Abstract
BackgroundChaperones 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.ResultsOf 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 μM) whereas a proinsulin molecule modified to prevent oligomerization showed good binding affinity (K'd 11.3 ± 7.8 μM).ConclusionsIntact 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.
Highlights
Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress
Peptide B11-23 represents a dominant autoantigen in type 1 diabetes
Under conditions of cell stress, such as heat stress or a high rate of protein synthesis, there is a higher amount of polypeptides misfolded, and in parallel there is a rapid increase of chaperone availability [1,2,3,4]
Summary
Chaperones facilitate proper folding of peptides and bind to misfolded proteins as occurring during periods of cell stress. As one of the dominant members of the chaperone family, heat shock protein (hsp) 70 shows strong and preferential upregulation in various cell populations. Insulin is a primary product of protein synthesis of pancreatic b-islet cells. Insulin represents a dominant antigen during the development of the immunological processes leading to pancreatic b-cell destruction and (insulindependent) type 1 diabetes. The hormone is a primary target of autoantibodies that emerge early in the prediabetic phase [16] the stimulation of cellmediated immune processes including the activation of insulin-reactive T-lymphocytes seems to be of major importance for the progression of b-cell-directed immune reactivity [17]. Insulin-specific T-cells can be isolated from human subjects both in the prediabetic phase and the onset of type 1 diabetes and are present in the diabetes-prone non-obese diabetic (NOD) mouse, an animal model of the human disease [18,19]
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