臺灣博碩士論文加值系統

在陰道滴蟲中，親環蛋白質對移除細胞膜上的Myb蛋白質並協助其進入細胞核起著至關重要的作用，之前的研究顯示，TvCyP1和TvCyP2可與Myb1和Myb3相互作用，並調節Myb1和Myb3蛋白質進入細胞核；與TvCyP1的二聚體結構相比，TvCyP2為單體結構並在N端多了一段延伸的片段，而且兩個蛋白質在調節Myb蛋白質運輸中發揮了不同的作用。我們解析了TvCyP2在不同條件下的四個X-ray晶體結構，所有結構均顯示N端延伸片段與相鄰TvCyP2的活性位點相互作用，這是一種不尋常的相互作用，所以也利用NMR技術來研究，結果顯示水溶液狀態下這種特殊的相互作用仍然存在，並且N端延伸片段似乎也可以和細胞膜發生作用，TvCyP2和TvCyP2-ΔN（不含N端延伸片段的TvCyP2）的細胞體內研究表明，兩種蛋白質確實有不同的亞細胞定位。為了深入了解陰道滴蟲中的蛋白質運輸，在這篇研究論文中，我們闡明了TvCyP1和TvCyP2與Myb3相互作用的分子基礎，我們解析出了TvCyP1/Myb3胜肽的複合物X-ray晶體結構，借助NMR和等溫滴定量熱法（ITC）研究，我們描繪出了Myb3的TvCyP2結合序列（Myb3 50-87），也了解Myb3與TvCyP1的結合比起與TvCyP2更具物理性；總之，這個研究提供了TvCyP1-Myb3相互作用的詳細結構分析以及TvCyP1和TvCyP2之間的差異研究，可以為設計藥物以治療抗藥菌株提供良好的基礎。

In Trichomonas vaginalis (T. vaginalis), cyclophilins play a vital role in dislodging Myb proteins from the membrane compartment and leading them to nuclear translocation. It is shown that in T. vaginalis, (TvCyp1) and (TvCyP2) interacts with Myb1 and Myb3 to regulate the nuclear translocation of Myb1 and Myb3 proteins. In comparison to TvCyp1, TvCyP2 containing an extended segment at the N-terminus (N-terminal segment) formed a monomer and showed a different role in regulating protein trafficking. Four X-ray structures of TvCyP2 were determined under various conditions, all showing the N-terminal segment interacting with the active site of a neighboring TvCyP2, an unusual interaction. NMR study revealed that this particular interaction exists in solution as well and also, the N-terminal segment seems to interact with the membrane. In vivo studies of TvCyP2 and TvCyP2-∆N (TvCyP2 without the N-terminal segment) indicated that both proteins have different subcellular localization.
To gain further insight into protein trafficking in the parasite, we also unraveled the molecular basis of the interaction of TvCyP1 and TvCyP2 with Myb3. We determined the X-ray structure of TvCyP1 in complexed with Myb3 peptide. With the aid of NMR and isothermal titration calorimetry (ITC) studies, we also mapped the TvCyP2-binding sequence of Myb3 (Myb350–87) as the binding motif. The study suggested that the binding of Myb3 with TvCyP1 is stronger than binding with TvCyP2. Together, the detailed structural insights on TvCyP1-Myb3 interaction and comparative study between TvCyP1 and TvCyP2 provided here could pave the way for newer drugs to treat drug-resistant strains.

Acknowledgment----------------------------------------------------------------------------------i
Abstract (English)------------------------------------------------------------------------------iv
Abstract (Chinese)------------------------------------------------------------------------------v
List of Contents--------------------------------------------------------------------------------vi
List of Abbreviation----------------------------------------------------------------------------xi
Chapter 1 Introduction--------------------------------------------------------------------------1
1.1 Cyclophilins--------------------------------------------------------------------------------1
1.1.1 Peptidyl Prolyl Isomerases--------------------------------------------------------------1
1.1.2 Human cyclophilins------------------------------------------------------------------------4
1.1.3 Cyclophilins and diseases-----------------------------------------------------------------6
1.2 Trichomonas vaginali------------------------------------------------------------------------8
1.2.1 The life cycle of T. vaginalis----------------------------------------------------------8
1.2.2 Clinical relevance of T. vaginalis------------------------------------------------------9
1.2.3 Cyclophilins in T. vaginalis------------------------------------------------------------10
1.2.4 Myb transcription factors in T. vaginalis-----------------------------------------------11
1.3 Purpose and outline of this study-----------------------------------------------------------12
Chapter 2 Materials and Methods-----------------------------------------------------------------14
2.1 Preparation of recombinants TvCyP1, TvCyP2, TvCyP2-∆N and Myb3 proteins---------------------14
2.2 Preparation of Myb3 and TvCyP2 peptides-----------------------------------------------------16
2.3 Analytical ultracentrifugation--------------------------------------------------------------16
2.4 Size-exclusion chromatography coupled to multi-angle static light scattering (SEC-MALS)-----17
2.5 Crystallization and data collection---------------------------------------------------------17
2.6 Refinement and structure determination------------------------------------------------------19
2.7 NMR Spectroscopy----------------------------------------------------------------------------20
2.8 Circular dichroism (CD) spectroscopy--------------------------------------------------------22
2.9 Immunofluorescence assay (IFA)--------------------------------------------------------------22
2.10 Isothermal Titration Calorimetry (ITC)-----------------------------------------------------23
Chapter 3 Results-------------------------------------------------------------------------------24
3.1 Structure and solution behavior TvCyP2----------------------------------------------------24
3.1.1 Biophysical behavior of TvCyP2 in solution------------------------------------------------24
3.1.2 PPIase activity of TvCyP2-----------------------------------------------------------------25
3.1.3 X-ray Crystal structure of TvCyP2---------------------------------------------------------26
3.1.4 Interaction of the N-terminal segment with the active site of TvCyP2 in X-ray structures--27
3.1.5 Interaction of the N-terminal segment with the active site of TvCyP2 in solution----------29
3.1.6 Effect of deletion of N-terminal segment residues-----------------------------------------30
3.1.7 Comparison of the thermostability of TvCyP2 with TvCyP1 and TvCyP2-∆N---------------------31
3.1.8 N-terminal segment function in protein Localization---------------------------------------31
3.1.9 Membrane interaction of the N-terminal segment--------------------------------------------32
3.1.10 Resonance assignments and secondary structure prediction of N-terminal segment-----------33
3.1.11 Comparison of the structures of TvCyP2 with other cyclophilins---------------------------34
3.2 Interaction of TvCyP1 with Myb3 peptides----------------------------------------------------35
3.2.1 Binding sequence in Myb3------------------------------------------------------------------35
3.2.2 Mapping the Myb3 peptide binding region in TvCyP1 peptide by NMR--------------------------36
3.2.3 X-ray crystal structure of TvCyP1 in complex with Myb3 peptide----------------------------37
3.2.4 Measuring the KD of interaction by ITC----------------------------------------------------39
3.3 Interaction of TvCyP2 with Myb3 peptides----------------------------------------------------40
3.3.1 Identification of Myb3 peptide interaction region in TvCyP2 by NMR------------------------40
3.3.2 Measuring the KD of interaction by ITC----------------------------------------------------41
3.4 Interaction of TvCyP1 and TvCyP2 with Myb3 protein------------------------------------------42
3.4.1 Identification of Myb3 protein interaction region in TvCyP1 and TvCyP2 by NMR-------------42
3.4.2 Measuring the KD of interaction by ITC----------------------------------------------------43
Chapter 4 Discussion----------------------------------------------------------------------------113
4.1 Structural and biophysical studies on TvCyP2------------------------------------------------113
4.2 Structural basis of interaction of TvCyP1 and TvCyP2 with Myb3------------------------------119
Chapter 5 Conclusion----------------------------------------------------------------------------123
References--------------------------------------------------------------------------------------125
Appendix----------------------------------------------------------------------------------------137
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