臺灣博碩士論文加值系統

寡聚體型轉錄因子的活性，能夠被一類具單一功能區塊的小型蛋白質，藉由干擾
轉錄因子的聚合進行調控。熱休克轉錄因子結合蛋白(HSBP)是一種分子量約10
kDa的小分子蛋白質，其本身與其目標蛋白─熱休克轉錄因子(HSF)的寡聚合區域
皆具有捲曲螺旋結構區塊；二者藉由此區塊的交互作用，HSBP在熱休克反應過
程中扮演負向調控者的角色。阿拉伯芥的HSBP是一種細胞質蛋白，然而在熱逆
境過後出現在細胞核中並和HSF進行交互作用，終止HSF的功能。為了揭露阿
拉伯芥HSBP在細胞質中交互作用的夥伴、以及與HSF的家族成員間交互作用概
況，我們分別分析HSBP免疫共沉澱樣本的質譜，以及進行HSBP與HSF的雙分
子螢光互補試驗。免疫共沉澱之質譜分析結果，呈現14個受熱處理後會和HSBP
具明顯交互作用的細胞質蛋白質。這份清單包含了細胞質型HSP70和整個MAG2
蛋白質複合體的蛋白質單元，而CIP1和KAC1是本清單中具最強證據力所支持
的兩個候選蛋白質；上述蛋白質皆被預測帶有一個以上的捲曲螺旋結構區塊。此
外、我們觀察到HSBP可與16個HSF進行交互作用，其大多數位於細胞核中。
這些HSFs被報導參與在包含了熱逆境、氧化逆境、以及種子成熟期的生理反應
中。我們推測做為一個小型的調控蛋白質，HSBP可透過與不同HSF、及含有捲
曲螺旋結構區的細胞質蛋白質發生交互作用，以因應眾多的逆境和生長發育上的
需求。

Transcription factors could be regulated by small single-domain proteins that disturb the formation of active oligomers. Heat shock factor (HSF) binding protein (HSBP) is a 10-kDa protein with a coiled-coil motif that interacts with the oligomerization domain of HSFs and is a negative regulatorof the heat stress response (HSR). In Arabidopsis (Arabidopsis thaliana), HSBP is a cytoplasmic protein that translocates to the nucleus after HS for HSF interaction. To uncover the cytoplasmic interacting proteins of HSBP and characterize the interaction with HSF members, we used mass spectrometry after HSBP co-immunoprecipitation (co-IP) and HSBP–HSF bimolecular fluorescence complementation assays. Mass spectrometry after co-IP revealed 14 cytoplasmic HSBP-interacting candidates responding to HS in Arabidopsis seedlings. The candidates were cytosolic HSP70s and all components of the MAIGO 2 complex; the most robust candidates were COP1-interactive protein 1 and kinesin-like protein for actin-based chloroplast movement 1, predicted to contain at least one coiled-coil domain. As well, 16 HSFs interacted with HSBP, predominantly in the nucleus, and included factors reported to be involved in the HS signaling cascade, antioxidant mechanisms, and seed maturation. As a small regulatory protein, HSBP interacts with HSFs and cytoplasmic coiled-coil–containing partners in response to numerous stress and developmental conditions.

Table of Contents

摘要 I
Abstract II
Abbreviations III

Introduction 1
Heat stress and heat shock response 1
Arabidopsis heat shock factors 2
microProteins 3
Heat shock factor binding proteins 3

Materials and Methods 6
Plants materials and growth condition 6
RNA extraction and RT-PCR 6
Western blot and immunoprecipitation (IP) analysis 7
Protein identification and database search 7
Bimolecular fluorescence complementation (BiFC) assays 8
Primers and sequence data 9

Results 10
Preparation of HSBP-3xFLAG and -GFP expression plants 10
Identi&;#64257;cation of candidate HSBP-interacting proteins 10
Confirmation of HSBP-interacting proteins 13
Interaction of HSBP with HSFs 13

Discussion 15
The action of HSBP during heat shock response involves cytoskeleton-associating proteins and endomembrane-localized proteins 15
HSBP interacts with HSP70 and HSF in the distinct cellular compartments 17
HSBP may regulate HSF function in antioxidant mechanisms and seed maturation, in addition to heat shock response 17
HSBP would have additional functions in the cytoplasm 19

Tables 20
Figures 22
References 27
Supplementary Data 35
Appendix 55

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