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Effects of various protease inhibitors on protein degradation of

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AN ABSTRACT OF THE THESIS OF
Paiyen Wu for the degree of Master of Science in Animal Science presented on March 18, 1996. Title: Effects of Various Protease Inhibitors on Protein Degradation of Cultured Myotubes

Abstract approved:

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Neil E\forsberg

Myofibrillar proteins constitute about 50-55% of total protein in muscle cells. Studies of the mechanism of turnover of these proteins under normal physiological conditions can be applied to 3 areas: meat production (muscle cell growth), meat industry (tenderization), and therapy of muscle diseases. Three intracellular proteolytic systems may be involved in myofibrillar protein turnover, including lysosomal cathepsins, calpains, and the multicatalytic protease complex (proteasome). Previous studies have indicated that lysosomal system is not important in myofibrillar protein turnover. In contrast, the calpain system appears to play an important role for intracellular myofibrillar protein turnover. However, the effect of the proteasome on turnover of this pool of protein remains uncertain. The goal of this study was to use protease inhibitors to distinguish the function of calpains and the proteasome muscle protein degradation.
In this study five different protease inhibitors were used in cultured rat muscle cells (L8 cell line), to assess the function of individual proteolytic species in myofibrillar protein degradation. All inhibitors, with the exception of PD150606, reduced total protein degradation (TPD) in cultured myotubes. At inhibitors' maximal effects, CI-II (75 [tM)

reduced TPD by 52%, CA reduced TPD by 44%, LACT reduced TPD by 26%, and ZIE reduced TPD by 53%. Although each of these inhibitors strongly reduced TPD, their abilities to reduce myofibrillar protein degradation were variable. At their maximal effects, CI-II reduced myofibrillar proteins degradation by 32%, ZIE decreased degradation of the same pool by 27%, and CA decreased degradation of this pool by 17%. LACT, a potent and specific proteasome inhibitor had no effect on myofibrillar protein degradation.
We found that inhibitors which inhibited calpains stabilized the myofibrillar pool yet proteasome specific inhibitor (lactacystin) had no effect on this pool, however lactacystin caused accumulations of ubiquitinated protein. CI-II stabilized the myofibrillar pool but did not caused Ub-protein complex accumulation. From this, it seems clear that calpain play key roles in coordination of myofibrillar protein degradation.
These data indicate that control of calpain activities in vivo may be useful in treating muscle diseases and in enhancing muscle growth of domestic animals.

Effects of Various Protease Inhibitors on Protein Degradation of Cultured Myotubes
by Paiyen Wu
A THESIS submitted to Oregon State University
in partial fulfillment of the requirements for the
degree of Master of Science
Completed March 18, 1996 Commencement June 1996

Master of Science thesis of Paiyen Wu presented on March 18, 1996. APPROVED:
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Major Professor, representing AnMal Science
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Dean of Graduate hool
I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of thesis to any reader upon request.
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Paiyen Wu, Author

Acknowledgements
I would like to express my sincere appreciation to my major professor, Dr. Neil E. Forsberg , for his encouragement, advice, and understanding during the course of my graduate study and the preparation of this thesis. I know very clearly that Dr. Forsberg is behind me all the time whenever or wherever I have problems.
I would also like to thank the members of my committee, Dr. C. Y. Hu and Dr. Phil McFadden for their advice and suggestions while servng on my graduate committee.
My thanks are extended to my colleagues, Mr. Bor-roung Ou, for his valuable suggestions and patient teaching, Mr. Jien-yia Huan and Mr. Roustem Nabioulem, for their sceintific discusstions and Ching-sun Chang, Jing Huang, Mei-chuan Wang, and Yoji Ueda for their encouragement and friendship, which are always important to me.
Special thanks to Oregon State University and the Department of Animal Sciences, for providing me an excellent learning and working environment during my graduate studies.

Dedication
This thesis is dedicated to my family: To my dearest husband, Shiun-long, for his endless love. To my parents, for their considerate care. To my parents in-laws, for their consistent patient. Which gave me the inspiration to continue.

TABLE OF CONTENTS

Page

INTRODUCTION

1

LITERATURE REVIEW

5

MATERIALS AND METHODS

26

RESULTS

33

DISCUSSION

44

CONCLUSION

52

BIBLIOGRAPHY

53

LIST OF FIGURES Figures 1. Effects of various inhibitors at different doses on total protein degradation

Page
34

2. Inhibitors' maximum effects on total protein degradation

36

3. Effects of various inhibitors on myofibrillar protein stability

38

4. Inhibitor's maximum effect on stabilization of myofibrillar proteins

38

5. Effects of various inhibitors on non-myofibrillar protein stability

39

6. Index of cytotoxicity

41

7. Ubiquitin Western blot

42

8. Autoradiography

43

LIST OF TABLES

Tables

Page

1. Effects of inhibitors on total protein degradation (TPD)

35

2. Effects of inhibitors on myofibrillar protein degradation

37

3. Specificities and actions of inhibitors

51

EFFECTS OF VARIOUS PROTEASE INHIBITORS ON PROTEIN DEGRADATION OF CULTURED MYOTUBES
INTRODUCTION
Myofibrillar proteins constitute about 50-55% of total protein in muscle cells (Goll et al., 1989). Studying the degradation of this pool is important to three different fields: meat production (muscle cell growth), the meat industry and therapeutic clinical usage. The basic knowledge of the involvement and regulation of three intracellular proteinase systems, including lysosomal cathepsins, Ca2+-dependent neutral protease (calpains), and multicatalytic protease complex (proteasome) are therefore of research and clinical interest.
Net growth of muscle tissue depends on both muscle protein synthesis and muscle protein degradation. In growing animals, synthesis and degradation tend to move in parallel, with synthesis rate exceeding degradation rate. It is estimated that to maintain muscle mass, the muscle would have to synthesize an amount of protein equivalent to 5 to 10 percent of its protein content on a daily basis (Allen, 1988). Hence inhibition of muscle protein degradation could cause a dramatic change in rate of muscle growth. Although cathepsins are found in muscle and have activities on myofibrillar protein substrates (Goll et al., 1983; Matsuishi et al., 1992), little evidence indicates correlation between lysosomal enzyme activities and myofibrillar protein degradation. Non-lysosomal proteases are therefore more likely to be responsible for myofibrillar protein degradation.
Summarizing from more than 30 various experiments, Goll et al (1989) have proposed a possible mechanism which indicates that calpain is responsible for at least the initial step or the rate-regulating step of myofibrillar protein degradation. The calpains can
InhibitorsMyofibrillar Protein DegradationTpdEffectsProtein Degradation