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: 5.18). 11/2010; 1(11):e102. DOI: 10.1038/cddis.2010.80
Source: PubMed

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.

Download full-text


Available from: Peter S Steyger, Aug 10, 2015
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND AND OBJECTIVE: Transtympanic administration of gentamicin is effective for treating patients with intractable vertigo. This study explored the spatial and temporal distribution of gentamicin in vestibular end-organs after transtympanic administration. METHODS: Thirty guinea pigs were transtympanically injected with gentamicin conjugated to Texas Red (GTTR) and their vestibular end-organs examined after various survival periods. Another 9 guinea pigs received GTTR at different doses. Nine animals received Texas Red only and served as controls. We used confocal microscopy to determine the cellular distribution of GTTR in semicircular canal cristae, as well as the utricular and saccular maculae. RESULTS: The most intense GTTR labeling was present in the saccule compared to other vestibular end-organs. GTTR fluorescence was detected predominantly in type I hair cells, type II hair cells and transitional cells after a single transtympanic dose of GTTR (0.1 mg/ml, 0.05 ml), while only weak fluorescence was observed in non-sensory cells such as supporting cells, dark cells and lumenal epithelial cells. Transitional cells displayed intense GTTR fluorescence in the supra-nuclear regions 24 hours after transtympanic injection that was retained for at least 4 weeks. A decreasing spatial gradient of GTTR fluorescence was observed sensory epithelial regions containing central type I to peripheral type I and then type II hair cells in the crista ampullaris, and from striolar to extra-striolar hair cells within the vestibular macula. GTTR fluorescence extended from being restricted to the apical cytoplasm at lower doses to the entire cell body of type I hair cells with increasing dose. GTTR fluorescence reached peak intensities for individual regions of interest within the cristae and maculae between 3 to 7 days after transtympanic injection. CONCLUSION: The saccular uptake of GTTR is greater than other vestibular end-organs after transtympanic injection. In the semi-circular canals.
    Hearing research 02/2013; 298. DOI:10.1016/j.heares.2013.01.010 · 2.85 Impact Factor
  • Source
    • "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. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Aminoglycosides like gentamicin are among the most commonly used antibiotics in clinical practice and are essential for treating life-threatening tuberculosis and Gram-negative bacterial infections. However, aminoglycosides are also nephrotoxic and ototoxic. Although a number of mechanisms have been proposed, it is still unclear how aminoglycosides induce cell death in auditory sensory epithelia and subsequent deafness. Aminoglycosides bind to various intracellular molecules, such as RNA and phosphoinositides. We hypothesized that aminoglycosides, based on their tissue-specific susceptibility, also bind to intracellular proteins that play a role in drug-induced ototoxicity. By conjugating an aminoglycoside, gentamicin, to agarose beads and conducting a gentamicin-agarose pull-down assay, we have isolated gentamicin-binding proteins (GBPs) from immortalized cells of mouse organ of Corti, HEI-OC1. Mass spectrometry identified calreticulin (CRT) as a GBP. Immunofluorescence revealed that CRT expression is concentrated in strial marginal cells and hair cell stereocilia, primary locations of drug uptake and cytotoxicity in the cochlea. In HEI-OC1 cells treated with gentamicin, reduction of CRT expression using small interfering RNA (siRNA) reduced intracellular drug levels. CRT-deficient mouse embryonic fibroblast (MEF) cells as well as CRT siRNA-transfected wild-type MEFs also had reduced cell viability after gentamicin treatment. A pull-down assay using deletion mutants of CRT determined that the carboxyl C-domain of CRT binds to gentamicin. HeLa cells transfected with CRT C-domain deletion mutant construct were more susceptible to gentamicin-induced cytotoxicity compared with cells transfected with full-length CRT or other deletion mutants. Therefore, we conclude that CRT binding to gentamicin is protective against gentamicin-induced cytotoxicity.
    Toxicological Sciences 07/2011; 124(2):378-87. DOI:10.1093/toxsci/kfr196 · 4.48 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Since introduction into clinical practice over 60 years ago, aminoglycoside antibiotics remain important drugs in the treatment of bacterial infections, cystic fibrosis and tuberculosis. However, the ototoxic and nephrotoxic properties of these drugs are still a major clinical problem. Recent advances in molecular biology and biochemistry have begun to uncover the intracellular actions of aminoglycosides that lead to cytotoxicity. In this review, we discuss intracellular binding targets of aminoglycosides, highlighting specific aminoglycoside-binding proteins (HSP73, calreticulin and CLIMP-63) and their potential for triggering caspases and Bcl-2 signalling cascades that are involved in aminoglycoside-induced cytotoxicity. We also discuss potential strategies to reduce aminoglycoside cytotoxicity, which are necessary for greater bactericidal efficacy during aminoglycoside pharmacotherapy.
    Integrative Biology 07/2011; 3(9):879-86. DOI:10.1039/c1ib00034a · 4.00 Impact Factor
Show more