臺灣博碩士論文加值系統

人體中大部分的正常體細胞無法無限制地進行細胞分裂，並最終會進入不可逆的生長停止階段，稱之為細胞老化。雖然目前已經有研究發現端粒縮短(telomere shortening)為誘發老化的主因，但仍有許多調控和維持細胞老化過程的未知因子。絲氨酸水解酶在人體蛋白質中佔了約1%的含量，且許多絲氨酸水解酶參與在重要的生理作用中，除此之外，許多絲氨酸水解酶被發現會影響細胞老化，所以本研究的目的為尋找會參與在老化過程的絲氨酸水解酶並進一步研究其特性。本研究利用以活性為基礎的化學蛋白質體分析方法，來鑑定在老化過程中活性有改變的絲氨酸水解酶。在本研究中，利用人類肺纖維母細胞IMR90作為研究細胞老化的平台，並以擁有ethyl-benzylphosphonofluoridates反應端的化學探針LCL08027來標定具有活性的絲氨酸水解酶。透過比較年輕和老化細胞中標定蛋白的差異，鑑定到一些絲氨酸水解酶在老化過程中會有活性的改變，其中tripeptidyl peptidase 2 (TPPII)和protein phosphatase methylesterase 1 (PPME1)的活性會隨著老化而降低。進一步利用small hairpin RNAs發現，當抑制TPPII和PPME1的基因表現時會誘導年輕的IMR90細胞進入老化，證實了這兩個絲氨酸水解酶會參與在老化過程中。總結以上，本研究成功利用以活性探針為基礎的蛋白質體分析法去鑑定到尚未被證明會被參與在老化過程的絲氨酸水解酶。

Most of normal human somatic cells cannot divide indefinitely and eventually enter a state of irreversible proliferative arrest termed replicative senescence. Although previous researches have shown that telomere shortening is the major reason for inducing senescence in human somatic cells, factors that mediate and maintain senescence are largely unknown. Serine hydrolases is a large enzyme family which constitutes about 1% of the human proteome and many of them are involved in important physiological processes. In addition, many serine hydrolases have been shown to affect cellular senescence. Thus, the goal of this research is to identify and characterize novel serine hydrolases that participate in senescence. An activity-probe based chemical proteomic approach was applied to identify serine hydrolases with their activities been altered during senescence. Here the human lung fibroblast IMR-90 cell was used as a model in this research. Chemical probe LCL8027, which has an ethyl-benzylphosphonofluoridates reactive group, is used to label active serine hydrolases. Through comparative labeling of both young and old cells, several serine hydrolases were identified that showed alteration of labeling activities during senescence. Among them, both the activities of tripeptidyl peptidase 2 (TPPII) and protein phosphatase methylesterase 1 (PPME1) were found to be decreased during senescence. Further analyses showed that knocking down TPPII or PPME1 using small hairpin RNAs induced young IMR90 cells into senescence, suggesting a role of these two serine hydrolases in senescence. Together, this study successfully applied an activity probe-based proteomic analysis to identify novel serine hydrolases that might be involved in cellular senescence.

目錄
目錄 i
表目錄 iii
圖目錄 iv
縮寫檢索表 v
摘要 vii
Abstract viii

第一章 緒論 1
1.1 細胞老化 (Cellular senescence) 1
1.1.1 複製老化 (Replicative senescence) 2
1.1.2 壓力所誘發之老化 (Stress-induced senescence) 2
1.2 絲氨酸水解酶 (Serine hydrolases) 3
1.3 蛋白質表現量與活性之監測 4
1.4 化學活性探針 (Activity-based chemical probe) 5
1.5 以Fluorophosphonate (FP)為反應端之化學活性探針 6
1.6 利用活性探針尋找參與在細胞老化中的絲氨酸水解酶 6

第二章 材料方法 8
2.1 細胞株培養 8
2.1.1 細胞株之培養 8
2.1.2 細胞株的來源與細胞培養液 8
2.2 細胞計數 8
2.3 生長曲線 (Population doubling curve) 9
2.4 蛋白質樣品處理與濃度測定 9
2.4.1 蛋白質樣品處理 9
2.4.2 蛋白質濃度測定 9
2.5 西方墨點法 (Western blotting analysis) 9
2.5.1 SDS聚丙烯醯胺凝膠電泳 (SDS-PAGE) 9
2.5.2 轉漬 (Transfer) 10
2.5.3 免疫轉漬分析 (Immunoblot) 10
2.6 考馬斯藍染色法 (Coomassie blue staining) 11
2.7 化學探針標定反應 11
2.7.1 LCL08027探針標定濃度測試 11
2.7.2 PD-10脫鹽管柱去除未反應LCL08027探針 11
2.7.3 LCL08027探針標定蛋白質之純化 11
2.8 液相層析串聯式質譜儀分析、蛋白身分鑑定及相對定量 12
2.8.1 質譜樣品前處理 12
2.8.2 液相層析串聯式質譜(LC-MS/MS)分析與蛋白身分鑑定及定量 13
2.9 大腸桿菌勝任細胞 (competent cells) 菌株與製備 13
2.9.1 菌株 13
2.9.2 製備 13
2.10 質體之來源、製備與轉殖作用 14
2.10.1 質體之來源 14
2.10.2 質體之製備 (Geneaid, PrestoTM mini plasmid kit) 14
2.10.3 轉殖作用 (Transformation) 14
2.11 轉染作用 (Transfection) 與慢病毒的製備 (Lentivirus production) 15
2.12 慢病毒效價測定 (Lentivirus determination) 15
2.13 免疫沉澱法 (Immunoprecipitation) 16
2.14 Senescence-associated beta-galactosidase staining 16
2.15 溴化去氧尿苷 (BrdU) 與免疫螢光染色 (Immunofluorescent staining) 16
2.16 全部訊息核糖核酸萃取與反轉錄聚合酶連鎖反應 (Reverse transcription-polymerase chain reaction; RT-PCR) 17
2.16.1 萃取全部訊息核糖核酸 17
2.16.2 反轉錄反應 (Reverse transcription) 18
2.17 即時定量聚合酶連鎖反應 (Real-time quantitative polymerase chain reaction；RT-Q-PCR) 18

第三章 實驗結果 19
3.1 分析IMR90細胞的老化現象 19
3.2 LCL08027探針標定IMR90細胞蛋白質萃取液之濃度探討 19
3.3 使用PD-10脫鹽管柱去除未反應探針之基本實驗條件探討 20
3.4 分析年輕細胞與老化細胞中絲氨酸水解酶之差異 21
3.5 利用蛋白質譜鑑定LCL08027探針標定之絲氨酸水解酶 21
3.6 利用西方墨點法確認特定蛋白標定的結果 22
3.7 利用免疫沉澱法分離絲氨酸水解酶進行分析 23
3.8 降低TPPII基因的表現導致IMR90細胞走向老化 24
3.9 降低PPME1基因的表現導致IMR90細胞走向老化 25

第四章 討論 26
參考文獻 33

表目錄
Tabel 1. shRNAs from National RNAi Core Facility 39
Tabel 2. Compare serine hydrolases between young and old IMR90 cell from mass spectrometry data 40
Tabel 3. Biological process of identified serine hydrolases by Ingenuity Pathway Analysis. 43
 
圖目錄
Figure 1. Structure and labeling mechanism of LCL08027.
44
Figure 2. Senescence phenotype of IMR90 cells. 45
Figure 3. Titration of LCL08027 probes in IMR90 cell lysates. 46
Figure 4. Schematic presentation of activity probe-based proteomic analysis using LCL08027. 47
Figure 5. Elution of probe-labeled proteins from PD-10 column. 48
Figure 6. Differences of probe-labeled serine hydrolases in young and old IMR90 cells. 49
Figure 7. Mass spectrometry analysis of probe-labeled serine hydrolases in young and old IMR90 cells. 50
Figure 8. The labeling of TPPII, TPPI, and PPME1 are altered in young and old IMR90 cells. 51
Figure 9. Schematic presentation of approach used to detect specific serine hydrolase activity. 52
Figure 10. Alteration of TPPII, PPME1, and TPPI activities during senescence. 54
Figure 11. The expression levels of TPPI, TPPII and PPME1 in young and senescent IMR90 cells. 55
Figure 12. Knock down TPPII in IMR90 cells caused senescence phenotype. 57
Figure 13. Knock down TPPII in IMR90 cells caused cell growth arrest. 60
Figure 14. Knock down PPME1 in IMR90 cells caused senescence phenotype. 61
Figure 15. Knock down TPPII in IMR90 cells caused cell growth arrest. 62
 

Hayflick L, Moorhead PS: The serial cultivation of human diploid cell strains. Exp Cell Res 1961, 25:585-621.
2.Hayflick L: The Limited in Vitro Lifetime of Human Diploid Cell Strains. Exp Cell Res 1965, 37:614-636.
3.Robbins E, Levine EM, Eagle H: Morphologic changes accompanying senescence of cultured human diploid cells. J Exp Med 1970, 131:1211-1222.
4.Kurz DJ, Decary S, Hong Y, Erusalimsky JD: Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells. J Cell Sci 2000, 113:3613-3622.
5.Dimri GP, Lee X, Basile G, Acosta M, Scott G, Roskelley C, Medrano EE, Linskens M, Rubelj I, Pereira-Smith O et al: A biomarker that identifies senescent human cells in culture and in aging skin in vivo. Proc Natl Acad Sci U S A 1995, 92:9363-9367.
6.Lee BY, Han JA, Im JS, Morrone A, Johung K, Goodwin EC, Kleijer WJ, DiMaio D, Hwang ES: Senescence-associated beta-galactosidase is lysosomal beta-galactosidase. Aging Cell 2006, 5:187-195.
7.Herbig U, Jobling WA, Chen BP, Chen DJ, Sedivy JM: Telomere shortening triggers senescence of human cells through a pathway involving ATM, p53, and p21(CIP1), but not p16(INK4a). Mol Cell 2004, 14:501-513.
8.Jackson JG, Pereira-Smith OM: p53 is preferentially recruited to the promoters of growth arrest genes p21 and GADD45 during replicative senescence of normal human fibroblasts. Cancer Res 2006, 66:8356-8360.
9.Narita M, Nuñez S, Heard E, Narita M, Lin AW, Hearn SA, Spector DL, Hannon GJ, Lowe SW: Rb-Mediated Heterochromatin Formation and Silencing of E2F Target Genes during Cellular Senescence. Cell 2003, 113:703-716.
10.Smith-Vikos T, Slack FJ: MicroRNAs and their roles in aging. J Cell Sci 2012, 125:7-17.
11.Zhang R, Chen W, Adams PD: Molecular Dissection of Formation of Senescence-Associated Heterochromatin Foci. Molecular and Cellular Biology 2007, 27:2343-2358.
12.Narita M, Narita M, Krizhanovsky V, Nuñez S, Chicas A, Hearn SA, Myers MP, Lowe SW: A Novel Role for High-Mobility Group A Proteins in Cellular Senescence and Heterochromatin Formation. Cell 2006, 126:503-514.
13.Pegoraro G, Kubben N, Wickert U, Gohler H, Hoffmann K, Misteli T: Ageing-related chromatin defects through loss of the NURD complex. Nat Cell Biol 2009, 11:1261-1267.
14.Pollina EA, Brunet A: Epigenetic regulation of aging stem cells. Oncogene 2011, 30:3105-3126.
15.Harley CB, Futcher AB, Greider CW: Telomeres shorten during ageing of human fibroblasts. Nature 1990, 345:458-460.
16.Rohme D: Evidence for a relationship between longevity of mammalian species and life spans of normal fibroblasts in vitro and erythrocytes in vivo. Proc Natl Acad Sci U S A 1981, 78:5009-5013.
17.Zakian VA: Telomeres: beginning to understand the end. Science 1995, 270:1601-1607.
18.Bodnar AG, Ouellette M, Frolkis M, Holt SE, Chiu CP, Morin GB, Harley CB, Shay JW, Lichtsteiner S, Wright WE: Extension of life-span by introduction of telomerase into normal human cells. Science 1998, 279:349-352.
19.Blasco MA: Telomere length, stem cells and aging. Nat Chem Biol 2007, 3:640-649.
20.Rothkamm K, Löbrich M: Evidence for a lack of DNA double-strand break repair in human cells exposed to very low x-ray doses. Proc Natl Acad Sci U S A 2003, 100:5057-5062.
21.Shay JW, Roninson IB: Hallmarks of senescence in carcinogenesis and cancer therapy. Oncogene 2004, 23:2919-2933.
22.Benanti JA, Galloway DA: The Normal Response to RAS: Senescence or Transformation? Cell Cycle 2004, 3:713-715.
23.Ferbeyre G, de Stanchina E, Lin AW, Querido E, McCurrach ME, J. Hannon G, Lowe SW: Oncogenic ras and p53 Cooperate To Induce Cellular Senescence. Mol Cell Biol 2002, 22:3497-3508.
24.Serrano M, Lin AW, McCurrach ME, Beach D, Lowe SW: Oncogenic ras Provokes Premature Cell Senescence Associated with Accumulation of p53 and p16INK4a. Cell 1997, 88:593-602.
25.Cravatt BF, Wright AT, Kozarich JW: Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu Rev Biochem 2008, 77:383-414.
26.Simon GM, Cravatt BF: Activity-based proteomics of enzyme superfamilies: serine hydrolases as a case study. J Biol Chem 2010, 285:11051-11055.
27.Fonovic M, Bogyo M: Activity-based probes as a tool for functional proteomic analysis of proteases. Expert Rev Proteomics 2008, 5:721-730.
28.Khan AR, James MN: Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes. Protein Sci 1998, 7:815-836.
29.Roberts RM, Mathialagan N, Duffy JY, Smith GW: Regulation and regulatory role of proteinase inhibitors. Crit Rev Eukaryot Gene Expr 1995, 5:385-436.
30.Whisstock J, Skinner R, Lesk AM: An atlas of serpin conformations. Trends Biochem Sci 1998, 23:63-67.
31.Lowe ME: Molecular mechanisms of rat and human pancreatic triglyceride lipases. J Nutr 1997, 127(4):549-557.
32.Licari LG, Kovacic JP: Thrombin physiology and pathophysiology. J Vet Emerg Crit Care (San Antonio) 2009, 19:11-22.
33.Hildenbrand R, Gandhari M, Stroebel P, Marx A, Allgayer H, Arens N: The urokinase-system--role of cell proliferation and apoptosis. Histol Histopathol 2008, 23:227-236.
34.Yoshida S, Shiosaka S: Plasticity-related serine proteases in the brain (review). Int J Mol Med 1999, 3:405-409.
35.Yanagida M: Functional proteomics; current achievements. J Chromatogr B Analyt Technol Biomed Life Sci 2002, 771:89-106.
36.Dove A: Proteomics: translating genomics into products? Nat Biotechnol 1999, 17:233-236.
37.Corthals GL, Wasinger VC, Hochstrasser DF, Sanchez JC: The dynamic range of protein expression: a challenge for proteomic research. Electrophoresis 2000, 21:1104-1115.
38.Gygi SP, Corthals GL, Zhang Y, Rochon Y, Aebersold R: Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology. Proc Natl Acad Sci U S A 2000, 97:9390-9395.
39.Gygi SP, Rist B, Gerber SA, Turecek F, Gelb MH, Aebersold R: Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat Biotechnol 1999, 17:994-999.
40.Wolters DA, Washburn MP, Yates JR, 3rd: An automated multidimensional protein identification technology for shotgun proteomics. Anal Chem 2001, 73:5683-5690.
41.Adam GC: Chemical Strategies for Functional Proteomics. Molecular & Cellular Proteomics 2002, 1:781-790.
42.Zhang H, Yan W, Aebersold R: Chemical probes and tandem mass spectrometry: a strategy for the quantitative analysis of proteomes and subproteomes. Curr Opin Chem Biol 2004, 8:66-75.
43.Jessani N, Cravatt BF: The development and application of methods for activity-based protein profiling. Curr Opin Chem Biol 2004, 8:54-59.
44.Evans MJ, Cravatt BF: Mechanism-based profiling of enzyme families. Chem Rev 2006, 106(8):3279-3301.
45.Jeffery DA, Bogyo M: Chemical proteomics and its application to drug discovery. Curr Opin Biotechnol 2003, 14:87-95.
46.Kalesh KA, Tan LP, Lu K, Gao L, Wang J, Yao SQ: Peptide-based activity-based probes (ABPs) for target-specific profiling of protein tyrosine phosphatases (PTPs). Chem Commun (Camb) 2010, 46:589-591.
47.Sadaghiani AM, Verhelst SHL, Bogyo M: Tagging and detection strategies for activity-based proteomics. Curr Opin Chem Biol 2007, 11:20-28.
48.Lo L-C, Pang T-L, Kuo C-H, Chiang Y-L, Wang H-Y, Lin J-J: Design and Synthesis of Class-Selective Activity Probes for Protein Tyrosine Phosphatases. J Proteome Res 2002, 1:35-40.
49.Greenbaum D, Medzihradszky KF, Burlingame A, Bogyo M: Epoxide electrophiles as activity-dependent cysteine protease profiling and discovery tools. Chem Biol 2000, 7:569-581.
50.Liu Y, Patricelli MP, Cravatt BF: Activity-based protein profiling: The serine hydrolases. Proc Natl Acad Sci U S A 1999, 96:14694-14699.
51.Hawkins RD, Mendel B: Selective inhibition of pseudo-cholinesterase by diisopropyl fluorophosphonate. Br J Pharmacol Chemother 1947, 2:173-180.
52.Dijkstra HP, Sprong H, Aerts BN, Kruithof CA, Egmond MR, Klein Gebbink RJ: Selective and diagnostic labelling of serine hydrolases with reactive phosphonate inhibitors. Org Biomol Chem 2008, 6:523-531.
53.Yoon IK, Kim HK, Kim YK, Song I-H, Kim W, Kim S, Baek S-H, Kim JH, Kim J-R: Exploration of replicative senescence-associated genes in human dermal fibroblasts by cDNA microarray technology. Exp Gerontol 2004, 39:1369-1378.
54.Chen YP: Utilization of Benzyl Fluorophosphonate-based Probes for Proteomic Applications. National Taiwan University; 2010.
55.Bartlett GJ, Porter CT, Borkakoti N, Thornton JM: Analysis of catalytic residues in enzyme active sites. J Mol Biol 2002, 324(1):105-121.
56.Ray SP, Deaton MK, Capodagli GC, Calkins LA, Sawle L, Ghosh K, Patterson D, Pegan SD: Structural and biochemical characterization of human adenylosuccinate lyase (ADSL) and the R303C ADSL deficiency-associated mutation. Biochemistry 2012, 51(33):6701-6713.
57.Milstein CP, Milstein C: A tryptic peptide containing a unique serine phosphate residue in rabbit phosphoglucomutase. Biochem J 1968, 109(1):93-99.
58.Mio T, Yamada-Okabe T, Arisawa M, Yamada-Okabe H: Functional cloning and mutational analysis of the human cDNA for phosphoacetylglucosamine mutase: identification of the amino acid residues essential for the catalysis. Biochim Biophys Acta 2000, 1492(2-3):369-376.
59.Fisher AB: Peroxiredoxin 6: a bifunctional enzyme with glutathione peroxidase and phospholipase A(2) activities. Antioxid Redox Signal 2011, 15(3):831-844.
60.D’Erchia AM, Tullo A, Lefkimmiatis K, Saccone C, Sbisà E: The Fatty Acid Synthase Gene is a Conserved p53 Family Target Gene from Worm to Human. Cell Cycle 2006, 5:750-758.
61.Ford JH: Saturated fatty acid metabolism is key link between cell division, cancer, and senescence in cellular and whole organism aging. Age 2010, 32:231-237.
62.Maeda M, Scaglia N, Igal RA: Regulation of fatty acid synthesis and Δ9-desaturation in senescence of human fibroblasts. Life Sci 2009, 84(3–4):119-124.
63.Macpherson E, Tomkinson B, Balow RM, Hoglund S, Zetterqvist O: Supramolecular structure of tripeptidyl peptidase II from human erythrocytes as studied by electron microscopy, and its correlation to enzyme activity. Biochem J 1987, 248(1):259-263.
64.Rockel B, Peters J, Kuhlmorgen B, Glaeser RM, Baumeister W: A giant protease with a twist: the TPP II complex from Drosophila studied by electron microscopy. EMBO J 2002, 21(22):5979-5984.
65.Hilbi H, Jozsa E, Tomkinson B: Identification of the catalytic triad in tripeptidyl-peptidase II through site-directed mutagenesis. Biochim Biophys Acta 2002, 1601:149-154.
66.Balow RM, Ragnarsson U, Zetterqvist O: Tripeptidyl aminopeptidase in the extralysosomal fraction of rat liver. J Biol Chem 1983, 258:11622-11628.
67.Stavropoulou V, Vasquez V, Cereser B, Freda E, Masucci MG: TPPII promotes genetic instability by allowing the escape from apoptosis of cells with activated mitotic checkpoints. Biochem Biophys Res Commun 2006, 346(2):415-425.
68.Huai J, Firat E, Nil A, Million D, Gaedicke S, Kanzler B, Freudenberg M, van Endert P, Kohler G, Pahl HL et al: Activation of cellular death programs associated with immunosenescence-like phenotype in TPPII knockout mice. Proc Natl Acad Sci U S A 2008, 105:5177-5182.
69.Hara K, Yonezawa K, Weng QP, Kozlowski MT, Belham C, Avruch J: Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. J Biol Chem 1998, 273(23):14484-14494.
70.Zoncu R, Efeyan A, Sabatini DM: mTOR: from growth signal integration to cancer, diabetes and ageing. Nat Rev Mol Cell Biol 2011, 12(1):21-35.
71.Hands SL, Proud CG, Wyttenbach A: mTOR''s role in ageing: protein synthesis or autophagy? Aging (Albany NY) 2009, 1(7):586-597.
72.Noda T, Ohsumi Y: Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast. J Biol Chem 1998, 273(7):3963-3966.
73.Young AR, Narita M, Ferreira M, Kirschner K, Sadaie M, Darot JF, Tavare S, Arakawa S, Shimizu S, Watt FM et al: Autophagy mediates the mitotic senescence transition. Genes Dev 2009, 23(7):798-803.
74.Wandzioch E, Pusey M, Werda A, Bail S, Bhaskar A, Nestor M, Yang J-J, Rice LM: PME-1 Modulates Protein Phosphatase 2A Activity to Promote the Malignant Phenotype of Endometrial Cancer Cells. Cancer Res 2014, 74(16):4295-4305.
75.Mannava S, Omilian AR, Wawrzyniak JA, Fink EE, Zhuang D, Miecznikowski JC, Marshall JR, Soengas MS, Sears RC, Morrison CD et al: PP2A-B56alpha controls oncogene-induced senescence in normal and tumor human melanocytic cells. Oncogene 2012, 31:1484-1492.