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.
Highlights
IntroductionAminoglycosides enter eukaryotic cells by endocytosis and are trafficked to lysosomes, the Golgi complex and endoplasmic reticulum (ER) before being released into the cytosol.[7,8,9] Aminoglycosides permeate non-selective cation channel directly into the cytoplasm.[10,11,12] In the cell, aminoglycosides can induce cytotoxicity that results in both apoptotic and non-apoptotic cell death by activating caspase-3, caspase-9, c-jun N-terminal kinases, calpains and cathespin D, or generating toxic levels of reactive oxygen species.[13,14,15,16,17]
We show that gentamicin binds to the lumenal domain and induces dimerization of CLIMP-63, that is dependent on palmitoylation
Aminoglycosides binding to ribosomal RNA could induce toxicity, but why do the kidney and cochlea have greater susceptibility to the drug? We hypothesized that differences in protein expression may be responsible for the susceptibility of particular cell types in the kidney and cochlea, and that aminoglycosides must interact with intracellular proteins to trigger cell death mechanisms, apoptosis
Summary
Aminoglycosides enter eukaryotic cells by endocytosis and are trafficked to lysosomes, the Golgi complex and endoplasmic reticulum (ER) before being released into the cytosol.[7,8,9] Aminoglycosides permeate non-selective cation channel directly into the cytoplasm.[10,11,12] In the cell, aminoglycosides can induce cytotoxicity that results in both apoptotic and non-apoptotic cell death by activating caspase-3, caspase-9, c-jun N-terminal kinases, calpains and cathespin D, or generating toxic levels of reactive oxygen species.[13,14,15,16,17]. We identified gentamicin-binding proteins (GBPs) from proximal tubule cells that may contribute to drug susceptibility in these cells. Of these proteins, only the cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) is localized in the ER membrane, and colocalized with gentamicin-Texas Red (GTTR). The physiological functions of CLIMP-63 have not been well documented, but it is thought to be an anchoring protein connecting the ER to the cytoskeleton.[18] The cytosolic domain of CLIMP-63 anchors the ER network to the microtubular cytoskeleton, and the lumenal domain of CLIMP-63 is required for its oligomerization and immobilization in the ER membrane.[18,19] We show that gentamicin binds to the lumenal domain and induces dimerization of CLIMP-63, that is dependent on palmitoylation. We identified 14-3-3 proteins as CLIMP-63-binding proteins, and that gentamicin enhances 14-3-3b binding to CLIMP-63 through 14-3-3y, having a major role in gentamicin-induced cytotoxicity
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