Site-Directed Mutagenesis Studies of Crammer for Structure, Stability and Inhibitory Potency

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研究生：	石敏芳 Shih, Min-Fang
論文名稱：	Site-Directed Mutagenesis Studies of Crammer for Structure, Stability and Inhibitory Potency 利用定點突變方法研究Crammer之結構，蛋白質特性及其酵素抑制性
指導教授：	呂平江 Lyu, Ping-Chiang 彭明德 Perng, Ming-Der
口試委員:	陳新
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2011
畢業學年度：	99
語文別：	中文
論文頁數：	62
中文關鍵詞：	crammer 、hydrophobic core
相關次數：	點閱：4 下載：0
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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

                                

1. Fox, T., de Miguel, E., Mort, J.S. & Storer, A.C. Potent slow-binding inhibition of cathepsin B by its propeptide. Biochemistry 31, 12571-6 (1992).
2. Taylor, M.A. et al. Recombinant pro-regions from papain and papaya proteinase IV-are selective high affinity inhibitors of the mature papaya enzymes. Protein Eng 8, 59-62 (1995).
3. Carmona, E. et al. Potency and selectivity of the cathepsin L propeptide as an inhibitor of cysteine proteases. Biochemistry 35, 8149-57 (1996).
4. Maubach, G. et al. The inhibition of cathepsin S by its propeptide--specificity and mechanism of action. Eur J Biochem 250, 745-50 (1997).
5. Lalmanach, G. et al. Inhibition of trypanosomal cysteine proteinases by their propeptides. J Biol Chem 273, 25112-6 (1998).
6. Billington, C.J., Mason, P., Magny, M.C. & Mort, J.S. The slow-binding inhibition of cathepsin K by its propeptide. Biochem Biophys Res Commun 276, 924-9 (2000).
7. Guay, J., Falgueyret, J.P., Ducret, A., Percival, M.D. & Mancini, J.A. Potency and selectivity of inhibition of cathepsin K, L and S by their respective propeptides. Eur J Biochem 267, 6311-8 (2000).
8. Jerala, R., Zerovnik, E., Kidric, J. & Turk, V. pH-induced conformational transitions of the propeptide of human cathepsin L. A role for a molten globule state in zymogen activation. J Biol Chem 273, 11498-504 (1998).
9. Denizot, F. et al. Novel structures CTLA-2 alpha and CTLA-2 beta expressed in mouse activated T cells and mast cells and homologous to cysteine proteinase proregions. Eur J Immunol 19, 631-5 (1989).
10. Delaria, K. et al. Inhibition of cathepsin L-like cysteine proteases by cytotoxic T-lymphocyte antigen-2 beta. J Biol Chem 269, 25172-7 (1994).
11. Kurata, M. et al. Expression, purification, and inhibitory activities of mouse cytotoxic T-lymphocyte antigen-2alpha. Protein Expr Purif 32, 119-25 (2003).
12. Karrer, K.M., Peiffer, S.L. & DiTomas, M.E. Two distinct gene subfamilies within the family of cysteine protease genes. Proc Natl Acad Sci U S A 90, 3063-7 (1993).
13. Barrett, A.J. Classification of peptidases. Methods Enzymol 244, 1-15 (1994).
14. Rawlings, N.D. & Barrett, A.J. Families of cysteine peptidases. Methods Enzymol 244, 461-86 (1994).
15. Rawlings, N.D. & Barrett, A.J. Evolutionary families of peptidases. Biochem J 290 ( Pt 1), 205-18 (1993).
16. Barrett, A.J. & Kirschke, H. Cathepsin B, Cathepsin H, and cathepsin L. Methods Enzymol 80 Pt C, 535-61 (1981).
17. Bond, J.S. & Butler, P.E. Intracellular proteases. Annu Rev Biochem 56, 333-64 (1987).
18. Fan, T.J., Han, L.H., Cong, R.S. & Liang, J. Caspase family proteases and apoptosis. Acta Biochim Biophys Sin (Shanghai) 37, 719-27 (2005).
19. Yamashima, T. Implication of cysteine proteases calpain, cathepsin and caspase in ischemic neuronal death of primates. Prog Neurobiol 62, 273-95 (2000).
20. Berti, P.J. & Storer, A.C. Alignment/phylogeny of the papain superfamily of cysteine proteases. J Mol Biol 246, 273-83 (1995).
21. Yamamoto, Y., Kurata, M., Watabe, S., Murakami, R. & Takahashi, S.Y. Novel cysteine proteinase inhibitors homologous to the proregions of cysteine proteinases. Curr Protein Pept Sci 3, 231-8 (2002).
22. Chapman, H.A., Riese, R.J. & Shi, G.P. Emerging roles for cysteine proteases in human biology. Annu Rev Physiol 59, 63-88 (1997).
23. Grzelakowska-Sztabert, B. [Molecular mechanisms of apoptosis induced by activation of membrane receptors from the TNF-R superfamily]. Postepy Biochem 44, 8-21 (1998).
24. McGrath, M.E. The lysosomal cysteine proteases. Annu Rev Biophys Biomol Struct 28, 181-204 (1999).
25. Turk, B., Turk, D. & Turk, V. Lysosomal cysteine proteases: more than scavengers. Biochim Biophys Acta 1477, 98-111 (2000).
26. Lennon-Dumenil, A.M., Bakker, A.H., Wolf-Bryant, P., Ploegh, H.L. & Lagaudriere-Gesbert, C. A closer look at proteolysis and MHC-class-II-restricted antigen presentation. Curr Opin Immunol 14, 15-21 (2002).
27. Vaes, G., Delaisse, J.M. & Eeckhout, Y. Relative roles of collagenase and lysosomal cysteine-proteinases in bone resorption. Matrix Suppl 1, 383-8 (1992).
28. Rzychon, M., Chmiel, D. & Stec-Niemczyk, J. Modes of inhibition of cysteine proteases. Acta Biochim Pol 51, 861-73 (2004).
29. Tao, K., Stearns, N.A., Dong, J., Wu, Q.L. & Sahagian, G.G. The proregion of cathepsin L is required for proper folding, stability, and ER exit. Arch Biochem Biophys 311, 19-27 (1994).
30. Hanewinkel, H., Glossl, J. & Kresse, H. Biosynthesis of cathepsin B in cultured normal and I-cell fibroblasts. J Biol Chem 262, 12351-5 (1987).
31. Cuozzo, J.W., Tao, K., Wu, Q.L., Young, W. & Sahagian, G.G. Lysine-based structure in the proregion of procathepsin L is the recognition site for mannose phosphorylation. J Biol Chem 270, 15611-9 (1995).
32. Groves, M.R., Coulombe, R., Jenkins, J. & Cygler, M. Structural basis for specificity of papain-like cysteine protease proregions toward their cognate enzymes. Proteins 32, 504-14 (1998).
33. Nishimura, Y., Kawabata, T. & Kato, K. Identification of latent procathepsins B and L in microsomal lumen: characterization of enzymatic activation and proteolytic processing in vitro. Arch Biochem Biophys 261, 64-71 (1988).
34. Rowan, A.D., Mason, P., Mach, L. & Mort, J.S. Rat procathepsin B. Proteolytic processing to the mature form in vitro. J Biol Chem 267, 15993-9 (1992).
35. Khan, A.R. & James, M.N. Molecular mechanisms for the conversion of zymogens to active proteolytic enzymes. Protein Sci 7, 815-36 (1998).
36. Podobnik, M., Kuhelj, R., Turk, V. & Turk, D. Crystal structure of the wild-type human procathepsin B at 2.5 A resolution reveals the native active site of a papain-like cysteine protease zymogen. J Mol Biol 271, 774-88 (1997).
37. Cygler, M. et al. Structure of rat procathepsin B: model for inhibition of cysteine protease activity by the proregion. Structure 4, 405-16 (1996).
38. Turk, D., Podobnik, M., Kuhelj, R., Dolinar, M. & Turk, V. Crystal structures of human procathepsin B at 3.2 and 3.3 Angstroms resolution reveal an interaction motif between a papain-like cysteine protease and its propeptide. FEBS Lett 384, 211-4 (1996).
39. Coulombe, R. et al. Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment. EMBO J 15, 5492-503 (1996).
40. Okano, H., Hirano, T. & Balaban, E. Learning and memory. Proc Natl Acad Sci U S A 97, 12403-4 (2000).
41. Tyler, V.E., Jr. & Malone, M.H. An investigation of the culture, constituents, and physiological activity of Panaeolus campanulatus. J Am Pharm Assoc Am Pharm Assoc 49, 23-7 (1960).
42. Quinn, W.G. & Greenspan, R.J. Learning and courtship in Drosophila: two stories with mutants. Annu Rev Neurosci 7, 67-93 (1984).
43. Davis, R.L. Physiology and biochemistry of Drosophila learning mutants. Physiol Rev 76, 299-317 (1996).
44. Waddell, S. & Quinn, W.G. Flies, genes, and learning. Annu Rev Neurosci 24, 1283-309 (2001).
45. Comas, D., Petit, F. & Preat, T. Drosophila long-term memory formation involves regulation of cathepsin activity. Nature 430, 460-3 (2004).
46. DeZazzo, J. & Tully, T. Dissection of memory formation: from behavioral pharmacology to molecular genetics. Trends Neurosci 18, 212-8 (1995).
47. Dubnau, J. & Tully, T. Gene discovery in Drosophila: new insights for learning and memory. Annu Rev Neurosci 21, 407-44 (1998).
48. Yin, J.C., Del Vecchio, M., Zhou, H. & Tully, T. CREB as a memory modulator: induced expression of a dCREB2 activator isoform enhances long-term memory in Drosophila. Cell 81, 107-15 (1995).
49. Tully, T. Discovery of genes involved with learning and memory: an experimental synthesis of Hirschian and Benzerian perspectives. Proc Natl Acad Sci U S A 93, 13460-7 (1996).
50. Davis, R.L. Olfactory memory formation in Drosophila: from molecular to systems neuroscience. Annu Rev Neurosci 28, 275-302 (2005).
51. Keene, A.C. & Waddell, S. Drosophila olfactory memory: single genes to complex neural circuits. Nat Rev Neurosci 8, 341-54 (2007).
52. Krashes, M.J. & Waddell, S. Rapid consolidation to a radish and protein synthesis-dependent long-term memory after single-session appetitive olfactory conditioning in Drosophila. J Neurosci 28, 3103-13 (2008).
53. Giot, L. et al. A protein interaction map of Drosophila melanogaster. Science 302, 1727-36 (2003).
54. Deshapriya, R.M. et al. Drosophila CTLA-2-like protein (D/CTLA-2) inhibits cysteine proteinase 1 (CP1), a cathepsin L-like enzyme. Zoolog Sci 24, 21-30 (2007).
55. Begg, G.E. & Speicher, D.W. Mass spectrometry detection and reduction of disulfide adducts between reducing agents and recombinant proteins with highly reactive cysteines. J Biomol Tech 10, 17-20 (1999).
56. Schagger, H. Tricine-SDS-PAGE. Nat Protoc 1, 16-22 (2006).
57. Smith, P.K. et al. Measurement of protein using bicinchoninic acid. Anal Biochem 150, 76-85 (1985).
58. D'Alessio K, J., McQueney, M.S., Brun, K.A., Orsini, M.J. & Debouck, C.M. Expression in Escherichia coli, refolding, and purification of human procathepsin K, an osteoclast-specific protease. Protein Expr Purif 15, 213-20 (1999).
59. Tobbell, D.A. et al. Identification of in vitro folding conditions for procathepsin S and cathepsin S using fractional factorial screens. Protein Expr Purif 24, 242-54 (2002).
60. Pauly, T.A. et al. Specificity determinants of human cathepsin s revealed by crystal structures of complexes. Biochemistry 42, 3203-13 (2003).
61. Barrett, A.J. Fluorimetric assays for cathepsin B and cathepsin H with methylcoumarylamide substrates. Biochem J 187, 909-12 (1980).
62. Barrett, A.J. et al. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J 201, 189-98 (1982).
63. Chen, R., Li, L. & Weng, Z. ZDOCK: an initial-stage protein-docking algorithm. Proteins 52, 80-7 (2003).
64. Schneidman-Duhovny, D., Inbar, Y., Nussinov, R. & Wolfson, H.J. PatchDock and SymmDock: servers for rigid and symmetric docking. Nucleic Acids Res 33, W363-7 (2005).
65. Sengupta, A., Mahalakshmi, R., Shamala, N. & Balaram, P. Aromatic interactions in tryptophan-containing peptides: crystal structures of model tryptophan peptides and phenylalanine analogs. J Pept Res 65, 113-29 (2005).
66. Deshapriya, R.M., Yuhashi, S., Usui, M., Kageyama, T. & Yamamoto, Y. Identification of essential residues of CTLA-2alpha for inhibitory potency. J Biochem 147, 393-404 (2010).

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