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

Oregon Hearing Research Center, Oregon Health & Science University, Portland, 97239, USA.
Cell Death & Disease (Impact Factor: 5.01). 11/2010; 1(11):e102. DOI: 10.1038/cddis.2010.80
Source: PubMed


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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Available from: Peter S Steyger
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    • "The mouse organ of Corti HEI-OC1 and stria vascularis SV-k1 cells have been previously described [24], [25] (generous gifts from Dr. Federico Kalinec, House Research Institute), and were maintained at 33°C with 10% CO2 [26]. Mouse kidney proximal tubule KPT2 and KPT11, and distal tubule KDT3 as previously described [26], [27], hepatocyte AML12 (ATCC), embryonic fibroblast K41 [28], and human embryonic kidney 293T cells (ATCC) were maintained at 37°C with 5% CO2. All cells were maintained in growth medium (DMEM with 10% FBS). "
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    • "This differential distribution pattern may be associated to the degree of drug entry into hair cells as described above. At lower doses, GTTR entering hair cells through the stereociliary transduction channels may be sequestered in the apical cytoplasm by gentamicin binding proteins (Karasawa et al., 2010, 2011). With increasing dose, the drug may saturate these binding proteins and is then present diffusely throughout the cytosol. "
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    • "However, our in vitro data in this study suggest that CRT could protect cells from gentamicin-induced cytotoxicity by binding the drug. This would reduce free gentamicin levels available for binding to other molecules, including CLIMP-63, a GBP also localized in the ER that promotes apoptosis upon binding to gentamicin (Karasawa et al., 2010). It is possible that gentamicin binding to CRT could disrupt function of this protein in vivo, which could be a major cause of gentamicin-induced ototoxicity. "
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