摘要
背景
热休克蛋白70(Heat shock protein70, HSP70)是热休克蛋白家族中被研究最多的成员,是一类与肿瘤密切相关的具有分子伴侣和免疫调节作用的蛋白。HSP70能帮助细胞适应各种应激反应,减轻环境有害因子对细胞的损害作用,从而增强机体细胞的生存能力。国内外许多研究已经表明,HSP70在许多肿瘤中高表达,并在其发生、发展、治疗及预后的整个疾病过程中都扮演着重要的角色。
煤焦沥青(Coal tar pitch, CTP)是煤炭在炼焦过程中产生的副产物,成分复杂,其所致肺癌是严重危害职业人群健康的恶性肿瘤之一。关于煤焦沥青致肿癌的研究很多,但其致癌机制尚未阐释清楚,有待进一步研究。
该研究采用中温煤焦沥青烟提取物(Coal tar pitch smoke extract)作为诱导剂,建立永生化的人支气管上皮细胞BEAS-2B的恶性转化模型,检测该细胞系在细胞发生恶性转化的过程中HSP70与核苷酸切除修复因子的表达情况,探索HSP70及核苷酸切除修复因子在细胞恶性转化过程中的作用及其可能的机制,为进一步研究HSP70的生物学作用和煤焦沥青的致癌机制提供一定的理论基础。
方法
1.细胞恶性转化模型的建立
实验分三组：即空白对照组、DMSO溶剂对照组、煤焦沥青组(煤焦沥青烟提取物溶液)。
制备煤焦沥青烟提取物作为染毒剂,通过细胞活力计数法确定合适的染毒剂量,并以此剂量来诱导BEAS-2B细胞恶性转化,然后观察细胞转化过程中细胞表型的改变情况。
2.细胞恶性转化过程的检测
用彗星实验检测染毒后BEAS-2B细胞损伤情况,并观察各代细胞形态的变化；用染色体核型分析和软琼脂集落形成实验检测细胞是否发生恶性转变。
3.HSP70蛋白表达水平与核苷酸切除修复因子表达水平的检测
Western blot法检测HSP70及核苷酸切除修复因子着色性干皮病互补基因蛋白质C(Xeroderma pigmentosum group, XPC)、切除修复交叉互补蛋白1(Excision repair cross complementing1, ERCC1)的表达,观察细胞受煤焦沥青烟提取物作用后,HSP70、XPC及ERCC1在传代过程中表达水平的动态变化。
4.统计学分析
采用SPSS12.0对数据进行统计学分析,符合正态分布的数据以x±s表示；多组数据比较采用单因素方差分析；两组数据比较采用t检验,构成比采用卡方检验：两两比较采用LSD法,检验水准a=0.05。
结果
1.细胞恶性转化试验
彗星实验结果显示,染毒组细胞出现DNA损伤,与对照组比较,染毒组细胞拖尾长度较对照组均显著增高,差异有统计学意义(P<0.05)；表明煤焦沥青烟提取物对BEAS-2B细胞具有遗传毒性。染毒组20代细胞发生形态学改变,细胞胞浆丰富,体积增大,类圆形、圆形比例增加,甚至部分细胞出现纤维细胞样改变；此代细胞核型出现明显异常,亚二倍体和超二倍体出现较多；但锚着力生长实验显示细胞仅有少数细胞能形成细胞集落,恶性程度不高。染毒组30代,胞体大小差异大,细胞内颗粒物增加,面积增大,大部分细胞外形变为类圆形、圆形,部分细胞膜边缘呈膜状向往扩展；细胞出现大量亚二倍体和超二倍体,畸变比率高；锚着力生长实验发现,此代细胞可以形成大细胞集落,无接触抑制,细胞发生了恶性转化。
2.BEAS-2B细胞中,蛋白质HSP70、XPC、ERCC1的表达
染毒组在0代和30代细胞HSP70的表达与对照组比较,表达水平均显著升高(P<0.05)；0代细胞XPC及ERCC1蛋白表达显著高于对照组(P<0.05)；30代细胞中XPC蛋白显著低于2个对照组(P<0.05), ERCC1蛋白表达显著高于对照组(P<0.05)；对照组各组之间各个蛋白的表达差异均无统计学意义(P>0.05)。
结论
HSP70参与煤焦沥青烟提取物所致的BEAS-2B细胞恶性转化过程；细胞恶变后,HSP70能抑制XPC的表达；在细胞染色体突变及恶性转化过程中,DNA损伤可能是其始动因素,HSP70蛋白和XPC及ERCC1蛋白功能异常可能是其重要原因。
Background Heat shock protein70(HSP70) is a class of protein with the role of molecular chaperone and immune regulatory, which is closely related with tumor and was studied most in recent years. HSP70can help cells adapt to all kinds of stress responses and reduce the cell damage by the harmful factors of environment, which contributes to the improved the survival ability of body's cells. Many researches had reported overexpressed HSP70in many tumors and sueggested that it was involved in tumor occurrence, development, treatment and prognosis at home and abroad.
Coal tar pitch(CTP), with complicated compositions, is the by-products of coal during the process of coking coal. CTP induced lung cancer is one of the malignant tumors that severely affect professional workers. Many Studies showed that CTP can cause lung cancer, but the mechanism was yet unknown,and the real mechanism needs further research.
This study tries to uncover the malignant transformation effects of coal tar pitch smoke extract on BEAS-2B in vitro and establish malignant transformation model of BEAS-2B induced by coal tar pitch extract, and then detect the expression of HSP70and nucleotide resection repair factor in BEAS-2B cell. To study the possible mechanism of HSP70and nucleotide resection repair factor during the process of malignant transformation, which will provide a basic tool for further studies on the biological function of HSP70and the mechanism of lung cancer induced by coal tar pitch.
Methods
1Establish malignant transformation model of BEAS-2B BEAS-2B cell were divided into three groups:a blank control group, DMSO solvent treated group, coal tar piteh smoke treated group (coal tar pitch smoke extract solution). Preparation of coal tar pitch smoke extract as dyeing agents and the appropriate dose was determined by the cell vitality count method; the change of cellular phenotype and the characteristics of the cellular biology were observed in the malignant transforxnation of BEAS-2B cells.
2Detection of the process of malignant transformation
The damage of BEAS-2B cells were detected by single cell gel electrophoresis and observed the change of cell forms in differents generation. Conventional chromosomal analysis and the soft agar colony formation test were used to detect whether cells have been induced to malignant transformation.3Detect the express level of HSP70and nucleotide resection repair factor
Observe the dynamic expression of xeroderma pigmentosum group C and excision repair cross complementing1in differents generation of BEAS-2B cells induced by coal tar pitch smoke extract.
4Statistic analysis
SPSS12.0was used for data analysis. Normal distribution data was expressed by x±s,and one-way ANOVA, t-test,χ2test,LSD were used to analyze the data(a=0.05).
Results
1Malignant transformation experiment
Comet assay results showed that coal tar pitch smoke extract may has genetoxic in BEAS-2B cell, and DNA damage of experiment group have longer tail length and showed significant statistical difference compared with control group(P<0.05). The20th generation cell of experiment group showed that cell had morphological changes, cell cytoplasm rich, cells became larger, the proportion of circular or similar to round shape cells rised, some cells even showed fibroblast-like changes. This generation cell in experiment group have significantly differents on cell's karyotype,appeared more sub-lipliod or hyperdiploid cell, but the soft agar colony formation test showed that only a few cells can form macroscopic colonies, degree of malignancy is not high.The30th generation cell of experiment group have signficant different on the cell body size, Cells particles increased, area increased, most of cell were circular or similar to round shape cells, part of cell membrane edge is membrane yearning extension; appeared a amount of sub-lipliod or hyperdiploid cell; high distortion rate, the soft agar colony formation test showed that cell can form macroscopic colonies, degree of malignancy is high. BEAS-2B cell have completed malignant transformation.
2The expression of HSP70、XPC and ERCC1.
In the0th and30th generation cell, the expression of HSP70in the experiment group,were notable higher than the contronl group(P<0.05); at the0th generation cell, the protein expression of XPC and ERCC1significant higher than contronl group(P <0.05); at the30th generation cell,the protein expression of XPC significantly lower than the two control group(P<0.05); the protein expression of ERCC1significantly higher than the two control group(P<0.05); there were no signifieant difference between Solvent control group and blank control group(P<0.05). Conclusion
HSP70take participate in the process of malignant trans formation induced by coal tar pitch smoke extract in BEAS-2B cell; HSP70can restrain the expression of XPC after malignant transformation of cells; The DNA damage may be the originating factor and the function abnormality of HSP70、XPC and ERCC1may be the important reasons in the process of chromosome mutation and malignant transformation in BEAS-2B cell.
引文
[1]邹小农.中国肺癌流行病学[J].中华肿瘤防治杂志.2007,14(12)：881-883
[2]陈万青,张思维,邹小农.中国肺癌的发病死亡的估计和流行趋势研究[J].中国肺癌杂志.2010,13(5)：488-493
[3]Radons J,Multhoff G. Immunostimulatory functions of membrane-bound and exported heat shock protein 70[J]. Exerc Immunol Rev.2005,11(11):17-33
[4]Didelot C, Lanneau D, Brunet M, et al. Anti-cancer therapeutic approaches based on intracellular and extracellular heat shock proteins[J]. Curr Med Chem.2007,14(27): 2839-2847
[5]Rohde M, Daugaard M, Jensen M H, et al. Members of the heat-shock protein 70 family promote cancer cell growth by distinct mechanisms[J]. Genes Dev.2005,19(5):570-582
[6]Giaginis C, Daskalopoulou S S, Vgenopoulou S, et al. Heat Shock Protein-27,-60 and-90 expression in gastric cancer:association with clinicopathological variables and patient survival[J]. BMC Gastroenterol.2009,9(5):14-17
[7]颜璐,张天宝.苯并(a)芘诱导BEAS-2B细胞恶性转化的不同阶段特征[J].毒理学杂志.2007,10(04)：328-329
[8]段燕英.热休克蛋白70在苯并(a)芘所致DNA损伤修复中的作用和交互作用蛋白分析[D].[博士学位论文].武汉：华中科技大学,2008
[9]郝巧玲,吴晓明,姚群峰等.核苷酸切除修复基因表达水平与肺癌易感性的关系[J].环境与职业医学.2004,12(03)：172-174
[10]Yang J, Liu X, Niu P, et al. Dynamic changes of XPA, XPC, XPF, XPG and ERCC1 protein expression and their correlations with levels of DNA damage in human bronchial epithelia cells exposed to benzo[a]pyrene[J]. Toxicology Letters.2007,174(1-3):10-17
[11]张少峰.NF-κB在肿瘤相关巨噬细胞促煤焦沥青烟提取物诱导BEAS-2B恶变过程中的表达[D].[硕士学位论文].郑州：郑州大学,2010
[12]卫止义,樊生才.煤焦油加工技术进展及产业化评述[J].煤化工.2007,2(1)：7-10
[13]Culp S J, Gaylor D W, Sheldon W G, et al. A comparison of the tumors induced by coal tar and benzo[a]pyrene in a 2-year bioassay[J]. Carcinogenesis.1998,19(1):117-124
[14]Lavoue J, Gerin M, Cote J, et al. Mortality and cancer experience of Quebec aluminum reduction plant workers. Part I:The reduction plants and coal tar pitch volatile (CTPV) exposure assessmen[J]. J Occup Environ Med.2007,49(9):997-1008
[15]Gibbs G W, Armstrong B,Sevigny M. Mortality and cancer experience of Quebec aluminum reduction plant workers. Part 2:mortality of three cohorts hired on or before January 1, 1951[J]. J Occup Environ Med.2007,49(10):1105-1123
[16]Armstrong B,Theriault G. Compensating lung cancer patients occupationally exposed to coal tar pitch volatiles[J]. Occup Environ Med.1996,53(3):160-167
[17]朱进国,吴庆深,孔令文.人支气管上皮细胞恶变过程中生物学特性及超微结构的研究 [J].四川肿瘤防治.2005,18(4)：209-212
[18]Li Z, Wu Y, Zhao Y, et al. Analysis of coal tar pitch and smoke extract components and their cytotoxicity on human bronchial epithelial cells[J]. J Hazard Mater.2011,186(2-3): 1277-1282
[19]Kong L, Wu Q,Zhu J. FHIT protein expression during malignant transformation of human bronchial epithelial cells induced by NNK[J]. Zhongguo Fei Ai Za Zhi.2006,9(2):167-172
[20]韩力薇,覃文新,王晨等.利用集落形成实验和成瘤实验初步研究新基因的成瘤作用[J].复旦学报(医学科学版).2001,10(04)：334-336+339
[21]Zhao P, Fu J, Yao B, et al. In vitro malignant transformation of human bronchial epithelial cells induced by benzo(a)pyrene[J]. Toxicol In Vitro.2012,26(2):362-368
[22]Gao A, Song S, Zuo X, et al. Epigenetic mediated transcriptional activation of PARP-1 participates in silica-associated malignant transformation of human bronchial epithelial cells[J]. Toxicology Letters.2010,193(3):236-241
[23]Ciocca D R.Calderwood S K. Heat shock proteins in cancer:diagnostic, prognostic, predictive, and treatment implications[J]. Cell Stress & Chaperones.2005,10(2):86-92
[24]Mosser D D,Morimoto R I. Molecular chaperones and the stress of oncogenesis[J]. Oncogene.2004,23(16):2907-2918
[25]Rutherford S L. Between genotype and phenotype:protein chaperones and evolvability[J]. Nature Reviews Genetics.2003,4(4):263-274
[26]Botzler C, Kolb H J, Issels R D, et al. Noncytotoxic alkyl-lysophospholipid treatment increases sensitivity of leukemic K562 cells to lysis by natural killer (NK) cells [J]. International Journal of Cancer.1996,65(5):633-638
[27]Multhoff G. Heat shock protein 72 (HSP72), hyperthermia-inducible immunogenic determinant on leukemic K562 and Ewing's Sarcoma cells[J]. International journal of hyperthermia.1997,13(1):39-48
[28]罗元明,刘彦信,郑德先.蛋白质组学研究方法及其在生物医学中的应用[J].基础医学与临床.2003,23(2)：113-118
[29]Lin H J, Wang C T, Niu K C, et al. Hypobaric hypoxia preconditioning attenuates acute lung injury during high-altitude exposure in rats via up-regulating heat-shock protein 70[J]. Clinical Science.2011,121(5):223-231
[30]Jiang J, Shi Y, Shan Z, et al. Bioaccumulation, oxidative stress and HSP70 expression in< i> Cyprinus carpio L. exposed to microcystin-LR under laboratory conditions[J]. Comparative Biochemistry and Physiology Part C:Toxicology & Pharmacology.2011, 15(12):58-61
[31]Wang J, Wu Y H,Yu C P. Single cell gel electrophoresis for detection of coal tar induced DNA injury of NIH/3T3 cells[J]. Zhonghua Lao Dong Wei Sheng Zhi Ye Bing Za Zhi.2009, 27(1):38-39
[32]Malhotra V, Kooy N W, Denenberg A G, et al. Ablation of the heat shock factor-1 increases susceptibility to hyperoxia-mediated cellular injury[J]. Exp Lung Res.2002,28(8):609-622
[33]Shevtsov M A, Khachatryan W A, Pozdnyakov A V, et al. Hsp70 Stress Protein is a Promising Tool in the Treatment of Brain Tumours in Children[J]. European Journal of Cancer.2011,47(12):S291-S291
[34]Aschkenasy G, Bromberg Z, Raj N, et al. Enhanced Hsp70 expression protects against acute lung injury by modulating apoptotic pathways[J]. Plos One.2011,6(11):e26956
[35]Thompson C B. Attacking cancer at its root[J]. Cell.2009,138(6):1051-1054
[36]李静.热休克蛋白70与P53相互作用在酵母双杂交系统中的检测及其对P53在细胞内定位的影响[D].[硕士学位论文].西安：中国人民解放军第四军医大学,2003
[37]Pakravan N, Soudi S,Hassan Z M. N-terminally fusion of Her2/neu to HSP70 decreases efficiency of Her2/neu DNA vaccine[J]. Cell Stress and Chaperones.2010,15(5):631-638
[38]Davis C D, Emenaker N J,Milner J A. Cellular proliferation, apoptosis and angiogenesis: molecular targets for nutritional preemption of cancer[J]. Semin Oncol.2010,37(3):243-257
[39]Tsan M F,Gao B. Heat shock proteins and immune system[J]. J Leukoc Biol.2009,85(6): 905-910
[40]Mukai T, Maeda Y, Tamura T, et al. Induction of cross-priming of naive CD8+T lymphocytes by recombinant bacillus Calmette-Guerin that secretes heat shock protein 70-major membrane protein-II fusion protein[J]. The Journal of Immunology.2009,183(10): 6561-6568
[41]Wang X P, Liu G Z, Song A L, et al. Expression and significance of heat shock protein 70 and glucose-regulated protein 94 in human esophageal carcinoma[J]. World J Gastroenterol. 2005,11(3):429-432
[42]工玉珍,吴逸明,周舫等.HSP70-1、HSP70-2基因多态性与肺癌易感性的相关性[J].环境与职业医学.2010,05(12)：305-309
[43]Kalikaki A, Kanaki M, Vassalou H, et al. DNA repair gene polymorphisms predict favorable clinical outcome in advanced non-small-cell lung cancer[J]. Clinical Lung Cancer.2009, 10(2):118-123
[44]Xiang T X, Li Y, Jiang Z, et al. RNA interference-mediated silencing of the Hsp70 gene inhibits human gastric cancer cell growth and induces apoptosis in vitro and in vivo[J]. Tumori.2008,94(4):539-550
[45]Jaattela M. Over-expression of hsp70 confers tumorigenicity to mouse fibrosarcoma cells[J]. International Journal of Cancer.1995,60(5):689-693
[46]Buraczynska M, Swatowski A, Buraczynska K, et al. Heat-shock protein gene polymorphisms and the risk of nephropathy in patients with Type 2 diabetes[J]. Clin Sci (Lond).2009,116(1):81-86
[47]He M, Guo H, Yang X, et al. Functional SNPs in HSPA1A gene predict risk of coronary heart disease[J]. Plos One.2009,4(3):e4851
[48]Li J X, Tang B P, Sun H P, et al. Interacting contribution of the five polymorphisms in three genes of Hsp70 family to essential hypertension in Uygur ethnicity[J]. Cell Stress Chaperones.2009,14(4):355-362
[49]Takahashi Y, Moriwaki S, Sugiyama Y, et al. Decreased gene expression responsible for post-ultraviolet DNA repair synthesis in aging:a possible mechanism of age-related reduction in DNA repair capacity[J]. Journal of Investigative Dermatology.2005,124(2): 435-442
[50]Tao G H, Yang L Q, Gong C M, et al. Effect of PARP-1 deficiency on DNA damage and repair in human bronchial epithelial cells exposed to Benzo(a)pyrene[J]. Molecular Biology Reports.2009,36(8):2413-2422
[51]Melis J P, Luijten M, Mullenders L H, et al. The role of XPC:implications in cancer and oxidative DNA damage[J].Mutat Res.2011,728(3):107-117
[52]李杰,刘洋,陈志文等.实时荧光定量PCR检测直肠癌XPC基因表达[J].山东医药.2008,48(014)：6-7
[53]杨瑾,刘晓勇,邹云锋等.B(a)P作用下的人支气管上皮细胞DNA损伤与XPA, XPC蛋白表达的关系[J].环境与职业医学.2007,24(4)：361-364
[54]吴晓明,周宜开,王志勇等.苯并(a)芘对肺癌细胞DNA损伤及修复基因表达水平的影响[J].中华劳动卫生职业病杂志.2002,12(06)：47-49
[55]Yin M, Yan J R, Voutsina A, et al. No evidence of an association of ERCC1 and ERCC2 polymorphisms with clinical outcomes of platinum-based chemotherapies in non-small cell lung cancer:A meta-analysis[J]. Lung Cancer.2011,72(3):370-377
[56]Hoffmann A C, Wild P, Leicht C, et al. MDR1 and ERCC1 Expression Predict Outcome of Patients with Locally Advanced Bladder Cancer Receiving Adjuvant Chemotherapy[J]. Neoplasia.2010,12(8):628-636
[57]Schneider S, Uchida K, Brabender J, et al. Downregulation of TS, DPD, ERCC1, GST-Pi, EGFR, and HER2 gene expression after neoadjuvant three-modality treatment in patients with esophageal cancer[J]. JOURNAL OF THE AMERICAN COLLEGE OF SURGEONS. 2005,200(3):336-344
[58]杨瑾,刘晓勇,邹云锋等.苯并芘处理对人支气管上皮细胞热休克蛋白70和着色性干皮病G蛋白表达的影响[J].毒理学杂志.2010,10(04)：267-270
[59]Tsan M F,Gao B. Heat shock proteins and immune system[J]. J Leukoc Biol.2009,85(6): 905-910
[60]宫智勇.热休克蛋白70在苯并(a)芘致细胞DNA损伤修复中的作用[D].[博士学位论文].武汉：华中科技大学,2006
[1]Kozeko L E. Heat shock proteins 90 kDa:diversity, structure, functions[J]. Tsitologiia.2010, 52(11):893-910
[2]Bosco D A, LaVoie M J, Petsko G A, et al. Proteostasis and Movement Disorders:Parkinson's Disease and Amyotrophic Lateral Sclerosis[J]. Cold Spring Harb Perspect Biol.2011,3(10): 65-68
[3]Saab S S, Lange H S,Maney D L. Gonadotrophin-releasing hormone neurons in a photoperiodic songbird express fos and egr-1 protein after a single long day[J]. J Neuroendocrinol.2010,22(3): 196-207
[4]Bartlett A I,Radford S E. An expanding arsenal of experimental methods yields an explosion of insights into protein folding mechanisms[J]. Nature Structural & Molecular Biology.2009,16(6): 582-588
[5]Dunker A K, Silman I, Uversky V N, et al. Function and structure of inherently disordered proteins[J]. Curr Opin Struct Biol.2008,18(6):756-764
[6]Powers E T, Morimoto R I, Dillin A, et al. Biological and chemical approaches to diseases of proteostasis deficiency[J]. Annual review of biochemistry.2009,78(12):959-991
[7]Morimoto R I. Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging[J]. Genes & Development.2008,22(11):1427-1438
[8]Balch W E, Morimoto R I, Dillin A, et al. Adapting proteostasis for disease intervention[J]. Science.2008,319(5865):916-919
[9]Serrano A L, Waegele M M,Gai F. Spectroscopic studies of protein folding:linear and nonlinear methods[J]. Protein Science.2012,21(2):157-170
[10]Kubelka J, Hofrichter J,Eaton W A. The protein folding speed limit'[J]. Curr Opin Struct Biol. 2004,14(1):76-88
[11]Herbst R, Schafer U,Seckler R. Equilibrium intermediates in the reversible unfolding of firefly (Photinus pyralis) luciferase[J]. Journal of Biological Chemistry.1997,272(11):7099-7105
[12]Ellis R J,Minton A P. Protein aggregation in crowded environments[J]. Biological Chemistry. 2006,387(5):485-497
[13]Mulligan V K, Hadley K C,Chakrabartty A. Analyzing complicated protein folding kinetics rapidly by analytical Laplace inversion using a Tikhonov regularization variant[J]. Anal Biochem. 2012,421(1):181-190
[14]Vabulas R M, Raychaudhuri S, Hayer-Hartl M, et al. Protein folding in the cytoplasm and the heat shock response[J]. Cold Spring Harb Perspect Biol.2010,2(12):a004390
[15]Vassilakos A, Cohen-Doyle M, Peterson P, et al. The molecular chaperone calnexin facilitates folding and assembly of class I histocompatibility molecules[J]. The EMBO journal.1996,15(7): 1495
[16]Stangl S, Themelis G, Friedrich L, et al. Detection of irradiation-induced, membrane heat shock protein 70 (Hsp70) in mouse tumors using Hsp70 Fab fragment[J]. Radiotherapy and Oncology. 2011,99(3):313-316
[17]陈劲松.热休克蛋白的分子遗传学研究进展[J].国外医学：遗传学分册.2001,24(003)：128-132
[18]Auluck P K, Chan H Y E, Trojanowski J Q, et al. Chaperone suppression of a-synuclein toxicity in a Drosophila model for Parkinson's disease[J]. Science.2002,295(5556):865-868
[19]Mayer M P. Gymnastics of molecular chaperones[J]. Molecular Cell.2010,39(3):321-331
[20]Kampinga H H,Craig E A. The HSP70 chaperone machinery:J proteins as drivers of functional specificity[J]. Nature Reviews Molecular Cell Biology.2010,11(8):579-592
[21]Rousseau F, Serrano L,Schymkowitz J W H. How evolutionary pressure against protein aggregation shaped chaperone specificity[J]. Journal of Molecular Biology.2006,355(5): 1037-1047
[22]Zhang C,Cuervo A M. Restoration of chaperone-mediated autophagy in aging liver improves cellular maintenance and hepatic function[J]. Nat Med.2008,14(9):959-965
[23]Sharma S K, De Los Rios P, Christen P, et al. The kinetic parameters and energy cost of the Hsp70 chaperone as a polypeptide unfoldase[J]. Nat Chem Biol.2010,6(12):914-920
[24]Frydman J. Folding of newly translated proteins in vivo:the role of molecular chaperones[J]. Annual review of biochemistry.2001,70(1):603-647
[25]Horwich AL. Chaperonin-Mediated Protein Folding[J]. Biophys J.2011,100(23):199-201
[26]Hartl F U, Bracher A,Hayer-Hartl M. Molecular chaperones in protein folding and proteostasis[J]. Nature.2011,475(7356):324-332
[27]Tartaglia G G, Dobson C M, Hartl F U, et al. Physicochemical determinants of chaperone requirements[J]. Journal of Molecular Biology.2010,400(3):579-588
[28]Hartl F U,Hayer-Hartl M. Converging concepts of protein folding in vitro and in vivo[J]. Nature Structural & Molecular Biology.2009,16(6):574-581
[29]Douglas N R, Reissmann S, Zhang J, et al. Dual action of ATP hydrolysis couples lid closure to substrate release into the group Ⅱ chaperonin chamber[J]. Cell.2011,144(2):240-252
[30]Zhang J, Baker M L, Schroder G F, et al. Mechanism of folding chamber closure in a group Ⅱ chaperonin[J]. Nature.2010,463(7279):379-383
[31]Munoz I G, Yebenes H, Zhou M, et al. Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin[J]. Nature Structural & Molecular Biology. 2010,18(1):14-19
[32]Kitamura A, Kubota H, Pack C G, et al. Cytosolic chaperonin prevents polyglutamine toxicity with altering the aggregation state[J]. Nature cell biology.2006,8(10):1163-1169
[33]Taipale M, Jarosz D F,Lindquist S. HSP90 at the hub of protein homeostasis:emerging mechanistic insights[J]. Nature Reviews Molecular Cell Biology.2010,11(7):515-528
[34]Wandinger S K, Richter K,Buchner J. The Hsp90 chaperone machinery[J]. Journal of Biological Chemistry.2008,283(27):18473-18477
[35]Shiau A K, Harris S F, Southworth D R, et al. Structural Analysis of< i> E. coli hsp90 Reveals Dramatic Nucleotide-Dependent Conformational Rearrangements[J]. Cell.2006,127(2): 329-340
[36]Chen B, Retzlaff M, Roos T, et al. Cellular strategies of protein quality control[J]. Cold Spring Harb Perspect Biol.2011,3(8):65-68
[37]Kamal A, Boehm M F,Burrows F J. Therapeutic and diagnostic implications of Hsp90 activation[J]. Trends in molecular medicine.2004,10(6):283-290
[38]成钢,胡维新.热激蛋白90的生物学特性[J].生命的化学.2009,25(005)：687-690
[39]Neckers L. Heat shock protein 90:the cancer chaperone[J]. Heat Shock Proteins in Cancer.2007, 25(26):231-252
[40]闫琪,孙文劫,肖旭华等.以热休克蛋白90为靶点的抗肿瘤药物研究进展[J].中国药物化学杂志.2010,5)：371-378
[41]Geller R, Vignuzzi M, Andino R, et al. Evolutionary constraints on chaperone-mediated folding provide an antiviral approach refractory to development of drug resistance[J]. Genes & Development.2007,21(2):195-199
[42]Eichmann C, Preissler S, Riek R, et al. Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy[J]. Proceedings of the National Academy of Sciences.2010,107(20):9111-9115
[43]Cabrita L D, Launay H, Dobson C M, et al. Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy[J]. Proceedings of the National Academy of Sciences.2009, 106(52):22239-22244
[44]O'Brien E P, Hsu S T D, Christodoulou J, et al. Transient tertiary structure formation within the ribosome exit port[J]. J Am Chem Soc.2010,12(15):56-60
[45]Ferbitz L, Maier T, Patzelt H, et al. Trigger factor in complex with the ribosome forms a molecular cradle for nascent proteins[J]. Nature.2004,431(7008):590-596
[46]Kaiser C M, Chang H C, Agashe V R, et al. Real-time observation of trigger factor function on translating ribosomes[J]. Nature.2006,444(7118):455-460
[47]Brandt F, Etchells S A, Ortiz J O, et al. The native 3D organization of bacterial polysomes[J], Cell.2009,136(2):261-271
[48]Brandt F, Carlson L A, Hartl F U, et al. The three-dimensional organization of polyribosomes in intact human cells[J]. Molecular Cell.2010,39(4):560-569
[49]Zhang G,Ignatova Z. Generic algorithm to predict the speed of translational elongation: implications for protein biogenesis[J]. Plos One.2009,4(4):e5036-e5039
[50]Vabulas R M,Hartl F U. Protein synthesis upon acute nutrient restriction relies on proteasome function[J]. Science.2005,310(5756):1960-1963
[51]Buchberger A, Bukau B,Sommer T. Protein quality control in the cytosol and the endoplasmic reticulum:brothers in arms[J]. Molecular Cell.2010,40(2):238-252
[52]Dagkessamanskaia A, Durand F, Uversky V N, et al. Functional dissection of an intrinsically disordered protein:Understanding the roles of different domains of Knr4 protein in protein-protein interactions[J]. Protein Science.2010,19(7):1376-1385
[53]Arndt V, Dick N, Tawo R, et al. Chaperone-assisted selective autophagy is essential for muscle maintenance[J]. Current Biology.2010,20(2):143-148
[54]Arndt V, Rogon C,H6hfeld J. To be, or not to be—molecular chaperones in protein degradation[J]. Cellular and Molecular Life Sciences.2007,64(19):2525-2541
[55]Kaganovich D, Kopito R,Frydman J. Misfolded proteins partition between two distinct quality control compartments[J]. Nature.2008,454(7208):1088-1095
[56]Shen D, Coleman J, Chan E, et al. Novel cell-and tissue-based assays for detecting misfolded and aggregated protein accumulation within aggresomes and inclusion bodies[J]. Cell Biochem Biophys.2011,60(3):173-185
[57]Neef D W, Jaeger A M,Thiele D J. Heat shock transcription factor 1 as a therapeutic target in neurodegenerative diseases[J]. Nature Reviews Drug Discovery.2011,10(12):930-944
