German Diabetes Centre, Leibniz Institute at Heinrich Heine University Düsseldorf, Institute of Clinical Diabetology, Auf'm Hennekamp 65, D-40225 Düsseldorf, Germany.
BMC Biochem. 2010 Nov 8;11:44. doi: 10.1186/1471-2091-11-44.
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.
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 μM) whereas a proinsulin molecule modified to prevent oligomerization showed good binding affinity (K'd 11.3 ± 7.8 μM).
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.
伴侣蛋白可促进肽段的正确折叠，并在细胞应激期间与错误折叠的蛋白质结合。肽与伴侣蛋白的复合物可诱导肽导向免疫。在这里，我们分析了（前）胰岛素原与热休克蛋白 70 家族中最具特征性的伴侣蛋白细菌 DnaK 的相互作用。
在一组重叠的 13 个人类前胰岛素原肽中，信号肽和每个胰岛素原结构域（A-链和 B-链、C-肽）中的一个进一步区域与 DnaK 具有高亲和力。在后一种情况下，覆盖 B-链区域 B11-23 大部分区域的肽表现出最强的结合，其结合亲和力与已知的 DnaK 高亲和力配体相当，解离平衡常数（K'd）为 2.2±0.4 μM。B-链区域 B11-23 位于两个胰岛素分子之间的界面处，在胰岛素寡聚体中不可用。事实上，天然胰岛素寡聚体与 DnaK 的亲和力非常低（K'd 67.8±20.8 μM），而修饰为防止寡聚化的胰岛素原分子则表现出良好的结合亲和力（K'd 11.3±7.8 μM）。
完整的胰岛素与热休克蛋白 70 伴侣蛋白 DnaK 只有较弱的相互作用，而单体胰岛素原和来自 3 个不同胰岛素原区域的肽则与伴侣蛋白有很强的结合。B-链肽 B11-23 的结合最强。有趣的是，肽 B11-23 是 1 型糖尿病中的主要自身抗原。
