73-kDa molecular chaperone HSP73 is a direct target of antibiotic gentamicin

J Biol Chem. 2004 Apr 23;279(17):17295-300. doi: 10.1074/jbc.M312217200. Epub 2004 Feb 13.

Abstract

Although gentamicin (GM) has been used widely as an antibiotic, the specific binding protein of the drug has not yet been understood sufficiently. Here we show that GM specifically associates with the 73-kDa molecular chaperone HSP73 and reduces its chaperone activity in vitro. In the present study, we investigated GM-specific binding proteins using a GM-affinity column and porcine kidney cytosol. After washing the column, only the 73-kDa protein was eluted from the column by the addition of 10 mm GM. None of the other proteins were found in the eluant. Upon immunoblotting, the protein was identical to HSP73. Upon CD spectrum analysis, the binding of GM to HSP73 resulted in a conformational change in the protein. Although HSP73 prevents aggregation of unfolded rhodanese in vitro, the chaperone activity of HSP73 was suppressed in the presence of GM. Using limited proteolysis of HSP73 by TPCK-trypsin, the GM binding site is a COOH-terminal for one third of the protein known to be a peptide-binding domain. During immunohistochemistry, HSP73 and GM were co-localized in enlarged lysosomes of rat kidneys with GM-induced acute tubular injury in vivo. Our results suggest that the specific association between HSP73 and GM may reduce the chaperone activity of HSP73 in vitro and/or in vivo, and this may have an interaction with GM toxicity in kidneys with GM-induced acute tubular injury.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylglucosaminidase / urine
  • Amino Acid Sequence
  • Animals
  • Anti-Bacterial Agents / pharmacology*
  • Binding Sites
  • Brain / metabolism
  • Carrier Proteins / metabolism*
  • Carrier Proteins / physiology*
  • Cattle
  • Chromatography
  • Circular Dichroism
  • Creatinine / blood
  • Cytosol / metabolism
  • Disease Models, Animal
  • Gentamicins / pharmacology*
  • HSC70 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins*
  • Immunoblotting
  • Immunohistochemistry
  • Kidney / metabolism
  • Kidney Tubules / metabolism
  • Lysosomes / metabolism
  • Male
  • Microscopy, Electron
  • Molecular Chaperones / pharmacology
  • Molecular Sequence Data
  • Nitrogen / blood
  • Peptides / chemistry
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Rats
  • Rats, Sprague-Dawley
  • Swine
  • Thiosulfate Sulfurtransferase / chemistry
  • Time Factors
  • Ultraviolet Rays

Substances

  • Anti-Bacterial Agents
  • Carrier Proteins
  • Gentamicins
  • HSC70 Heat-Shock Proteins
  • HSP70 Heat-Shock Proteins
  • Hspa8 protein, rat
  • Molecular Chaperones
  • Peptides
  • Creatinine
  • Thiosulfate Sulfurtransferase
  • Acetylglucosaminidase
  • Nitrogen