Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2008 Activation Profiles and Regulatory Cascades of the Human Kallikrein-Related Peptidases Hyesook Yoon Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected] FLORIDA STATE UNIVERSITY COLLEGE OF ARTS AND SCIENCES ACTIVATION PROFILES AND REGULATORY CASCADES OF THE HUMAN KALLIKREIN-RELATED PEPTIDASES By HYESOOK YOON A Dissertation submitted to the Department of Chemistry and Biochemistry in partial fulfillment of the requirements for the degree of Doctor of Philosophy Degree Awarded: Fall Semester, 2008 The members of the Committee approve the dissertation of Hyesook Yoon defended on July 10th, 2008. ________________________ Michael Blaber Professor Directing Dissertation ________________________ Hengli Tang Outside Committee Member ________________________ Brian Miller Committee Member ________________________ Oliver Steinbock Committee Member Approved: ____________________________________________________________ Joseph B. Schlenoff, Chair, Department of Chemistry and Biochemistry The Office of Graduate Studies has verified and approved the above named committee members. ii ACKNOWLEDGMENTS I would like to dedicate this dissertation to my parents for all your support, and my sister and brother. I would also like to give great thank my advisor, Dr. Blaber for his patience, guidance. Without him, I could never make this achievement. I would like to thank to all the members in Blaber lab. They are just like family to me and I deeply appreciate their kindness, consideration and supports. I specially like to thank to Mrs. Sachiko Blaber for her endless guidance and encouragement. I would like to thank Dr Jihun Lee, Margaret Seavy, Rani and Doris Terry for helpful discussions and supports. Finally I would like to thank and dedicate this work to my friends at Florida State University. This work was supported by grant NIH R15NS057771 and NMSS PP1113 (National Multilple Sclerosis Society). iii TABLE OF CONTENTS List of Tables ............................................................................................................. vi List of Figures ............................................................................................................ viii Abstract ......................................................................................................................xiv 1. INTRODUCTION ................................................................................................. 1 1.1 Serine proteases ....................................................................................... 1 1.2 A History of Human Kallikrein ............................................................... 2 1.3 Function and Association with diseases .................................................. 3 1.4 Known Kallikrein Activation Cascades ................................................... 6 1.5 Main Research Goals ............................................................................... 8 2. ACTIVATION PROFILES AND REGULATORY CASCADES OF THE HUMAN KALLIKREIN RELATED PEPTIDASES 2.1 Introduction ............................................................................................. 9 2.2 Materials and Methods ............................................................................ 13 2.2.1 Cloning of pro-KLK fusion proteins ......................................... 13 2.2.2 Expression of pro-KLK fusion proteins ................................... 15 2.2.3 Production of mature KLK proteins .......................................... 16 2.2.4 Production of pro-KLK proteins ............................................... 16 2.2.5 Pro-KLK fusion protein hydrolysis assay ................................ 17 2.2.6 Auto-activation assay of pro-KLK3, 11, 12 and 15 .................. 17 2.2.7 Activation assay of pro-KLK’s ................................................. 19 2.3 Results ..................................................................................................... 20 2.3.1 Purification of pro-KLK fusion proteins .................................. 20 2.3.2 Proteolysis of pro-KLK fusion protein by mature KLK’s ........ 20 2.3.3 Activation studies of pro-KLK’s ............................................... 75 2.3.4 Auto-activation of pro-KLK’s .................................................. 75 2.3.5 Cross-activation of pro-KLK’s ................................................. 77 2.4 Discussion ................................................................................................ 81 3. ACTIVATION PROFILES OF HUMAN KALLIKREIN-RELATED PEPTIDASES BY THROMBOSTASIS PROTEASES 3.1 Introduction ............................................................................................. 92 3.2 Materials and Methods ............................................................................ 93 3.2.1 Production of pro-KLK fusion proteins .................................... 93 3.2.2 Production of pro-KLK proteins ............................................... 93 3.2.3 Pro-KLK fusion protein hydrolysis assay ................................ 93 iv 3.2.4 Activation assay of pro-KLKs .................................................. 93 3.3 Results ..................................................................................................... 94 3.3.1 Purificaiton of pro-KLK fusion proteins .................................. 94 3.3.2 Proteolysis of pro-KLK fusion proteins by thrombostasis proteases ...................................................... 94 3.3.3 Activation studies of recombinant pro-KLKs ........................... 123 3.4 Discussion ................................................................................................ 123 4. CONCLUSIONS AND FUTURE STUDIES ........................................................ 132 REFERENCES .......................................................................................................... 134 BIOGRAPHICAL SKETCH ..................................................................................... 146 v LIST OF TABLES 2.1 Summary of mass spectrometry and N-terminal sequencing data for the 24hr of KLK11 hydrolysis of the pro-KLK (1-15) fusion protein at pH7.4 ............. 64 2.2 Mass spectrometry of pro-KLK1 fusion protein sample exhibiting proteolysis After incubation with mature KLKs .............................................................. 65 2.3 Mass spectrometry of pro-KLK2 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 65 2.4 Mass spectrometry of pro-KLK3 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 66 2.5 Mass spectrometry of pro-KLK4 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 66 2.6 Mass spectrometry of pro-KLK5 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 67 2.7 Mass spectrometry of pro-KLK6 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 67 2.8 Mass spectrometry of pro-KLK7 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 68 2.9 Mass spectrometry of pro-KLK8 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 68 2.10 Mass spectrometry of pro-KLK9 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 69 2.11 Mass spectrometry of pro-KLK10 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 69 2.12 Mass spectrometry of pro-KLK11 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 70 2.13 Mass spectrometry of pro-KLK12 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 70 vi 2.14 Mass spectrometry of pro-KLK13 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 71 2.15 Mass spectrometry of pro-KLK15 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 71 2.16 Mass spectrometry of pro-KLK16 fusion protein sample exhibiting proteolysis after incubation with mature KLKs ............................................................... 72 2.17 Mass spectrometry and N-terminal sequence analyses of pro-KLK fusion protein samples exhibiting additional or unique proteolyses after incubation with mature KLKs at pH 6.0 .............................................................................................. 72 2.18 Percent hydrolysis of pro-KLK (1-15) fusion proteins for 100:1 molar ratio incubation with mature KLKs at pH 6.0/7.4 for 1h at 37 °C .......................... 73 2.19 Percent hydrolysis of pro-KLK (1-15) fusion proteins for 100:1 molar ratio incubation with