錯誤褶疊蛋白質誘導之擬熱休克反應機制之探討

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陳銘澤(Ming-Tse Chen) 查詢紙本館藏

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生命科學系

論文名稱

錯誤褶疊蛋白質誘導之擬熱休克反應機制之探討
(Understanding the mechnism of misfolded protein induced- heat-shock-like response)

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摘要(中)

本研究旨在探討水稻小分子量熱休克蛋白質基因Oshsp18.0受脯胺酸(Proline)類似物Azetidine-2-carboxylate (AZC)之誘導模式及其相關調控機制。生物藉由誘導熱休克基因來抵抗外界環境溫度的改變。除溫度改變外，環境中存在之重金屬離子、化學物質或胺基酸類似物，甚至僅酸鹼度的改變皆可誘導熱休克基因之表現。熱休克基因的表現導致細胞內熱休克蛋白質大量累積，有助於增加細胞對嚴苛環境之耐受性，因此植物體於逆境時常累積大量小分子量熱休克蛋白質。將水稻白化幼苗處理致死高溫(48℃)或AZC後可見嚴重離子滲漏之現象。離子滲漏現象可因預先處理非致死性高溫(41℃)而緩和。水稻染色體中存在九個第一族小分子量熱休克蛋白質基因成員。我們針對Oshsp18.0進行詳細研究；發現此基因受高溫、銅、鎘、砷、AZC、酸鹼變化誘導。AZC會取代Proline之位置結合至寡胜肽並使寡胜肽之正常褶疊受到抑制。細胞誘導unfolded protein response (UPR) 以消除AZC引起之不正常蛋白質累積。將水稻白化苗預先處理蛋白質生合成抑制劑(cycloheximide)後，Oshsp18.0對AZC有反應延遲之現象；相對地，Oshsp18.0於高溫逆境並未有反應延遲現象。基因之表現受到轉錄因子及轉錄元素之間交互作用調控；且已有研究顯示，Oshsp18.0及Oshsp17.3二者重疊之啟動子區域存在一段包含九個核苷酸之序列(AZRE)與AZC具有專一性之關聯。綜合以上結果，推論存在特定轉錄因子會與AZRE進行專一性反應。
為獲得此轉錄因子之序列，我們利用yeast one-hybrid之技術釣取此轉錄因子之基因。由篩選所得之基因中，確有參與在UPR相關之基因(包括促進胜肽褶疊、運送、降解及終止蛋白質的合成)。我們選取一具高度重覆性之基因－10-kDa chaperonin－進行gel retardation實驗。結果顯示，此蛋白質並未能在in vitro情況下與AZRE進行鍵結；因此有必要繼續由所篩選所得之基因中挑選其他候選者進行分析。

摘要(英)

We focused on studying the mechanism of gene induction of Oshsp18.0 under treatment of L-azetidine-2-carboxylate (AZC), a proline analog, in this study. Organisms respond to elevated temperature by up-regulating heat shock genes. Not only in temperature stress, but also in various kinds of chemical irritations and pH change induces expression of heat shock genes. Induction of heat shock genes results in accumulation of heat shock proteins. Accumulated heat shock proteins enhances thermotolerance or resistance to following severe stress. AZC induces expression of small heat shock protein genes, however, it causes sever ion-leakage to a comparable level of lethal heat stress treatment. By pre-treatment of nonlethal heat stress, the ion-leakage dropped. Hence, acclimation enhances thermotolerance while AZC causes severe ion-leakage. We chose one of the nine class I small heat shock protein genes in rice, Oshsp18.0, for detailed analysis via RT-PCR. Oshsp18.0 is expressed under heat stress, AZC, Cu2+, Cd2+ or As3+. Oshsp18.0 is expressed at a higher level under heat stress, AZC, As3+ and lower level under Cu2+ and Cd2+. Even change of pH slightly induces expression of Oshsp18.0. As a prline analog, AZC is likely to incorporate into polypeptides which causes misfolded protein and unfolded protein response. Pre-treatment of inhibitor of protein de novo synthesis, cycloheximide, gene expression of Oshsp18.0 delayed under AZC treatment which was not observed in other treatments. This implies different gene regulation mechanism is involved under AZC treatment. Induction of heat shock genes depends on the interaction of transcription factor and regulatory element located on the promoter region of heat shock genes. A 9-base-pair element, AZRE (5’ -CGTCCAGGAC- 3’), was previously identified in the promoters of Oshsp18.0 and Oshsp17.3. AZRE responded to AZC treatment specifically. To identify the transcription factor(s) that specifically interact with AZRE, we used yeast one-hybrid technology to screen cDNA library which was prepared from AZC treatment. Of the candidates, genes involved in the unfolded protein response were obtained. For its repetition, 10-kDa chaperonin (10Cpn), was selected for further analysis. 10Cpn is a constitutively expressed gene as shown via RT-PCR. In vitro binding assay showed no interaction with AZRE. Other candidate genes must be tested in the future.

關鍵字(中)

★ 脯胺酸類似物
★ 錯誤褶疊蛋白質
★ 熱休克反應
★ 小分子量熱休克蛋白質
★ 錯誤褶疊蛋白質反應
★ 內質網逆境

關鍵字(英)

★ Oshsp18.0
★ misfolded protein
★ heat shock response
★ small heat shock protein
★ proline analog
★ azetidine-2-carboxylic acid
★ AZC
★ ER stress
★ unfolded protein response

論文目次

中文摘要 I
Abstract II
Abbreviations list IV
Introduction 1
Materials and Methods 7
Results 12
Discussions 17
References 22
Figure and Table 28
Table 1. Primer list 28
Table 2. Yeast one-hybrid screened data 29-32
Figure 1. Ion leakage analysis uner treatment of 5 mM
AZC 33
Figure 2a. Acclimation with mild heat stress prior to
severe stress 34
Figure 2b. Statistic analysis 35
Figure 3. Gene expression of Oshsp18.0 under high
temperature stress 36
Figure 4. Gene expression of Oshsp18.0 under treatment
of AZC 37
Figure 5. Gene expression of Oshsp18.0 under treatment
of heavy metals 38
Figure 6. Gene expression of Oshsp18.0 under treatment
of arsenite (III) 39
Figure 7. Gene expression of Oshsp18.0 under change of
pH value 40
Figure 8. Gene expression of Oshsp18.0 under control and
low temperature 41
Figure 9a. Gene expression of Oshsp18.0 under pre-
treatment cycloheximide 42
Figure 9b. Gene expression of Oshsp18.0 under pre-
treatment of cycloheximide 43
Figure 10. Gene expression of Oshsp18.0 under various
conc. of cycloheximide 44
Figure 11. Gene expression of 10-kDa chaperonin gene
(10Cpn) 45
Figure 11b. Gel retardation analysis 46
Appendix 47
Appendix 1. Yeast one-hybrid procedure 47
Appendix 2. Blast data of 10CPN toward DRTF 48-49
Appendix 3a. cDNA sequence alignment of OsProT & OsAaP 50-54
Appendix 3b. Amino acid alignment of OsProT & OsAaP 55
Appendix 3c. Hydrophobicity profile of OsProT & OsAaP 56
Appendix 4. Gene expression of OsProT & OsAaP 57

參考文獻

1.Muhuddin Rajin Anwar, et al. (2007) Climate change
impact on rainfed wheat in south-eastern Australia.
Field Crops Res 104: 139-147.
2.Tadashi Kumagai, et al. (2001) Effects of supplemental
UV-B radiation on the growth and yield of two cultivars
of Japanese lowland rice (Oryza sativa L.) under the
field in a cool rice-growing region of Japan.
Agriculture, Ecosystems and Environ 83: 201-208.
3.Boyer, J. S. (1982) Plant Productivity and Environment.
Science 218: 443-448.
4.P. Krishnan, et al. (2007) Impact of elevated CO2 and
temperature on rice yield and methods of adaptation as
evaluated by crop simulation studies. Agriculture,
Ecosystems and Environ 122: 233-242.
5.F. Ritossa (1962) A new puffing pattern induced by
temperature shock and DNP in Drosophila. Experientia
18: 571-573.
6.Tissieres, A., Mitchell, H. K., and Tracy, U. M. (1974)
Protein synthesis in salivary glands of Drosophila
melanogaster: Relation to chromosome puffs. J Mol Biol
85: 389-98.
7.N. Škreb and Frank, Z. (1963) Developmental
abnormalities in the rat induced by heat shock. J
Embryol expMorph 11: 445-457.
8.J. Brachet (1949) Les effets morphogénétiques,
cytochimiques et biochimiques d'un choc thermique au
stade gastrula chez les Amphibiens. Times Journées Cyto-
embryologiques belgonéerlandaises Gand 50-55.
9.Malik, M. K., et al. (1999) Modified expression of a
carrot small heat shock protein gene, hsp17. 7, results
in increased or decreased thermotolerancedouble dagger.
Plant J 20: 89-99.
10.Harndahl, U., et al. (1999) The chloroplast small heat
shock protein undergoes oxidation-dependent
conformational changes and may protect plants from
oxidative stress. Cell Stress Chaperones 4: 129-38.
11.Sun, W., et al. (2001) At-HSP17.6A, encoding a small
heat-shock protein in Arabidopsis, can enhance
osmotolerance upon overexpression. Plant J 27: 407-15.
12.Sugino, M., et al. (1999) Overexpression of DnaK from a
halotolerant cyanobacterium Aphanothece halophytica
acquires resistance to salt stress in transgenic tobacco
plants. Plant Sci 146: 81-88.
13.Guan, J. C., et al. (2004) Characterization of the
genomic structures and selective expression profiles of
nine class I small heat shock protein genes clustered
on two chromosomes in rice (Oryza sativa L.). Plant Mol
Biol 56: 795-809.
14.Alexandrov, V. J., Ouchakov, B. P., and Poljansky, G.
I. (1961) The thermal death of cells in relation to
the problem of the adaptation of organisms to the
temperature of the environment. Pathol Biol (Paris) 9:
849-54.
15.Vierling, E. (1991) The role of heat shock proteins in
plants. Annu Rev Plant Physiol Plant Mol Biol 42: 579-
620.
16.Larkindale, J., et al. (2005) Heat stress phenotypes of
Arabidopsis mutants implicate multiple signaling
pathways in the acquisition of thermotolerance. Plant
Physiol 138: 882-97.
17.Wangxia Wang, et al. (2004) Role of plant heat-shock
proteins and molecular chaperones in the abiotic stress
response. TRENDS in Plant Sci 9: 244-252.
18.Christine Queitsch, et al. (2000) Heat shock protein
101 plays a crucial role in thermotolerance in
Arabidopsis. Plant Cell 12: 479-492.
19.Yeh, C. H., et al. (1997) Expression of a gene encoding
a 16.9-kDa heat-shock protein, Oshsp16.9, in
Escherichia coli enhances thermotolerance. Proc Natl
Acad Sci U S A 94: 10967-72.
20.Ung Lee, et al. (2006) The Arabidopsis ClpB/Hsp100
family of proteins: chaperones for stress and
chloroplast development. Plant J 49: 115-127.
21.Eva Czarnecka, Paul C. Fox, and Gurley, William B.
(1990) In vitro interaction of nuclear proteins with the
promoter of soybean heat shock gene Gmhsp17.5E. Plant
Physiol 94: 935-943.
22.Nover, L., et al. (1996) The Hsf world: classification
and properties of plant heat stress transcription
factors. Cell Stress Chaperones 1: 215-23.
23.Nakai, A. (1999) New aspects in the vertebrate heat
shock factor system: Hsf3 and Hsf4. Cell Stress
Chaperones 4: 86-93.
24.Pascal von Koskull-Döring, Klaus-Dieter Scharf, and
Nover, Lutz (2007) The diversity of plant heat stress
transcription factors. TRENDS in Plant Sci 12: 452-457.
25.Lutz Nover, et al. (2001) Arabidopsis and the heat
stress transcription factor world: how many heat stress
transcription factors do we need? Cell Stress &
Chaperones 6: 177-189.
26.Jingkang Guo, et al. (2008) Genome-wide analysis of
heat shock transcription factor families in rice and
Arabidopsis. J Genet and Genomics 35: 105-118.
27.Franziska Schramm, et al. (2008) A cascade of
transcription factor DREB2A and heat stress
transcription factor HsfA3 regulates the heat stress
response of Arabidopsis. Plant J 53: 264-274.
28.Takumi Yoshida, et al. (2008) Functional analysis of an
Arabidopsis heat-shock transcription factor HsfA3 in the
transcriptional cascade downstream of the DREB2A stress-
regulatory system. Biochem Biophysic Res Commun 368:
512-521.
29.Wolfgang Busch, Markus Wunderlich, and Schöffl, Fritz
(2005) Identification of novel heat shock factor-
dependent genes and biochemical pathways in Arabidopsis
thaliana. Plant J 41: 1-14.
30.Yee-yung Charng, et al. (2007) A heat-inducible
transcription factor, HsfA2, is required for extension
of acquired thermotolerance in Arabidopsis. Plant
Physiol 143: 251-262.
31.Ayako Nishizawa, et al. (2006) Arabidopsis heat shock
transcription factor A2 as a key regulator in response
to several types of environmental stress. Plant J 48:
535-547.
32.Naoki Yokotani, et al. (2008) Expression of rice heat
stress transcription factor OsHsfA2e enhances tolerance
to environmental stresses in transgenic Arabidopsis.
Planta 227: 957-967.
33.Concepción Almoguera, Pilar Prieto-Dapena, and Jordano,
Juan (1998) Dual regulation of a heat shock promoter
during embryogenesis: stage-dependent role of heat shock
elements. Plant J 13: 437-446.
34.Coca, M. A., et al. (1996) Differential regulation of
small heat-shock genes in plants: analysis of a water-
stress-inducible and developmentally activated sunflower
promoter. Plant Mol Biol 31: 863-876.
35.Almoguera, C., et al. (2002) A seed-specific heat-shock
transcription factor involved in developmental
regulation during embryogenesis in sunflower. J Biol
Chem 277: 43866-43872.
36.Kadowaki, H., Nishitoh, H., and Ichijo, H. (2004)
Survival and apoptosis signals in ER stress: the role of
protein kinases. J Chem Neuroanat 28: 93-100.
37.Bonilla, M., Nastase, K. K., and Cunningham, K. W.
(2002) Essential role of calcineurin in response to
endoplasmic reticulum stress. EMBO J 21: 2343-53.
38.Kozutsumi, Yasunori, et al. (1988) The presence of
malfolded proteins in the endoplasmic reticulum signals
the induction of glucose-regulated proteins. Nature
332: 462-464.
39.Hampton, Randolph Y. (2000) ER stress response: Getting
the UPR hand on misfolded proteins. Current Biol 10:
R518-R521.
40.Mori, K. (2000) Tripartite management of unfolded
proteins in the endoplasmic reticulum. Cell 101: 451-
454.
41.Immaculada M. Martínez and Chrispeels, Maarten J.
(2003) Genomic analysis of the unfolded protein response
in Arabidopsis shows its connection to important
cellular processes. Plant Cell 15: 561-576.
42.Shinya Kamauchi, et al. (2005) Gene expression in
response to endoplasmic reticulum stress in Arabidopsis
thaliana. FEBS J 272: 3461-3476.
43.Liu, J. X., et al. (2007) An endoplasmic reticulum
stress response in Arabidopsis is mediated by
proteolytic processing and nuclear relocation of a
membrane-associated transcription factor, bZIP28. Plant
Cell 19: 4111-4119.
44.Iwata, Y. and Koizumi, N. (2005) An Arabidopsis
transcription factor, AtbZIP60, regulates the
endoplasmic reticulum stress response in a manner unique
to plants. Proc Natl Acad Sci USA 102: 5280-5285.
45.Reyes, F., et al. (2006) AtUTr1, a UDP-glucose/UDP-
galactose transporter from Arabidopsis thaliana, is
located in the endoplasmic reticulum and up-regulated by
the unfolded protein response. J Biol Chem 281: 9145-
9151.
46.Kirst, M. E., et al. (2005) Identification and
characterization of endoplasmic reticulum-associated
degradation proteins differentially affected by
endoplasmic reticulum stress. Plant Physiol 138: 218-
231.
47.Koizumi, N., et al. (1999) Overexpression of a gene
that encodes the first enzyme in the biosynthesis of
asparagine-linked glycans makes plants resistant to
tunicamycin and obviates the tunicamycin-induced
unfolded protein response. Plant Physiol 121: 353-361.
48.Urade, R. (2007) Cellular response to unfolded proteins
in the endoplasmic reticulum of plants. FEBS J 274:
1152-1171.
49.L. Fowden (1956) Azetidine-2-carboxylic acid: a new
cyclic imino acid occurring in plants. Biochem J 64:
323-332.
50.M. H. Richmond (1962) The effect of amino acid
analogues on growth and protein synthesis in
microorganisms. Bacteriol Rev 26: 398-420.
51.S. I. Yokota, et al. (2000) Upregulation of cytosolic
chaperonin CCT subunits during recovery from chemical
stress that causes accumulation of unfolded proteins.
Eur J Biochem 267: 1658-1664.
52.Michael G. Pitman, et al. (1977) Effect of azetidine 2-
carboxylic acid on ion uptake and ion release to the
xylem of excised barley roots. Plant Physiol 60: 240-
246.
53.Liu, Y. and Chang, A. (2008) Heat shock response
relieves ER stress. EMBO J 27: 1049-1059.
54.Lin, C. Y., Roberts, J. K., and Key, J. L. (1984)
Acquisition of Thermotolerance in Soybean Seedlings :
Synthesis and Accumulation of Heat Shock Proteins and
their Cellular Localization. Plant Physiol 74: 152-160.
55.Zhu, Y. Y., et al. (2001) Reverse transcriptase
template switching: a SMART approach for full-length
cDNA library construction. Biotechniques 30: 892-897.
56.Huang, B., Jin, L., and Liu, J. Y. (2008)
Identification and characterization of the novel gene
GhDBP2 encoding a DRE-binding protein from cotton
(Gossypium hirsutum). J Plant Physiol 165: 214-233.
57.Lin, Y.P. (2006) Promoter and functional assay of
stress proteins in plants. Master Thesis, Grauduate
schoolof Life Science, National Central University
58.Gregersen, N., et al. (2006) Protein misfolding and
human disease. Annu Rev Genomics Hum Genet 7: 103-24.
59.Chiti, F. and Dobson, C. M. (2006) Protein misfolding,
functional amyloid, and human disease. Annu Rev Biochem
75: 333-66.
60.Dobson, C. M. (2006) Protein aggregation and its
consequences for human disease. Protein Pept Lett 13:
219-27.
61.Yeh, C.H., et al. (2007) Physiological effects of
azetidine on cellular leakage in soybean seedlings.
Plant Sci 172: 1124-1130.
62.Ueda, A., et al. (2008) Altered expression of barley
proline transporter causes different growth responses in
Arabidopsis. Planta 227: 277-86.
63.Claes Andréasson, Etienne P. A. Neve, and Ljungdahl,
Per O. (2004) Four permeases import proline and the
toxic proline analogue azetidine-2-carboxylate into
yeast. Yeast 21: 193-199.
64.Schwacke, R., et al. (1999) LeProT1, a transporter for
proline, glycine betaine, and gamma-amino butyric acid
in tomato pollen. Plant Cell 11: 377-92.
65.Ueda, A., et al. (2001) Functional analysis of salt-
inducible proline transporter of barley roots. Plant
Cell Physiol 42: 1282-9.
66.Wallace, I. S., Choi, W. G., and Roberts, D. M. (2006)
The structure, function and regulation of the nodulin 26-
like intrinsic protein family of plant
aquaglyceroporins. Biochim Biophys Acta 1758: 1165-75.
67.Isayenkov, S. V. and Maathuis, F. J. (2008) The
Arabidopsis thaliana aquaglyceroporin AtNIP7;1 is a
pathway for arsenite uptake. FEBS Lett 582: 1625-8.
68.Cape, J. N. (1993) Direct damage to vegetation caused
by acid rain and polluted cloud: definition of critical
levels for forest trees. Environ Pollut 82: 167-80.
69.Elliott, C. L., Eberhardt, J. C., and Brennan, E. G.
(1987) The effect of ambient ozone pollution and acidic
rain on the growth and chlorophyll content of green and
white ash. Environ Pollut 44: 61-70.
70.Mehdi Mollapour, Andy Shepherd Peter W. Piper (2008)
Novel stress responses facilitate Saccharomyces
cerevisiae growth in the presence of the monocarboxylate
preservatives. Yeast 25: 169-177.
71.Wilson, Derek, et al. (2008) DBD--taxonomically broad
transcription factor predictions: new content and
functionality. Nucl Acids Res 36: D88-92.
72.Joung, J. K., Ramm, E. I., and Pabo, C. O. (2000) A
bacterial two-hybrid selection system for studying
protein-DNA and protein-protein interactions. Proc Natl
Acad Sci USA 97: 7382-7.
73.M. Shichiri, et al. (2001) A novel acetyltransferase
found in Saccharomyces cerevisiae S1278b that detoxifies
a proline analogue, azetidine-2-carboxylic acid. J Biol
Chem 276: 41998-42002.
74.M. Nomura and Takagi, H. (2004) Role of the yeast
acetyltransferase Mpr1 in oxidative stress: regulation
of oxygen reactive species caused by a toxic proline
catabolism intermediate. Proc Natl Acad Sci USA 101:
12616-12621.
75.Yoko Okushima, et al. (2002) Isolation and
Characterization of a putative transducer of endoplasmic
reticulum stress in Oryza sativa. Plant Cell Physiol
43: 532-539.
76.Kenji Yamada, et al. (2007) Cytosolic HSP90 regulates
the heat shock response that is responsible for heat
acclimation in Arabidopsis thaliana. J Biol Chem 282:
37794-37804.
77.Jane Larkindale, et al. (2005) Heat stress phenotypes
of Arabidopsis mutants implicate multiple signaling
pathways in the acquisition of thermotolerance. Plant
Physiol 138: 882-897.
78.Zhi-shui He, et al. (2008) Maturation of the nodule-
specific transcript MsHSF1c in Medicago sativa may
involve interallelic trans-splicing. Genomics
79.J. Lynn Zimmerman, et al. (1989) Novel regulation of
heat shock genes during carrot somatic embryo
development. Plant Cell 1: 1137-1146.
80.Yunlai Tang, et al. (2007) Heat stress induces an
aggregation of the light-harvesting complex of
photosystem II in spinach plants. Plant Physiol 143:
629-638.
81.Vinocur, B. and Altman, A. (2005) Recent advances in
engineering plant tolerance to abiotic stress:
achievements and limitations. Curr Opinion in Biotech
16: 123-132.
82.Mittler, R. (2006) Abiotic stress, the field
environment and stress combination. TRENDS in Plant Sci
11: 15-19.
83.Kyte, J and Doolittle, R.F. (1982) A simple method for
displaying the dydropathic character of a protein. J Mol
Biol 157: 105-132

指導教授

葉靖輝(Ching-Hui Yeh)

審核日期

2008-7-24

推文