[HTML][HTML] CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

T Karasawa, Q Wang, LL David, PS Steyger - Cell death & disease, 2010 - nature.com
T Karasawa, Q Wang, LL David, PS Steyger
Cell death & disease, 2010nature.com
Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To
understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney
proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from
mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass
spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells,
cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized …
Abstract
Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63.
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