Background. Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. Complexes of peptides with chaperones induce peptide-directed immunity. Here we analyzed the interaction of (pre)proinsulin with the best characterized chaperone of the hsp70 family, bacterial DnaK. Results. Of a set of overlapping 13-mer peptides of human preproinsulin high affinity binding to DnaK was found for the signal peptide and one further region in each proinsulin domain (A- and B-chain, C-peptide). Among the latter, peptides covering most of the B-chain region B11-23 exhibited strongest binding, which was in the range of known high-affinity DnaK ligands, dissociation equilibrium constant (K'd) of 2.2 ± 0.4 μM. The B-chain region B11-23 is located at the interface between two insulin molecules and not accessible in insulin oligomers. Indeed, native insulin oligomers showed very low DnaK affinity (K'd 67.8 20.8 M) whereas a proinsulin molecule modified to prevent oligomerization showed good binding affinity (K'd 11.3 ± 7.8 μM). Conclusions. Intact insulin only weakly interacts with the hsp70 chaperone DnaK whereas monomeric proinsulin and peptides from 3 distinct proinsulin regions show substantial chaperone binding. Strongest binding was seen for the B-chain peptide B 11-23. Interestingly, peptide B11-23 represents a dominant autoantigen in type 1 diabetes. © 2010 Burkart et al; licensee BioMed Central Ltd.
Burkart, V., Siegenthaler, R. K., Blasius, E., Vandenbroeck, K., Alloza, I., Fingberg, W., … Kolb, H. (2010). High affinity binding of hydrophobic and autoantigenic regions of proinsulin to the 70 kDa chaperone DnaK. BMC Biochemistry, 11(1). https://doi.org/10.1186/1471-2091-11-44