Responsive image
博碩士論文 etd-0801116-023742 詳細資訊
Title page for etd-0801116-023742
論文名稱
Title
幽門螺旋桿菌感染下SUMO蛋白媒介p38及磷酸化p38蛋白的質核轉移
SUMOs mediate the nuclear transfer of p38 and p-p38 during Helicobacter pylori infection
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
82
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2016-06-16
繳交日期
Date of Submission
2016-09-01
關鍵字
Keywords
p38、質核轉移、幽門螺旋桿菌、SUMO-2、SUMO-1、SIM
SUMO-1, SIM, Helicobacter pylori, p38, nuclear translocalization, SUMO-2
統計
Statistics
本論文已被瀏覽 5673 次,被下載 0
The thesis/dissertation has been browsed 5673 times, has been downloaded 0 times.
中文摘要
細胞遭受各種環境壓力、細胞激素、或者DNA 損害,p38 蛋白激酶的反應調節對細胞非常的重要。p38 分布於細胞質與細胞核。存在於細胞質的p38 會因為各種壓力觸發而往細胞核移動,只是這其中反應機制的認知依舊有著相當大的模糊地帶。細胞遭受壓力的其中一項反饋即是增進SUMO 蛋白的表現。SUMO的眾多調控機制其中之一便是幫助目標蛋白的質核轉移。我們研究證明幽門螺旋桿菌能觸發SUMO 的表現以及p38 的活化。SUMO 與磷酸化及無磷酸化的p38都能以非共價鍵的方式結合,這種結合方式還存在著濃度依賴的關係。當細胞遭受幽門螺旋桿菌的感染時, SUMO-1 及SUMO-2 可將存在於細胞質的p38 送入細胞核內,其中,SUMO-2 對p38 的親和力明顯優於SUMO-1 對p38。SUMO 對p38 的親和性研究中, 我們還發現把p38 的SIM3 位置作突變之後即會大幅度的降低與SUMO 的結合進而影響SUMO 幫助p38 質核轉移的態勢。我們的研究證
實SUMO 是p38 的質核轉移的調控者,以非共價結合的方式調控並且不因p38有無磷酸化而受到影響。
Abstract
p38 mitogen-activated protein kinase (MAPK) is of the essence in the cell’s response to environmental stresses, cytokines and DNA damage. p38 is distributed both in the cytosol and nucleus, and cytosolic p38 translocates into the nucleus in response to various stimuli, yet the exact mechanisms remain largely unclear. One response to cellular stress is the elevated expression of SUMO proteins. SUMOs have many roles in cellular biology including promoting nuclear translocalization. In our study, we have demonstrated that exposure of human cells to Helicobacter pylori (Hp) induces the expression of SUMOs and the activation of p38. A non-covalent interaction between SUMOs and both non-phosphorylated and phosphorylated p38 (p38 and p-p38) was identified, and the interaction was found to be SUMO concentration dependent. We found that upon Hp stimulation cytosolic p38 could be translocated into the nucleus by both SUMO-1 and SUMO-2, although both p38 and p-p38 have a stronger binding affinity for SUMO-2 than for SUMO-1. Mutation of SUMO interacting motif 3 (SIM3) of p38 abolished its binding to SUMOs and decreased SUMO-dependent nuclear transfer of p38. This study demonstrates that SUMOs serve as novel regulators of p38 and p-p38 nuclear translocation through a non-covalent SUMO-p38 interaction, independent of the phosphorylation state of p38.
目次 Table of Contents
學位論文審定書........................................................................................................ i
誌謝…..……………………………………………………....………...................... ii
中文摘要……………………………………..................…………………….…….. iii
Abstract in English……............…………………………………………………….. iv
Content………………………………………………………..…………..…………. v
Abbreviations……………………………………………..………………………… ix
Introduction……………………………………………………………………….. 1
1. Small ubiquitin-related modifier (SUMO)………………………... 1
1.1. SUMOylation……………………………………………………… 2
1.2. SUMO-interaction…………………………………………………. 3
1.3. SUMO regulates the nuclear transport…………………………….. 3
2. Mitogen activated protein kinase 14 (MAPK14), p38 4
2.1. The stresses stimulated the activation of p38……………………... 4
2.2. Nuclear translocation of p38………………………………………. 5
3. Helicobacter pylori (H. pylori or Hp)…………………………….. 6
3.1. Hp-infection induced p38-related apoptosis………………………. 6
Objectives………………………………………………………………………… 8
Materials and methods……………………………………………………………. 9-19
1. Cell culture and Hp infection……………………………………… 9
2. SUMOylation site prediction……………………………………… 9
3. SUMO-interacting motif (SIM) prediction………………………... 9
4. Plasmids……………………………………………………………. 10
5. Reverse-transcription-PCR (RT-PCR)…………………………….. 13
6. Transfections………………………………………………………. 14
7. Yeast two hybrid and β-Galactosidase assay……………………… 14
8. Western blots………………………………………………………. 15
9. Antibodies………………………………………………………….. 16
10. Cell viability assay………………………………………………… 16
11. In vitro p38 phosphorylation……………………………………… 17
12. In vitro pull-down assay…………………………………………… 17
13. In vitro SUMOylation assay……………………………………….. 18
14. Immunofluorescence assay………………………………………… 18
15. Nuclear and cytosolic isolation……………………………………. 19
Results……………………………………………………………………………… 20-32
1. p38 interacts with SUMOs………………………………………….. 23
2. Hp infection induces the expression of p38 and SUMOs in AGS cells…………………………………………………………………..
23
3 p38-mediated apoptosis is associated with SUMOs during Hp infection………………………………………………………………
24
4 The nuclear localization of endogenous p38 and p-p38 is dependent on the levels of SUMOs……………………………………………
25
5. SUMOs mediated nuclear localization of p38 is independent of p38 phosphorylation…………………………………………………….
27
6. SUMO-2 binds more strongly than SUMO-1 to p38-DN, p38WT and p-p38……………………………………………………………..
30
7. SUMO mediated nuclear transfer of p38 is SIM dependent………… 31
Table 1. Yeast two hybrid results demonstrating the interactions between p38 and SUMOs…………………………………………………………..
33
Fig. 1-1. Pull-down assays between GST-p38 and His-SUMOs……………… 34-5
Fig. 1-2. In vitro SUMOylation assays show there are no covalent interactions between p38 and SUMOs………………………………..………….
36-7
Fig. 2. Hp-infection induces the expression of p38, p-p38, SUMO-1 and SUMO-2 in AGS cells……………………………………………….
38-9
Fig. 3. p38-mediated apoptosis is associated with SUMOs during Hp infection………………………………………………………………
40-1
Fig. 4. SUMO-2 is more efficient than SUMO-1 in regulating nuclear p38 and p-p38 during Hp infection……………………………………….
42-3
Fig. 5-1. p38-WT and non-activated p38-DN both colocalize with SUMOs in the Nucleus…………………………………………………………..
44-5
Fig. 5-2-1. SUMOs positively regulate the nuclear transport of p-p38, p38-WT and p38-DN in response to Hp infection…………………………….
46-7
Fig. 5-2-2. Hp-induced apoptosis was related to SUMOs-regulated p38.…………………………………………….…………………….
48-9
Fig. 6. SUMO-2 binds more strongly than SUMO-1 to p38-DN, p38WT and p-p38…………………………………………………………………
50-1
Fig. 7. Binding affinities for SUMOs, and nuclear transfer of p38 are decreased in SIM mutants……………………………………………
52-3
Fig. 8. A model for the nuclear translocation of non-covalent interaction between p38 and SUMOs……………………………………………..
54-5
Discussion…………………………………………………………………………… 56-60
References……………………………………………………………………………. 61-6
Selected publications………………………………………………………………… 67
參考文獻 References
Adachi M, Fukuda M, Nishida E. Two co-existing mechanisms for nuclear import of MAP kinase: passive diffusion of a monomer and active transport of a dimer. EMBO J 1999;18:5347-58.
Charruyer A, Grazide S, Bezombes C, et al. UV-C light induces raft-associated acid sphingomyelinase and JNK activation and translocation independently on a nuclear signal. J Biol Chem 2005;280:19196-204.
Chen A, Wang PY, Yang YC, et al. SUMO regulates the cytoplasmonuclear transport of its target protein Daxx. J Cell Biochem 2006;98:895-911.
Chuderland D, Konson A, Seger R. Identification and characterization of a general nuclear translocation signal in signaling proteins. Mol Cell 2008;31:850-61.
Comerford KM, Leonard MO, Karhausen J, et al. Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia. Proc. Natl. Acad. Sci. U.S.A. 2003; 100:986-91.
Derijard B, Raingeaud J, Barrett T, et al. Independent human MAP-kinase signal transduction pathways defined by MEK and MKK isoforms. Science 1995;267:682-5.
Ding SZ, Minohara Y, Fan XJ, et al. Helicobacter pylori infection induces oxidative stress and programmed cell death in human gastric epithelial cells. Infect Immun 2007;75:4030-9.
Dohmen RJ. SUMO protein modification. Biochim Biophys Acta 2004;1695:113-31.
Ernst PB, Peura DA, Crowe SE. The translation of Helicobacter pylori basic research to patient care. Gastroenterology. 2006; 130: 188–206.
Everett RD, Freemont P, Saitoh H, et al. The disruption of ND10 during herpes simplex virus infection correlates with the Vmw110- and proteasome-dependent loss of several PML isoforms. J Virol 1998;72:6581-91.
Ferrigno P, Posas F, Koepp D, et al. Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1. EMBO J 1998; 17:5606-14.
Gong X, Ming X, Deng P, et al. Mechanisms regulating the nuclear translocation of p38 MAP kinase. J Cell Biochem 2010;110:1420-9.
Han J, Jiang Y, Li Z, et al. Activation of the transcription factor MEF2C by the MAP kinase p38 in inflammation. Nature 1997;386:296-9.
Hannich JT, Lewis A, Kroetz MB, et al. Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae. J Biol Chem 2005;280:4102-10.
Hay RT. SUMO-specific proteases: a twist in the tail, Trends Cell Biol. 2007; 17: 370-76.
Hecker CM, Rabiller M, Haglund K, et al. Specification of SUMO1- and SUMO2-interacting motifs. J Biol Chem 2006; 281: 16117-27.
Jones NL, Shannon PT, Cutz E, et al. Increase in proliferation and apoptosis of gastric epithelial cells early in the natural history of Helicobacter pylori infection. Am J Pathol 1997;151:1695-703.
Johnson ES. Protein modification by SUMO. Annu Rev Biochem 2004; 73:355-82.
Keates S, Keates AC, Warny M, et al. Differential activation of mitogen-activated protein kinases in AGS gastric epithelial cells by cag+ and cag- Helicobacter pylori. J Immunol 1999;163:5552-9.
Kerscher O. SUMO junction-what's your function? New insights through SUMO-interacting motifs. EMBO Rep 2007;8:550-5.
Khokhlatchev AV, Canagarajah B, Wilsbacher J, et al. Phosphorylation of the MAP kinase ERK2 promotes its homodimerization and nuclear translocation. Cell 1998;93:605-15.
Ki MR, Lee HR, Goo MJ, et al. Differential regulation of ERK1/2 and p38 MAP kinases in VacA-induced apoptosis of gastric epithelial cells. Am J Physiol Gastrointest Liver Physiol 2008;294:G635-47.
Knipscheer P, Flotho A, Klug H. et al. Ubc9 Sumoylation regulates SUMO target discrimination. Mol Cell 2008; 31: 371-82.
Kyriakis JM, Avruch J. Mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation. Physiol Rev 2001;81:807-69.
Lin DY, Huang YS, Jeng JC. Et al. Role of SUMO-interacting motif in Daxx SUMO modification, subnuclear localization, and repression of sumoylated transcription factors. Mol Cell 2006; 24: 341-54.
Manza LL, Codreanu SG, Stamer SL, et al. Global shifts in protein sumoylation in response to electrophile and oxidative stress. Chem Res Toxicol 2004;17:1706-15.
Peek RM Jr, Blaser MJ. Helicobacter pylori and gastrointestinal tract adenocarcinomas. Nat. Rev. Cancer 2002; 2: 28-37.
Pomorski T, Meyer TF, Naumann M. Helicobacter pylori-induced prostaglandin E(2) synthesis involves activation of cytosolic phospholipase A(2) in epithelial cells. J Biol Chem 2001;276:804-10.
Powell LM, Chen A, Huang YC, et al. The SUMO pathway promotes basic helix-loop-helix proneural factor activity via a direct effect on the Zn finger protein senseless. Mol Cell Biol 2012;32:2849-60.
Raingeaud J, Gupta S, Rogers JS, et al. Pro-inflammatory cytokines and environmental stress cause p38 mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine. J Biol Chem 1995;270:7420-6.
Ryu SW, Chae SK, Kim E. Interaction of Daxx, a Fas binding protein, with sentrin and Ubc9. Biochem. Biophys. Res. Commun. 2000; 279:6-10.
Saitoh H, Hinchey J. Functional heterogeneity of small ubiquitin-related protein modifiers SUMO-1 versus SUMO-2/3. J Biol Chem 2000;275:6252-8.
Sehat B, Tofigh A, Lin Y, et al. SUMOylation mediates the nuclear translocation
and signaling of the IGF-1 receptor. Sci Signal 2010; 3 (108): ra10.
Song J, Zhang Z, Hu W, et al. Small ubiquitin-like modifier (SUMO) recognition of a SUMO binding motif: a reversal of the bound orientation. J Biol Chem 2005;280:40122-9
Tatham MH, Jaffray E, Vaughan OA, et al. Polymeric chains of SUMO-2 and SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9. J Biol Chem 2001;276:35368-74.
Truong K, Lee TD, Li B, et al. Sumoylation of SAE2 C terminus regulates SAE nuclear localization. J Biol Chem 2012;287:42611-9.
Um JW, Chung KC. Functional modulation of parkin through physical interaction with SUMO-1. J Neurosci Res 2006;84:1543-54.
Yang SH, Sharrocks AD. The SUMO E3 ligase activity of Pc2 is coordinated through a SUMO interaction motif. Mol. Cell Biol. 2007; 30:2193-205.
Yeh JJ, Tsai S, Wu DC, et al. P-selectin-dependent platelet aggregation and apoptosis may explain the decrease in platelet count during Helicobacter pylori infection. Blood 2010;115:4247-53.
Zhu J, Zhu S, Guzzo CM, et al. Small ubiquitin-related modifier (SUMO) binding determines substrate recognition and paralog-selective SUMO modification. J Biol Chem 2008;283:29405-15.
電子全文 Fulltext
本電子全文僅授權使用者為學術研究之目的,進行個人非營利性質之檢索、閱讀、列印。請遵守中華民國著作權法之相關規定,切勿任意重製、散佈、改作、轉貼、播送,以免觸法。
論文使用權限 Thesis access permission:自定論文開放時間 user define
開放時間 Available:
校內 Campus:永不公開 not available
校外 Off-campus:永不公開 not available

您的 IP(校外) 位址是 100.26.35.111
論文開放下載的時間是 校外不公開

Your IP address is 100.26.35.111
This thesis will be available to you on Indicate off-campus access is not available.

紙本論文 Printed copies
紙本論文的公開資訊在102學年度以後相對較為完整。如果需要查詢101學年度以前的紙本論文公開資訊,請聯繫圖資處紙本論文服務櫃台。如有不便之處敬請見諒。
開放時間 available 永不公開 not available

QR Code