臺灣博碩士論文加值系統

Drosophila melanogaster crammer is a small protein with 79 amino acids. The primary sequence of crammer is similar to that of the propeptide of cathepsin, therefore, crammer has been considered as a novel propeptide-like inhibitor. Recently, crammer has been reported to involve in the formation of long term memory by regulating the activity of cathepsin, but its regulatory mechanism is still unclear. In this study, site directed mutagenesis was applied to explore the hot spot residues of crammer in inhibitory potency against cathepsin. Meanwhile, biochemical and biophysical methods were used to clarify structural and functional relationships. Our inhibitory assay reveals that the conserved aromatic residues with other organisms in crammer play an important role in inhibitory potency. Alanine substitutions at these positions lose more than 20% inhibitory ability. Moreover, most of mutants have no obvious influences in protein structure, except for W9A, Y12A and Y20A. Trp9 in crammer made close contacts with Y12, F16 and Y20 by pi-pi stacking and alanine replacement disrupts this interaction, thus resulting in the structural unfolded. On the other hand, the functional loss of W53 is resulted from disrupting the interaction between cathepin B and crammer. Taken together, this study identified the hot spot residues of crammer in cathepsin inhibition, which will expand the potential of pharmaceutical theapry Alzheimer’s disease.

Abstract 1
中文摘要 2
Abbreviations 3
Chapter 1. Introduction 4
1.1 Cysteine protease inhibitor 4
1.2 Cysteine protease 5
1.3 Learning and memory 6
1.4 The theme of this thesis 7
Chapter 2. Materials and Methods 9
2.1 Construction of recombinant crammer mutant 9
2.2 Protein expression and purification 9
2.3 MALDI-TOF MASS analysis 10
2.4 Tricine SDS PAGE 11
2.5 Quantification of protein concentration 12
2.7 Expression of fly procathepsin B 13
2.8 Isolation and solubilization of procathepsin B inclusion bodies 13
2.9 In vitro folding and autoprocessing of proCTSB 14
2.10 Enzymatic assay of cathepsin B 14
2.11 Quantification of cathepsin B concentration by E-64 15
2.12 Inhibitory assay of cathepsin B or CTSX with mutant proteins 16
2.13 Circular Dichroism Spectroscopy 17
2.14 Fluorescence measurements 18
2.15 NMR spectroscopy 18
2.16 Molecular modeling and docking 19
Chapter 3. Results and Discussion 20
3.1 Determination of structurally and functionally important residues 20
3.2 Protein expression and purification 21
3.3 Inhibitory assay of mutant proteins against cathepsin B from Drosophila melanogaster 22
3.4 Comparison of circular dichroism spectra between wild type and mutant proteins 24
3.5 Intrinsic fluorescence assay 25
3.6 Investigate the protein folding through 1H-15N HSQC spectra 28
Chapter 4. Conclusions 30
Figures 31
Figure 1 Multiple sequence alignments 31
Figure 2 Three dimensional structure of procathepsin L 32
Figure 3 Experimental strategy 33
Figure 4 Construction of the expression plasmid, pAED4 34
Figure 5 Standard curve for quantifying protein concentration 35
36
Figure 6 Possible functional important residues in crammer. 36
Figure 7 Amino acid residues in the crammer protein which may be important for protein structure and function. 37
Figure 8 Expression profiles of mutant proteins. 38
Figure 9 HPLC profile for purification of mutant proteins. 39
Figure 10 Mass spectra for identifying the molecular weight of mutant proteins. 40
Figure 11 The inhibitory activity comparison of mutant proteins analyzed by different strategies. 41
Figure 12 The inhibitory activity of wild type crammer toward cathepsin B. 42
Figure 13 The dose dependent inhibitory assay of wild type crammer and mutant proteins. 43
Figure 14 The comparison of inhibitory potency between single mutants and double mutants. 44
Figure 15 Circular dichroism spectra of C72S and double mutant proteins. 45
Figure 16 Thermostability comparisons upon different pH. 46
Figure 17 Hydrophobic cores in crammer. 47
Figure 18 The aromatic residues in core 1 affect protein packing. 48
Figure 19 The impact on protein packing by aromatic residues in core 2. 49
Figure 20 1H 15N HSQC spectra 50
Figure 21 The interactions between W9 and the surrounding amino acids. 51
Figure 22 Protein-protein docking model between crammer and cathepsin B. 52
Tables 53
Table 1 Sequence of oligonucleotide used for site directed mutagenesis in this study. 53
Table 2 The constructs of mutant proteins in this study. 54
Table 3 The theoretical molecular weight and observed molecular weight of mutant proteins. 55
Appendix 56
Appendix I. Catalytic mechanism of cysteine protease 57
Reference 58

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