jìn)膽固醇流出，抑制炎癥體活化從而抑制動脈粥樣硬化進(jìn)展。在進(jìn)展期斑塊內(nèi)，巨噬細(xì)胞自噬水平降低，斑塊易損性增高，極易導(dǎo)致斑塊破裂，引起急性冠脈綜合征。因此，調(diào)控巨噬細(xì)胞自噬已成為心血管領(lǐng)域的關(guān)注熱點(diǎn)，深入探究其機(jī)理將為動脈粥樣硬化的防治提供新思路。

關(guān)鍵詞：自噬；巨噬細(xì)胞；膽固醇流出；炎癥體；動脈粥樣硬化

來源出版物：中國動脈硬化雜志, 2016, 24(1): 97-100

聯(lián)系郵箱：田野，yetian@ems.hrbmu.edu.cn

支氣管哮喘與細(xì)胞自噬

馬龍艷，吳琦

摘要：近年來，隨著人們對細(xì)胞生物學(xué)、遺傳學(xué)研究的不斷深入，細(xì)胞自噬在支氣管哮喘（以下簡稱為哮喘）中的作用日趨明朗，特別在遺傳因素、氧化應(yīng)激和環(huán)境等方面，自噬調(diào)控可能直接影響哮喘的發(fā)生和發(fā)展，這將有望成為哮喘治療的新方向。本文就細(xì)胞自噬、細(xì)胞自噬與哮喘的關(guān)系進(jìn)行論述。

關(guān)鍵詞：哮喘；自噬；氧化性應(yīng)激；遺傳學(xué)；環(huán)境

來源出版物：天津醫(yī)藥, 2016, 44(1): 3-4

聯(lián)系郵箱：吳琦，wq572004@163.com

白藜蘆醇誘導(dǎo)細(xì)胞自噬在神經(jīng)退行性疾病進(jìn)展中的作用

董雯，王蓉

摘要：細(xì)胞自噬是清除自身異常蛋白和受損細(xì)胞器的過程，在調(diào)節(jié)細(xì)胞內(nèi)環(huán)境穩(wěn)態(tài)、細(xì)胞生長、發(fā)育和衰老以及疾病發(fā)生發(fā)展中起重要作用。自噬功能障礙與神經(jīng)退行性疾病如阿爾茨海默病、帕金森病、亨廷頓病等密切相關(guān)。這些疾病的大腦神經(jīng)元內(nèi)大多存在特定的病理性蛋白的異常聚集。白藜蘆醇對自噬具有調(diào)節(jié)作用，能促進(jìn)自噬流的發(fā)生，有效清除易形成聚集體的病理性蛋白，對神經(jīng)退行性疾病具有一定的防治潛力。本文綜述了白藜蘆醇調(diào)節(jié)細(xì)胞自噬方面的功能及在神經(jīng)退行性疾病防治的應(yīng)用。

關(guān)鍵詞：白藜蘆醇；沉默信息調(diào)控因子1；去乙?；蛔允?；神經(jīng)退行性疾?。痪奂w

來源出版物：藥學(xué)學(xué)報(bào), 2016, 51(1): 18-22

聯(lián)系郵箱：王蓉，rong_wang72@aliyun.com

不同強(qiáng)度不同時(shí)間耐力訓(xùn)練對于大鼠心肌細(xì)胞自噬發(fā)生程度的影響

馬曉雯，常蕓，王世強(qiáng)，等

摘要：目的：探討不同強(qiáng)度不同時(shí)間耐力訓(xùn)練對大鼠心肌細(xì)胞自噬程度的影響，為運(yùn)動強(qiáng)度和時(shí)間對心肌細(xì)胞自噬發(fā)生機(jī)制的探討提供依據(jù)。方法：選用48只健康成年雄性SD大鼠，隨機(jī)分為安靜對照組、中等強(qiáng)度訓(xùn)練組和大強(qiáng)度訓(xùn)練組，每組16只。分別以15.2 m/min速度5°坡度和28 m/min速度10°坡度進(jìn)行跑臺訓(xùn)練，每周訓(xùn)練5天，每次訓(xùn)練1 h。于第8周和16周分別取各組大鼠8只進(jìn)行體重稱量、超聲心動圖檢測，計(jì)算心臟重量指數(shù)（HWI）；取左心室壁用于HE染色，觀察心肌組織形態(tài)；制備超薄切片進(jìn)行透射電鏡觀察，檢測自噬發(fā)生程度。結(jié)果：運(yùn)動訓(xùn)練16周后，大強(qiáng)度組 HWI顯著低于同期對照組及同組訓(xùn)練8周后（P＜0.05）。超聲心動圖顯示，訓(xùn)練8周后，中等強(qiáng)度組 EF值顯著高于同期對照組（P＜0.05）；訓(xùn)練16周后，中等強(qiáng)度組LVPWD、LVPWS、EF均顯著高于同組訓(xùn)練8周后，大強(qiáng)度組 EF值顯著低于同期對照組（P＜0.05）。HE染色結(jié)果顯示，大強(qiáng)度組心肌細(xì)胞損傷嚴(yán)重。透射電鏡檢測顯示，長時(shí)間大強(qiáng)度耐力訓(xùn)練使自噬小體增多，自噬程度增加。結(jié)論：耐力訓(xùn)練可引起心肌組織細(xì)胞良好的適應(yīng)性重塑，但大強(qiáng)度長時(shí)間耐力訓(xùn)練會導(dǎo)致心肌纖維形態(tài)異常，線粒體損傷聚變，自噬體增多等病理現(xiàn)象，構(gòu)成心肌損傷的結(jié)構(gòu)基礎(chǔ)。

關(guān)鍵詞：運(yùn)動強(qiáng)度；心肌細(xì)胞；自噬；耐力訓(xùn)練

來源出版物：中國運(yùn)動醫(yī)學(xué)雜志, 2016, 35(1): 27-31

聯(lián)系郵箱：常蕓，changyun@ciss.cn

雷帕霉素誘導(dǎo)細(xì)胞自噬在衰老相關(guān)疾病中的作用

吳伯艷，劉新光，陳維春

摘要：哺乳動物雷帕霉素靶蛋白（mammalian target of rapamycin，mTOR）是衰老和衰老相關(guān)疾病的一個(gè)關(guān)鍵調(diào)節(jié)因子。雷帕霉素（rapamycin，RAPA）可通過抑制

m TOR通路，誘導(dǎo)和促進(jìn)細(xì)胞自噬的發(fā)生。細(xì)胞自噬是維持細(xì)胞內(nèi)穩(wěn)態(tài)的主要方式與途徑，通過降解多余的、受損的及衰老的蛋白與細(xì)胞器，為細(xì)胞重建、再生和修復(fù)提供必需原料。早老癥（hutchinson-gilford progeria syndrome，HGPS）患者細(xì)胞中伴隨早老蛋白（progerin）的異常聚集；此外，諸如亨廷頓病、帕金森病、阿爾茨海默病等神經(jīng)退行性疾病細(xì)胞內(nèi)同樣出現(xiàn)異常蛋白質(zhì)的聚集，而這些異常蛋白的清除正依賴于細(xì)胞的自噬作用。由此可見，雷帕霉素是潛在的抗衰老、治療早老癥及衰老相關(guān)疾病的重要藥物。該文主要闡述雷帕霉素促進(jìn)細(xì)胞自噬方面的功能及在HGPS、神經(jīng)退行性疾病方面的應(yīng)用。

關(guān)鍵詞：雷帕霉素；雷帕霉素靶蛋白；自噬；早老癥；早老蛋白；神經(jīng)退行性疾病

來源出版物：中國藥理學(xué)通報(bào), 2015, 31(1): 11-14

聯(lián)系郵箱：吳伯艷，chenwchun@126.com

細(xì)胞自噬分子機(jī)制的進(jìn)展

馮文之，陳揚(yáng)，俞立

摘要：細(xì)胞自噬是一類依賴于溶酶體的蛋白質(zhì)降解途徑，在真核生物中非常保守。自噬能夠感受細(xì)胞所處環(huán)境的各種信號，如氨基酸、糖等營養(yǎng)物質(zhì)的缺乏、pH值或滲透壓的改變等，使細(xì)胞做出應(yīng)激反應(yīng)，在惡劣環(huán)境下存活。同時(shí)，自噬過程會清除細(xì)胞內(nèi)錯(cuò)誤折疊或聚集的蛋白質(zhì)，受損或老化細(xì)胞器以維持細(xì)胞內(nèi)部穩(wěn)態(tài)。自噬發(fā)生時(shí)，細(xì)胞內(nèi)部的胞質(zhì)組分被包裹在自噬體中，自噬體與溶酶體融合進(jìn)行降解，產(chǎn)生新的小分子，如氨基酸等供細(xì)胞重新利用。一系列研究發(fā)現(xiàn)自噬的信號通路非常復(fù)雜，已報(bào)道有40個(gè)自噬相關(guān)蛋白（Atg蛋白）參與了自噬體的形成過程。Atg蛋白按照一定步驟發(fā)揮功能，同時(shí)相互影響，利用內(nèi)膜系統(tǒng)構(gòu)建成一個(gè)閉合的雙層膜結(jié)構(gòu)。將對細(xì)胞自噬研究的歷史、自噬分子機(jī)制的前沿進(jìn)展進(jìn)行綜述。

關(guān)鍵詞：細(xì)胞自噬；自噬體；溶酶體；Atg蛋白；蛋白質(zhì)降解

來源出版物：生命科學(xué), 2015, 27(7): 859-866

聯(lián)系郵箱：俞立，liyulab@mail.tsinghua.edu.cn

細(xì)胞自噬與腫瘤的關(guān)系研究進(jìn)展

楊晨，李萍，梁廷明

摘要：細(xì)胞自噬（autophagy）在腫瘤的發(fā)生發(fā)展過程中扮演著非常重要的角色。自噬作用是細(xì)胞的一種自我保護(hù)機(jī)制，是真核細(xì)胞用于清除細(xì)胞內(nèi)聚物及受損細(xì)胞器，進(jìn)而維持細(xì)胞內(nèi)穩(wěn)態(tài)的一種蛋白質(zhì)降解途徑。從細(xì)胞自噬的類型及其形成，細(xì)胞自噬的分子調(diào)控機(jī)制，自噬對腫瘤發(fā)生及發(fā)展、以及治療耐藥等惡性行為的影響，腫瘤中自噬與預(yù)后的關(guān)聯(lián)，干預(yù)自噬對腫瘤治療的影響和細(xì)胞自噬的研究方法等方面進(jìn)行綜述，以期為腫瘤的治療提供新思路。

關(guān)鍵詞：細(xì)胞自噬；腫瘤；信號通路

來源出版物：生命科學(xué), 2015, 27(002): 151-160

聯(lián)系郵箱：梁廷明，tmliang@njnu.edu.cn

脂多糖通過PI3K/Akt/mTOR通路調(diào)控巨噬細(xì)胞自噬

杜濤，黃海，陳欣，等

摘要：目的：觀察脂多糖對巨噬細(xì)胞自噬活化的影響及相關(guān)信號通路的探討。方法：體外培養(yǎng)巨噬細(xì)胞株RAW264.7，分為對照組、饑餓狀態(tài)激活自噬組、單純脂多糖（LPS）刺激組、LPS+PI3K抑制劑（hVps34）組和 LPS+mTOR抑制劑（雷帕霉素）組。構(gòu)建熒光真核表達(dá)載體pcDNA3.1-GFP-LC3，轉(zhuǎn)染巨噬細(xì)胞，通過熒光顯微鏡觀察各組細(xì)胞中自噬體形成情況。qRT-PCR方法檢測各組中與細(xì)胞自噬相關(guān)的Atg5、Atg7、LC3-II和Bnip3 mRNA表達(dá)水平的改變。利用Western blotting檢測LC3-II、p-Akt和p-mTOR蛋白在各組中的表達(dá)情況，以評價(jià)LPS激活巨噬細(xì)胞自噬的分子通路。結(jié)果：成功構(gòu)建穩(wěn)定表達(dá)GFP-LC3的巨噬細(xì)胞，在熒光顯微鏡下可以觀察到自噬在饑餓狀態(tài)組、LPS+hVps34組和LPS+雷帕霉素組均有明顯增強(qiáng)；qRT-PCR檢測到Atg5、LC3-II和 Bnip3 mRNA的表達(dá)在饑餓狀態(tài)組、LPS+ hVps34組和 LPS+雷帕霉素組均有明顯增強(qiáng)，而在 LPS組中略微下降；Western blotting檢測發(fā)現(xiàn)p-Akt在饑餓狀態(tài)組、LPS組和LPS+雷帕霉素組中表達(dá)明顯升高；p-mTOR在饑餓狀態(tài)組、LPS+hVps34組和LPS+雷帕霉素組表達(dá)明顯下降；LC3-II的表達(dá)在饑餓狀態(tài)組、LPS+ hVps34組和 LPS+雷帕霉素組中表達(dá)要高于對照組和LPS組。結(jié)論：LPS參與巨噬細(xì)胞自噬的調(diào)控，其可能的信號通路為PI3K/Akt/mTOR通路，但仍存在其它有效的調(diào)控通路。

關(guān)鍵詞：脂多糖類；自噬；巨噬細(xì)胞；Akt

來源出版物：中國病理生理雜志, 2014, 30(4): 675-680

聯(lián)系郵箱：陳慧，zheling76@163.com

營養(yǎng)缺乏對人肺癌細(xì)胞自噬的誘導(dǎo)作用

郭倩倩，劉志燕，姜麗麗，等

摘要：目的：觀察營養(yǎng)缺乏對人非小細(xì)胞肺癌 A549及95D細(xì)胞自噬活性的影響，建立A549及95D細(xì)胞的自噬模型。方法：以EBSS緩沖液代替1640完全培養(yǎng)基，饑餓誘導(dǎo)處于對數(shù)生長期的A549及95D細(xì)胞0、1、2、3、4、5 h后，采用單丹磺酰戊二胺（MDC）熒光染色法檢測細(xì)胞內(nèi)自噬泡的形成，并采用Western blot方法檢測自噬特異性基因微管相關(guān)蛋白1輕鏈3（LC3）和自噬相關(guān)基因Beclin1的蛋白表達(dá)水平。結(jié)果：饑餓處理A549及95D細(xì)胞后，胞內(nèi)MDC熒光顆粒逐漸增多，饑餓4 h達(dá)峰值;Beclin1的表達(dá)及LC3-II與LC3-I蛋白表達(dá)量的比值（LC3-II/LC3-I）隨著饑餓時(shí)間的延長逐漸增加，分別在饑餓3 h和4 h達(dá)峰值。結(jié)論：營養(yǎng)缺乏可以誘導(dǎo)增強(qiáng)A549及95D細(xì)胞的自噬活性，在饑餓4 h時(shí)細(xì)胞自噬水平達(dá)峰值；成功構(gòu)建了營養(yǎng)缺乏誘導(dǎo)的人肺癌細(xì)胞自噬模型，為深入研究自噬在非小細(xì)胞肺癌發(fā)生發(fā)展過程中的作用及其機(jī)制奠定了良好的基礎(chǔ)。

關(guān)鍵詞：半導(dǎo)體激光器；驅(qū)動電路；恒流源

來源出版物：光電技術(shù)應(yīng)用, 2013, 28(6): 71-73

細(xì)胞自噬在肝臟疾病中的作用

黃蘭蔚，徐列明

摘要：近年來，越來越多的證據(jù)表明細(xì)胞自噬在慢性肝炎病毒感染、酒精性肝病、脂肪肝等各種類型的肝臟疾病的發(fā)生發(fā)展中起到重要作用，成為關(guān)注和研究的新焦點(diǎn)。自噬是指細(xì)胞利用溶酶體大范圍降解長壽命蛋白質(zhì)、大分子物質(zhì)、核糖體及受損細(xì)胞器的過程。簡述了各種肝臟疾病與細(xì)胞自噬的關(guān)系，認(rèn)為探索細(xì)胞自噬在肝病機(jī)制中所扮演的角色，將可能成為治療肝臟疾病的一個(gè)新靶點(diǎn)。

關(guān)鍵詞：自噬；肝疾??；綜述

來源出版物：臨床肝膽病雜志, 2014, 2: 186-188

Crosstalk between endoplasmic reticulum stress, oxidative stress, and autophagy: Potential therapeutic targets for acute CNS injuries

Nakka, VP; Prakash-babu, P; Vemuganti R

Abstract: Endoplasmic reticulum (ER) stress induces a variety of neuronal cell death pathways that play a critical role in the pathophysiology of stroke. ER stress occurs when unfolded/misfolded proteins accumulate and the folding capacity of ER chaperones exceeds the capacity of ER lumen to facilitate their disposal. As a consequence, a complex set of signaling pathways will be induced that transmit from ER to cytosol and nucleus to compensate damage and to restore the normal cellular homeostasis, collectively known as unfolded protein response (UPR). However, failure of UPR due to severe or prolonged stress leads to cell death. Following acute CNS injuries, chronic disturbances in protein folding and oxidative stress prolong ER stress leading to sustained ER dysfunction and neuronal cell death. While ER stress responses have been well studied after stroke, there is an emerging need to study the association of ER stress with other cell pathways that exacerbate neuronal death after an injury. In this review, we summarize the current understanding of the role for ER stress in acute brain injuries, highlighting the diverse molecular mechanisms associated with ER stress and its relation to oxidative stress and autophagy. We also discussed the existing and developing therapeutic options aimed to reduce ER stress to protect the CNS after acute injuries.

關(guān)鍵詞：ER stress; oxidative stress; autophagy; crosstalk; acute CNS injury

來源出版物：Molecular Neurobiology, 2016, 53(1): 532-544

聯(lián)系郵箱：Vemuganti, R; vemuganti@neurosurgery.wisc.edu

CXC chemokine receptor 3 promotes steatohepatitis in mice through mediating inflammatory cytokines, macrophages and autophagy

Zhang, X; Han, JQ; Man, K; et al.

Abstract: Background & Aims: CXC chemokine receptor 3 (CXCR3) is involved in virus-related chronic liver inflammation. However, the role of CXCR3 in non-alcoholic steatohepatitis (NASH) remains unclear. We aimed to investigate the role of CXCR3 in NASH. Methods: Human liver tissues were obtained from 24 non-alcoholic fatty liver disease (NAFLD) patients and 20 control subjects. CXCR3 knockout (CXCR3-/-), obese db/db mice and their wild-type (WT) littermates were used in both methionine-and-choline-deficient (MCD) diet and high-fat high-carbohydrate high-cholesterol (HFHC) diet-induced NASH models. In addition, MCD-fed WT mice were administrated with CXCR3 specific antagonists. Results: CXCR3 was significantly upregulated in liver tissues of patients with NAFLD and in dietary-induced NASH animal models. Compared with WT littermates, CXCR3-/-mice were more resistant to both MCD and

HFHC diet-induced steatohepatitis. Induction of CXCR3 in dietary-induced steatohepatitis was associated with the increased expression of hepatic pro-inflammatory cytokines, activation of NF-kappa B, macrophage infiltration and T lymphocytes accumulation (Th1 and Th17 immune response). CXCR3 was also linked to steatosis through inducing hepatic lipogenic genes. Moreover, CXCR3 is associated with autophagosome-lysosome impairment and endoplasmic reticulum (ER) stress in steatohepatitis as evidenced by LC3-II and p62/SQSTM1 accumulation and the induction of GRP78, phospho-PERK and phospho-eIF2 alpha. Inhibition of CXCR3 using CXCR3 antagonist significantly suppressed MCD-induced steatosis and hepatocytes injury in AML-12 hepatocytes. Blockade of CXCR3 using CXCR3 antagonists in mice reversed the established steatohepatitis. Conclusions: CXCR3 plays a pivotal role in NASH development by inducing production of cytokines, macrophage infiltration, fatty acid synthesis and causing autophagy deficiency and ER stress.

來源出版物：Journal of Hepatology, 2016, 64(1): 160-170

聯(lián)系郵箱：Yu, J; junyu@cuhk.edu.hk

Homeostatic control of innate lung inflammation by vici syndrome gene Epg5 and additional autophagy genes promotes influenza pathogenesis

Lu, Q; Yokoyama, CC; Williams, JW; et al.

Abstract: Mutations in the autophagy gene EPG5 are linked to the multisystem human disease Vici syndrome, which is characterized in part by pulmonary abnormalities, including recurrent infections. We found that Epg5-deficient mice exhibited elevated baseline innate immune cellular and cytokine-based lung inflammation and were resistant to lethal influenza virus infection. Lung transcriptomics, bone marrow transplantation experiments, and analysis of cellular cytokine expression indicated that Epg5 plays a role in lung physiology through its function in macrophages. Deletion of other autophagy genes including Atg14, Fip200, Atg5, and Atg7 in myeloid cells also led to elevated basal lung inflammation and influenza resistance. This suggests that Epg5 and other Atg genes function in macrophages to limit innate immune inflammation in the lung. Disruption of this normal homeostatic dampening of lung inflammation results in increased resistance to influenza, suggesting that normal homeostatic mechanisms that limit basal tissue inflammation support some infectious diseases.

來源出版物：Cell Host & Microbe, 2016, 19(1): 102-113

聯(lián)系郵箱：Virgin, HW; virgin@wustl.edu

The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy

Lazarou, M; Sliter, DA; Kane, LA; et al.

Abstract: Protein aggregates and damaged organelles are tagged with ubiquitin chains to trigger selective autophagy. To initiate mitophagy, the ubiquitin kinase PINK1 phosphorylates ubiquitin to activate the ubiquitin ligase parkin, which builds ubiquitin chains on mitochondrial outer membrane proteins, where they act to recruit autophagy receptors. Using genome editing to knockout five autophagy receptors in HeLa cells, here we show that two receptors previously linked to xenophagy, NDP52 and optineurin, are the primary receptors for PINK1- and parkin-mediated mitophagy. PINK1 recruits NDP52 and optineurin, but not p62, to mitochondria to activate mitophagy directly, independently of parkin. Once recruited to mitochondria, NDP52 and optineurin recruit the autophagy factors ULK1, DFCP1 and WIPI1 to focal spots proximal to mitochondria, revealing a function for these autophagy receptors upstream of LC3. This supports a new model in which PINK1-generated phospho-ubiquitin serves as the autophagy signal on mitochondria, and parkin then acts to amplify this signal. This work also suggests direct and broader roles for ubiquitin phosphorylation in other autophagy pathways.

來源出版物：Nature, 2015, 524(7565): 309-314

聯(lián)系郵箱：Youle, RJ; youler@ninds.nih.gov

Lysosomal calcium signalling regulates autophagy through calcineurin and TFEB

Medina, DL; Di, Paola S; Peluso, I; et al.

Abstract: The view of the lysosome as the terminal end of cellular catabolic pathways has been challenged by recent studies showing a central role of this organelle in the control of cell function. Here we show that a lysosomal Ca2+signalling mechanism controls the activities of the phosphatase calcineurin and of its substrate TFEB, a master transcriptional regulator of lysosomal biogenesis and autophagy. Lysosomal Ca2+release through mucolipin 1 (MCOLN1) activates calcineurin, which binds and dephosphorylates TFEB, thus promoting its nuclear translocation. Genetic and pharmacological inhibition of calcineurin suppressed TFEB activity during starvation and physical exercise, while calcineurin overexpression and constitutive activation had the opposite effect. Induction of autophagy and lysosomal biogenesis through TFEB required MCOLN1-mediated calcineurin activation. These

data link lysosomal calcium signalling to both calcineurin regulation and autophagy induction and identify the lysosome as a hub for the signalling pathways that regulate cellular homeostasis.

來源出版物：Nature Cell Biology, 2015, 17(3): 288-299

聯(lián)系郵箱：Medina, DL; medina@tigem.it

Endogenous Drp1 mediates mitochondrial autophagy and protects the heart against energy stress

Ikeda, Y; Shirakabe, A; Maejima, Y; et al.

Abstract: Rationale: Both fusion and fission contribute to mitochondrial quality control. How unopposed fusion affects survival of cardiomyocytes and left ventricular function in the heart is poorly understood. Objective: We investigated the role of dynamin-related protein 1 (Drp1), a GTPase that mediates mitochondrial fission, in mediating mitochondrial autophagy, ventricular function, and stress resistance in the heart. Methods and Results: Drp1 downregulation induced mitochondrial elongation, accumulation of damaged mitochondria, and increased apoptosis in cardiomyocytes at baseline. Drp1 downregulation also suppressed autophagosome formation and autophagic flux at baseline and in response to glucose deprivation in cardiomyocytes. The lack of lysosomal translocation of mitochondrially targeted Keima indicates that Drp1 downregulation suppressed mitochondrial autophagy. Mitochondrial elongation and accumulation of damaged mitochondria were also observed in tamoxifen- inducible cardiac-specific Drp1 knockout mice. After Drp1 downregulation, cardiac-specific Drp1 knockout mice developed left ventricular dysfunction, preceded by mitochondrial dysfunction, and died within 13 weeks. Autophagic flux is significantly suppressed in cardiacspecific Drp1 knockout mice. Although left ventricular function in cardiac-specific Drp1 heterozygous knockout mice was normal at 12 weeks of age, left ventricular function decreased more severely after 48 hours of fasting, and the infarct size/area at risk after ischemia/reperfusion was significantly greater in cardiac-specific Drp1 heterozygous knockout than in control mice. Conclusions: Disruption of Drp1 induces mitochondrial elongation, inhibits mitochondrial autophagy, and causes mitochondrial dysfunction, thereby promoting cardiac dysfunction and increased susceptibility to ischemia/reperfusion.

關(guān) 鍵 詞 ： autophagy; Drp1 protein, mouse; heart; ischemia/reperfusion injury; mitochondria

來源出版物：Circulation Research, 2015, 116(2): 264-278

聯(lián)系郵箱：Sadoshima, J, sadoshju@njmsrutgers.edu

The cytosolic sensor cGAS detects Mycobacterium tuberculosis DNA to induce type I interferons and activate autophagy

Watson, RO; Bell, SL; MacDuff, DA; et al.

Abstract: Type I interferons (IFNs) are critical mediators of antiviral defense, but their elicitation by bacterial pathogens can be detrimental to hosts. Many intracellular bacterial pathogens, including Mycobacterium tuberculosis, induce type I IFNs following phagosomal membrane perturbations. Cytosolic M. tuberculosis DNA has been implicated as a trigger for IFN production, but the mechanisms remain obscure. We report that the cytosolic DNA sensor, cyclic GMP-AMP synthase (cGAS), is required for activating IFN production via the STING/ TBK1/IRF3 pathway during M. tuberculosis and L. pneumophila infection of macrophages, whereas L. monocytogenes short-circuits this pathway by producing the STING agonist, c-di-AMP. Upon sensing cytosolic DNA, cGAS also activates cell-intrinsic antibacterial defenses, promoting autophagic targeting of M. tuberculosis. Importantly, we show that cGAS binds M. tuberculosis DNA during infection, providing direct evidence that this unique host-pathogen interaction occurs in vivo. These data uncover a mechanism by which IFN is likely elicited during active human infections.

來源出版物：Cell Host & Microbe, 2015, 17(6): 811-819

聯(lián)系郵箱：Cox, JS; jeffery.cox@ucsf.edu

Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation

Wong, YC; Holzbaur, ELF

Abstract: Mitophagy is a cellular quality control pathway in which the E3 ubiquitin ligase parkin targets damaged mitochondria for degradation by autophagosomes. We examined the role of optineurin in mitophagy, as mutations in optineurin are causative for amyotrophic lateral sclerosis (ALS) and glaucoma, diseases in which mitochondrial dysfunction has been implicated. Using live cell imaging, we demonstrate the parkin-dependent recruitment of optineurin to mitochondria damaged by depolarization or reactive oxygen species. Parkin’s E3 ubiquitin ligase activity is required to ubiquitinate outer mitochondrial membrane proteins, allowing optineurin to stably associate

with ubiquitinated mitochondria via its ubiquitin binding domain; in the absence of parkin, optineurin transiently localizes to damaged mitochondrial tips. Following optineurin recruitment, the omegasome protein double FYVE-containing protein 1 (DFCP1) transiently localizes to damaged mitochondria to initialize autophagosome formation and the recruitment of microtubule-associated protein light chain 3 (LC3). Optineurin then induces auto- phagosome formation around damaged mitochondria via its LC3 interaction region (LIR) domain. Depletion of endogenous optineurin inhibits LC3 recruitment to mitochondria and inhibits mitochondrial degradation. These defects are rescued by expression of siRNA-resistant wild-type optineurin, but not by an ALS-associated mutant in the ubiquitin binding domain (E478G), or by optineurin with a mutation in the LIR domain. Optineurin and p62/SQSTM1 are independently recruited to separate domains on damaged mitochondria, and p62 is not required for the recruitment of either optineurin or LC3 to damaged mitochondria. Thus, our study establishes an important role for optineurin as an autophagy receptor in parkin-mediated mitophagy and demonstrates that defects in a single pathway can lead to neurodegenerative diseases with distinct pathologies.

關(guān) 鍵 詞 ： mitophagy; autophagosome; optineurin; amyotrophic lateral sclerosis; Parkinson’s disease

來源出版物：Proceedings of the National Academy of Sciences, 2014, 111(42): E4439-E4448

聯(lián)系郵箱：Holzbaur, ELF; holzbaur@mail.med.upenn.edu

The return of the nucleus: Transcriptional and epigenetic control of autophagy

Fullgrabe, J; Klionsky, DJ; Joseph, B

Abstract: Autophagy is a conserved process by which cytoplasmic components are degraded by the lysosome. It is commonly seen as a cytoplasmic event and, until now, nuclear events were not considered of primary importance for this process. However, recent studies have unveiled a transcriptional and epigenetic network that regulates autophagy. The identification of tightly controlled transcription factors (such as TFEB and ZKSCAN3), microRNAs and histone marks (especially acetylated Lys16 of histone 4 (H4K16ac) and dimethylated H3K9 (H3K9me2)) associated with the autophagic process offers an attractive conceptual framework to understand the short-term transcriptional response and potential long-term responses to autophagy.

來源出版物：Nature Reviews Molecular Cell Biology, 2014, 15(1): 65-74

聯(lián)系郵箱：Joseph, B; bertrand.joseph@ki.se

Autophagy and apoptosis: Where do they meet?

Mukhopadhyay, S; Panda, PK; Sinha, N; et al.

Abstract: Autophagy and apoptosis are two important cellular processes with complex and intersecting protein networks; as such, they have been the subjects of intense investigation. Recent advances have elucidated the key players and their molecular circuitry. For instance, the discovery of Beclin-1’s interacting partners has resulted in the identification of Bcl-2 as a central regulator of autophagy and apoptosis, which functions by interacting with both Beclin-1 and Bax/Bak respectively. When localized to the endoplasmic reticulum and mitochondria, Bcl-2 inhibits autophagy. Cellular stress causes the displacement of Bcl-2 from Beclin-1 and Bax, thereby triggering autophagy and apoptosis, respectively. The induction of autophagy or apoptosis results in disruption of complexes by BH3-only proteins and through post-translational modification. The mechanisms linking autophagy and apoptosis are not fully defined; however, recent discoveries have revealed that several apoptotic proteins (e.g., PUMA, Noxa, Nix, Bax, XIAP, and Bim) modulate autophagy. Moreover, autophagic proteins that control nucleation and elongation regulate intrinsic apoptosis through calpain- and caspase-mediated cleavage of autophagy-related proteins, which switches the cellular program from autophagy to apoptosis. Similarly, several autophagic proteins are implicated in extrinsic apoptosis. This highlights a dual cellular role for autophagy. On one hand, autophagy degrades damaged mitochondria and caspases, and on the other hand, it provides a membrane-based intracellular platform for caspase processing in the regulation of apoptosis. In this review, we highlight the crucial factors governing the crosstalk between autophagy and apoptosis and describe the mechanisms controlling cell survival and cell death.

關(guān)鍵詞：autophagy; apoptosis; crosstalk; Bcl-2; Beclin-1; BH3-only proteins

來源出版物：Apoptosis, 2014, 19(4): 555-566

聯(lián)系郵箱：Bhutia, SK; sujitb@nitrkl.ac.in

編輯：王微

Isolation and characterization of autophagy-defective mutants of Saccharomyces cerevisiae

Tsukada, M; Ohsumi, Y

Autophagy in the yeast is similar to that in mammalian cells. A mutant designated as apg1 (autophagy) defective in accumulation of autophagic bodies in the vacuoles was isolated by selection using a light microscope from a mutagenized proteinase-deficient strain. In the apg1 strain, which has normal vacuolar proteinases, nitrogen starvation did not induce protein degradation. The apg1 mutant lost its viability faster than wild-type cells during nitrogen starvation. By using the loss of viability as a first screening test, 75 other apg mutants were selected. These apg mutants including apg1 fell into 15 complementation groups. Genetic analyses of representative apg mutants revealed that they all had single recessive chromosomal mutations. Strains with each apg mutation were defective in protein degradation in the vacuoles induced by nitrogen starvation and homozygous diploids for each apg mutation did not sporulate. These results on the apg mutants suggest that autophagy via autophagic bodies is indispensable for protein degradation in the vacuoles under starvation conditions, and that at least 15 APG genes are involved in autophagy in yeast.