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Structural and biophysical study on the interaction of IP3 receptor with huntingtin and its effect on the huntingtin fibrillation

초록/요약

Huntington’s disease (HD) is a neurodegenerative disorder caused by expanding CAG repeats (i.e., coding polyglutamine) in the first exon (Exon1) of huntingtin protein (Htt). Both normal (Htt) and expanded huntingtin (Httexp) proteins are known to bind to the C-terminal region of inositol 1,4,5 trisphosphate receptor (IP3R), which is involved in the pathogenesis of HD. IP3R is an intracellular calcium (Ca2+) release channel that plays an important role in maintaining Ca2+ homeostasis. To understand the binding mechanism between huntingtin protein and IP3R, we prepared the normal and expanded Htt proteins (Htt17Q and Htt46Q, respectively) and the C-terminal region of IP3R (IP3R-CTD). For binding assay, we have employed surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR results revealed that the expanded Htt (Htt46Q) binds to IP3R-CTD with higher binding affinity compared to the normal Htt (Htt17Q). Also, we designed the peptide derived from IP3R (hereinafter referred to as IP3R-CTDLH) with a much narrower range of peptides than the known IC10 peptides that bind to huntingtin proteins, and the binding between IP3R-CTDLH and huntingtin proteins was confirmed by ITC. To understand the role of coiled-coil conformation for binding, we prepared coil-breaking variants of IP3R-CTD by disrupting the charged interaction between lysine and glutamate (i.e., KK and EE mutants). In order to investigate whether coiled-coil maintenance affects the interaction between Htt and IP3R, NMR backbone assignment of Htt17Q protein was performed. Then, IP3R-CTDLH variants (WT, KK and EE) were titrated onto 15N-labeled Htt17Q. The spectra showed that IP3R-CTDLH-WT binding caused the severe line broadening of most residues of Htt17Q. However, there were little spectral changes of Htt17Q in the presence of high concentrations IP3R-CTDLH-KK. Interestingly, the addition of IP3R-CTDLH-EE caused the chemical shift changes as well as line broadening of Htt17Q. Taken together, the maintenance of the coiled-coil structure of CTDLH plays an important role in the binding of huntingtin. Additionally, we found that NMR spectra of Htt17Q were gradually changed in a concentration dependent manner, and two major conformations were observed, one at high concentration (~100 μM) and the other at low concentration (~20 μM). Interestingly, fibrillation of Htt17Q occurred rapidly above 100 μM, and the spectrum of Htt17Q after fibrillation was completely identical to that of low-concentration Htt17Q. To monitor the fibrillation of pathogenic Htt and the effects of IP3R-CTDLH on the fibrillation, we prepared MBP-fused Htt17Q and 46Q. Unlike Htt17Q, Htt46Q was found to produce fibril immediately after MBP cleavage, and the formation of fibril was demonstrated by thioflavin T assay. IP3R-CTDLH peptides, including coil-breaking variants, affect the fibrillation of Htt46Q in a concentration dependent manner. In addition, Htt46Q fibril morphology was significantly altered in the presence of the IP3R-CTDLH peptides. In this study, we demonstrated that IP3R-CTDLH peptides, much smaller than the known IC10, can bind directly to huntingtin proteins via coiled-coil interaction and these interactions affect amyloidogenesis of pathogenic huntingtin.

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목차

General Introduction 1
Chapter 1 Purification and structural characterization of the exon1 region of huntingtin and the C-terminal domain of IP3R 5
1. Introduction 5
2. Experimental Procedures 8
2.1 Materials 8
2.2 Cloning, overexpression, and purification of huntingtin proteins 8
2.3 Cloning, overexpression, and purification of IP3R-CTDs 10
2.4 Surface plasmon resonance (SPR) 15
2.5 Multi Angle Light Scattering (MALS) 15
2.6 Far-UV Circular Dichroism (CD) spectroscopy 16
2.7 Isothermal titration calorimetry (ITC) 16
2.8 Co-crystallization 17
2.9 X-ray diffraction 17
3. Results and discussion 18
3.1 Expression and purification of huntingtin proteins 18
3.2 Expression and purification of the IP3R-CTD variants 25
3.3 Oligomeric states of IP3R-CTD and its variants 31
3.4 Secondary structure and thermal stability of IP3R-CTD variants using Circular Dichroism (CD) 34
3.5 Binding differences between normal and abnormal huntingtins with IP3R-CTD using SPR analysis 37
3.6 Isothermal Titration Calorimetry (ITC) 39
3.7 Co-crystallization of MBP-Htt and IP3R-CTD complex 43
4. Conclusion 45
Chapter 2 NMR study of the interaction between Huntingtin and IP3R-CTD variants and its effects on the huntingtin fibrillation 46
1. Introduction 46
2. Experiment procedures 48
2.1 Materials 48
2.2 Preparation of [13C and 15N]-labeled Htt17Q 48
2.3 Backbone assignment of Htt17Q 48
2.4 NMR titration 49
2.5 Thioflavin T fluorescence assay 49
3. Results and discussion 50
3.1 Conformation change of Htt17Q depending on concentration and temperature 50
3.2 NMR backbone assignments of Htt17Q 54
3.3 NMR titration unlabled-IP3R-CTDLH to Htt17Q 56
3.4 NMR titration of huntingtin to IP3R-CTD variants 64
3.5 Fibrillation of normal and expanded huntingtin Exon1 using Thioflavin T analysis 67
3.6 TEM images of normal and expanded huntingtin proteins after fibrillization 68
3.7 Effect on different fibril morphology of Htt46Q by IP3R-CTDLH variants 70
4. Conclusion 74
References 77
국문 초록 83

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