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Identification of Monoubiquitinated LDH in Skeletal Muscle Cells Exposed to Oxidative Stress and Spaceflight  K. Hirasaka1, Y. Onishi1, H. Furouchi1, T. Ogawa2, N. Suzue2, K. Kishi1 and T. Nikawa1  1Dept of Nutrition, Univ of Tokushima School of Medicine, Tokushima; 2Dept of Orthopaedics, Univ of Tokushima School of Medicine, Tokushima, JAPAN.

?Background? We previously reported that unloading, such as spaceflight and tail-suspension, induced protein-ubiquitination in skeletal muscle in association with oxidative stress, suggesting that oxidative stress may play an important role in unloading-mediated muscle atrophy.  To elucidate how oxidative stress induces protein-ubiquitination in skeletal muscle cells, we examined the protein-ubiquitination profile in rat myoblastic L6 cells treated with hydrogen peroxide.  ?Results and Discussion? Hydrogen peroxide ubiquitinated many proteins with low molecular masses (less than 60 kDa) as well as high molecular masses (more than 160 kDa).  In these ubiquitinated proteins, a 42-kDa-ubiquitinated protein was abundantly accumulated and immediately disappeared after a hydrogen peroxide treatment.  Microsequencing analysis revealed that the 42-kDa-protein was identical to mono-ubiquitinated form of rat lactate dehydrogenase A (LDH-A).  A proteasome inhibitor, epoxomicin, did not change the amount of mono-ubiquitinated LDH-A and prevent the decline of LDH activity in L6 cells treated with hydrogen peroxide.  In addition, a mono-ubiquitinated LDH-A protein was observed in gastrocnemius muscles of spaceflight or tail-suspended rats.  Since mono-ubiquitination functions as a potent sorting signal to lysosome, hydrogen peroxide and unloading may stimulate LDH degradation in lysosome. ?Conclusion? Oxidative stress preferentially induced mono-ubiquitination of LDH in skeletal muscle cells. 

(Supported by MEXT, Japan)