Huang Kun, Dong Jian, Phillips Nelson B, Carey Paul R, Weiss Michael A
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
J Biol Chem. 2005 Dec 23;280(51):42345-55. doi: 10.1074/jbc.M507110200. Epub 2005 Oct 20.
Insulin is susceptible to fibrillation, a misfolding process leading to well ordered cross-beta assembly. Protection from fibrillation in beta cells is provided by sequestration of the susceptible monomer within zinc hexamers. We demonstrate that proinsulin is refractory to fibrillation under conditions that promote the rapid fibrillation of zinc-free insulin. Proinsulin fibrils, as probed by Raman microscopy, are nonetheless similar in structure to insulin fibrils. The connecting peptide, although not well ordered in native proinsulin, participates in a fibril-specific beta-sheet. Native insulin and proinsulin exhibit similar free energies of unfolding as inferred from guanidine denaturation studies: relative amyloidogenicities are thus not correlated with global stability. Strikingly, the susceptibility of proinsulin to fibrillation is increased by scission of the connecting peptide at single sites. We thus propose that the connecting peptide constrains a large scale conformational change in the misfolded protein. A tethering mechanism is proposed based on a model of an insulin protofilament derived from electron-microscopic image reconstruction. The proposed relationship between cross-beta assembly and protein topology is supported by studies of single-chain analogs (mini-proinsulin and insulin-like growth factor I) in which foreshortened connecting peptides further retard fibrillation. In addition to its classic function to facilitate disulfide pairing, the connecting peptide may protect beta cells from toxic protein misfolding in the endoplasmic reticulum.
胰岛素易发生纤维化，这是一个导致形成有序交叉β组装的错误折叠过程。β细胞中对纤维化的保护是通过将易感单体隔离在六聚体锌中来实现的。我们证明，在促进无锌胰岛素快速纤维化的条件下，胰岛素原对纤维化具有抗性。通过拉曼显微镜检测，胰岛素原纤维的结构与胰岛素纤维相似。连接肽虽然在天然胰岛素原中无序，但参与了纤维特异性β折叠。从胍变性研究推断，天然胰岛素和胰岛素原具有相似的解折叠自由能：因此，相对淀粉样蛋白生成性与整体稳定性无关。引人注目的是，连接肽在单个位点的切割会增加胰岛素原对纤维化的敏感性。因此，我们提出连接肽限制了错误折叠蛋白中的大规模构象变化。基于从电子显微镜图像重建得到的胰岛素原丝模型，提出了一种拴系机制。单链类似物（迷你胰岛素原和胰岛素样生长因子I）的研究支持了所提出的交叉β组装与蛋白质拓扑结构之间的关系，其中缩短的连接肽进一步延缓了纤维化。除了其促进二硫键配对的经典功能外，连接肽还可以保护β细胞免受内质网中有毒蛋白质错误折叠的影响。
