Abstract
Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins.
Highlights
Assistance of folding and restructuring of polypeptide chains is the main feature of folding helper proteins that provide support for the maintenance of correctly folded client proteins in the cell
To identify a selective small-molecule inhibitor of the amide peptide bond cis/trans isomerase (APIase) site of heat shock proteins (Hsps)[70], a compound library was screened for inhibition of the Hsp-assisted refolding of denatured firefly luciferase in vitro
We investigated if recombinant Hsp[70] binds 1 via its C-terminal peptide binding site which is identical to the APIase catalytic cleft in the Hsp[70] chaperone DnaK14
Summary
Assistance of folding and restructuring of polypeptide chains is the main feature of folding helper proteins that provide support for the maintenance of correctly folded client proteins in the cell. For the major players of the PPIase families, the cyclophilin and FK506-binding proteins (FKBPs), cyclosporins and FK506 derivatives, respectively, serve as high affinity, low molecular mass inhibitors of the PPIases function of these enzymes[8,9] They offer versatile tools to assess the physiological role of the protein folding network in living cells providing a clear indication for the chemical mode of action of these enzymes in the cell[4,10,11]. The bacterial Hsp[70] protein DnaK was identified as a secondary amide peptide bond cis/trans isomerase (APIase), which selectively accelerates the cis/trans isomerization of non-proline peptide bonds[14] This activity resides in the substrate binding domain and is considered to assist folding processes by increasing the peptide chain flexibility around the rigid secondary amide functionality. The direct identification of this role of Hsp[70] has been hampered by the lack of specific Hsp[70] inhibitors
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