Identification of mono-ubiquitinated LDH-A in skeletal muscle cells exposed to oxidative stress.
We previously reported that oxidative stress is associated with unloading-mediated ubiquitination of muscle proteins. To further elucidate the involvement of oxidative stress in ubiquitination, we examined the ubiquitination profile in rat myoblastic L6 cells after treatment with hydrogen peroxide. Hydrogen peroxide induced many ubiquitinated proteins with low molecular masses (less than 60 kDa) as well as high molecular masses (more than 160 kDa). Among them, a 42-kDa-ubiquitinated protein was abundantly accumulated and immediately disappeared after the treatment. Microsequencing revealed that the 42-kDa-protein was identical to the mono-ubiquitinated form of rat lactate dehydrogenase A (LDH-A), and we confirmed that hydrogen peroxide induced the mono-ubiquitination of LDH-A in COS7 cells overexpressing LDH-A and ubiquitin. Under unloading conditions, such as tail-suspension and spaceflight, mono-ubiquitinated LDH was accumulated in gastrocnemius muscle. Interestingly, E-64-d plus pepstatin, lysosomal protease inhibitors, further accumulated mono-ubiquitinated LDH-A in the cells after treatment with hydrogen peroxide, while they did not affect the amount of poly-ubiquitinated LDH. In contrast, epoxomicin, a potent proteasome inhibitor, did not change the amount of mono-ubiquitinated LDH-A in L6 cells treated with hydrogen peroxide, although it significantly increased the amount of poly-ubiquitinated LDH. Our results suggest that oxidative stress induces not only poly-ubiquitination but also mono-ubiquitination of LDH-A, which may be involved in its lysosomal degradation during unloading.
Onishi
Yuko
Y
Department of Nutrition, The University of Tokushima School of Medicine, Tokushima 770-8503, Japan.
Hirasaka
Katsuya
K
Ishihara
Ibuki
I
Oarada
Motoko
M
Goto
Jumpei
J
Ogawa
Takayuki
T
Suzue
Naoto
N
Nakano
Shunji
S
Furochi
Harumi
H
Ishidoh
Kazumi
K
Kishi
Kyoichi
K
Nikawa
Takeshi
T
D000818:Animals; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D000080864:Lactate Dehydrogenase 5; D018482:Muscle, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018384:Oxidative Stress; D010736:Phosphoglycerate Mutase / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D014452:Ubiquitins / Q000378:metabolism*; D014893:Weightlessness
D007527:Isoenzymes; D011480:Protease Inhibitors; D014452:Ubiquitins; D006861:Hydrogen Peroxide; D007770:L-Lactate Dehydrogenase; D000080864:Lactate Dehydrogenase 5; D010736:Phosphoglycerate Mutase