아주대학교

목차

General Introduction 1
Neurodegenerative disease 1
Huntington's disease 2
Alzheimer's disease 7
Chapter 1 (Huntington's disease) 13
1. Introduction 13
2. Experimental Procedures 15
2.1. Construct design, protein expression and purification 15
2.1.1. Cloning and protein expression 15
2.1.2 Purification of HttEx1-N17 and17Q 15
2.1.3 Purification of HttEx1-46Q 16
2.2. ThT fluorescence assay 16
2.3. Cryo-EM 17
2.4. Transmission electron microscopy (TEM) 17
2.5. CD spectroscopy 18
2.6. NMR spectroscopy 18
2.7. Atomic force microscopy (AFM) 19
3. Results 21
3.1. Expression and purification of HttEx1-N17,17Q and46Q 21
3.2. Concentration-dependent structural transitions and fibrillation of HttEx1-17Q 23
3.2.1. ThT fluorescence assay 23
3.2.2. Cryo-EM imaging 27
3.2.3. CD spectroscopy and secondary structure prediction 29
3.2.4. Monomer concentration-dependent NMR analysis 32
3.2.5. Time-course NMR analysis 40
3.2.6. Proposed model 45
3.3. Effect of salt concentration on fibrillation of HttEx1-17Q 47
3.3.1. ThT fluorescence assay 47
3.3.2. Negative staining (NS)-TEM imaging 51
3.3.3. CD spectroscopy and secondary structure prediction 53
3.4. Effect of other factors on fibrillation of HttEx1-17Q 55
3.4.1. Alcohol effect 55
3.4.2. pH effect 60
3.4.3. Cross-seeding 62
3.5. Comparison of HttEx1-17Q and46Q fibrils 64
4. Discussion 66
Chapter 2 (Alzheimer's disease) 68
1. Introduction 68
2. Experimental Procedures 69
2.1. Peptide synthesis 69
2.1.1. Synthesis of N(a)-[(9H-fluoren-9-ylmethoxy)carbonyl]-N(s)- (triphenylmethyl)-L-histidine methyl ester 69
2.1.2. General procedure I for alkylation of histidine methyl ester N(a)-[(9H-Fluoren-9-ylmethoxy)carbonyl]-N(p), N(s)- bis(cyclohexyl)- L-histidine methyl ester 69
2.1.3. General procedure II for the hydrolysis of esters N(a)-[(9H- Fluoren-9-ylmethoxy)carbonyl]-N(p), N(s)-bis(cyclohexyl)-L-histidine 70
2.1.4. General procedures for peptide synthesis 70
2.2. Protein preparation 73
2.2.1. Tau purification 73
2.2.2. Aβ42 oligomer preparation 73
2.3. ThT fluorescence assay 74
2.4. TEM 75
2.5. NMR spectroscopy 75
2.6. Cytotoxicity assay 76
2.7. Seeding assay 76
3. Results 78
3.1. Inhibitory effect of novel peptides on aggregation of Aβ42 78
3.1.1. Preparation of Aβ42 78
3.1.2. ThT fluorescence assay 79
3.1.3. NS-TEM imaging 84
3.1.4. HSQC titration analysis 86
3.1.5. NMR analysis after induced fibrillation 88
3.1.6. 1D NMR spectroscopy 90
3.1.7. Cytotoxicity assay 92
3.2. Inhibitory effect of novel peptides on aggregation of Tau 95
3.2.1. Expression and purification of Tau 95
3.2.2. ThT fluorescence assay 97
3.2.3. NS-TEM imaging 100
3.2.4. HSQC titration analysis 102
3.2.5. NMR analysis after induced fibrillation 104
3.2.6. Residue-specific analysis of Tau 107
3.2.7. Seeding assay 112
4. Discussion 114
Conclusion 115
References 116
국문 초록 129