Huang Kun, Maiti Nakul C, Phillips Nelson B, Carey Paul R, Weiss Michael A
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Biochemistry. 2006 Aug 29;45(34):10278-93. doi: 10.1021/bi060879g.
Systemic amyloidoses, an important class of protein misfolding diseases, are often due to fibrillation of disulfide-cross-linked globular proteins otherwise unrelated in sequence or structure. Although cross-beta assembly is regarded as a universal property of polypeptides, it is not understood how such amyloids accommodate diverse disulfide connectivities. Does amyloidogenicity depend on protein topology? A model is provided by insulin, a two-chain protein containing three disulfide bridges. The importance of chain topology is demonstrated by mini-proinsulin (MP), a single-chain analogue in which the C-terminus of the B chain (residue B30) is tethered to the N-terminus of the A chain (A1). The B30-A1 tether impedes the fiber-specific alpha --> beta transition, leading to slow formation of a structurally nonuniform amorphous precipitate. Conversely, fibrillation is robust to interchange of disulfide bridges. Whereas native insulin exhibits pairings [A6-A11, A7-B7, and A20-B19], metastable isomers with alternative pairings [A6-B7, A7-A11, A20-B19] or [A6-A7, A11-B7, A20-B1] readily undergo fibrillation with essentially identical alpha --> beta transitions. Respective pairing schemes are in each case retained. Isomeric fibrils and the amorphous MP precipitate are each able to seed the fibrillation of wild-type insulin, suggesting a structural correspondence between respective nuclei or modes of assembly. Together, our results demonstrate that effects of polypeptide topology on amyloidogenicity depend on structural context. Although the native structures and stabilities of single-chain insulin analogues are similar to those of wild-type insulin, the interchain tether constrains the extent of conformational distortion at elevated temperature, retards initial non-native aggregation, and is apparently incompatible with the mature structure of an insulin protofilament. We speculate that the general danger of fibrillation has imposed a constraint in protein evolution, selecting for topologies unfavorable to amyloid formation.
系统性淀粉样变性是一类重要的蛋白质错误折叠疾病，通常由二硫键交联的球状蛋白质发生纤维化引起，这些蛋白质在序列或结构上原本并无关联。尽管交叉β组装被认为是多肽的普遍特性，但尚不清楚此类淀粉样蛋白如何容纳多样的二硫键连接方式。淀粉样变性是否取决于蛋白质拓扑结构？胰岛素提供了一个模型，它是一种含三条二硫键的双链蛋白质。迷你胰岛素原（MP）是一种单链类似物，其中B链的C末端（残基B30）与A链的N末端（A1）相连，它证明了链拓扑结构的重要性。B30 - A1连接阻碍了纤维特异性的α→β转变，导致结构不均匀的无定形沉淀形成缓慢。相反，二硫键的交换对纤维化影响不大。天然胰岛素的二硫键配对为[A6 - A11、A7 - B7和A20 - B19]，具有替代配对[A6 - B7、A7 - A11、A20 - B19]或[A6 - A7、A11 - B7、A20 - B1]的亚稳异构体很容易发生纤维化，且α→β转变基本相同。每种情况下各自的配对方案都得以保留。异构纤维和无定形MP沉淀都能够引发野生型胰岛素的纤维化，这表明各自的核或组装模式之间存在结构对应关系。总之，我们的结果表明多肽拓扑结构对淀粉样变性的影响取决于结构背景。尽管单链胰岛素类似物的天然结构和稳定性与野生型胰岛素相似，但链间连接在高温下限制了构象扭曲的程度，延缓了初始的非天然聚集，并且显然与胰岛素原纤维的成熟结构不相容。我们推测纤维化的普遍风险在蛋白质进化过程中施加了一种限制，选择了不利于淀粉样蛋白形成的拓扑结构。
