Gentamicin inhibits HSP70‐assisted protein folding by interfering with substrate recognition

Abstract

Edited by Miguel De la RosaKeywords: Molecular chaperone Heat-shock protein with subunit molecular masses of 70 kDa Antibiotics Gentamicin a b s t r a c tWe previously reported that gentamicin (GM) specifically binds to heat-shock protein with subunit molecular masses of 70 kDa (HSP70). In the present study, we have investigated the effects of GM binding on HSP70-assisted protein folding in vitro. The C-terminal, and not the N-terminal of HSP70 was found to bind to GM. GM significantly suppressed refolding of firefly luciferase in the presence of HSP70 and HSP40, although the ATPase activity of HSP70 was unaffected by GM. A surface plasmon resonance analysis revealed that GM specifically interferes with the binding of HSP70 to a model peptide that mimics the exposed hydrophobic surface of the folding intermediates. These results indicated that GM inhibits the chaperone activity of HSP70 and may suppress protein folding via inhibition of HSP70 in vivo.

Gut epithelial inducible heat-shock proteins and their modulation by diet and the microbiota

“…82 The latter effect may have resulted from the gentamicin-specific inhibition of HSP70's chaperoning activity, thus promoting a positive feedback response. 83,84 In normal pigs, oral administration of the broad-spectrum antibiotic amoxicillin to mothers around parturition was found to affect the mother's fecal and the offspring's gut microbiota as well as the offspring's gut epithelial iHSPs: small intestinal iHSP27 remained unaffected, while iHSP70 decreased transiently, and both iHSP proteins increased in colonic mucosa. 45,63 Data were interpreted as being essentially related to amoxicillin-induced changes in maternal microbiota and subsequent microbiota transfer to offspring after birth.…”

Aminoglycosides Suppress the Protein Folding Activity of the Molecular Chaperone HSC70: Implication of a Structure-Activity Relationship

“…We previously demonstrated that gentamicin suppresses the protein refolding activity of HSC70 [29,30]. To examine whether other AGs have similar effects on HSC70 activity, the refolding of denatured firefly luciferase was used as a model substrate.…”

“…We previously demonstrated that gentamicin specifically inhibits the HSC70/substrate interaction, but not the HSC70/HSP40 interaction [30]. To examine whether other AGs have the same effect, a substrate binding assay of HSC70 using F-RCMLA as a model substrate was performed [31,32].…”

“…HSC70 from porcine brain and recombinant human HSP40 (DNAJB1) expressed in Escherichia coli were purified as described previously [30]. Protein concentrations were determined via the bicinchoninic acid method using a BCA assay kit (Pierce, Rockford, Ill., USA) and bovine serum albumin as a standard.…”

“…In addition, we reported that gentamicin suppresses the refolding activity of the HSC70/HSP40 chaperone complex by inhibiting substrate binding to the carboxyl-terminal SBD. These observations suggest that gentamicin specifically inhibits the activity of HSC70 in the kidney [29,30]. …”