Aggregation and fibrillization of the recombinant human prion protein huPrP90-231
Aggregation and fibrillization of the recombinant human prion protein huPrP90-231
复制标题
DOI：
10.1021/bi991967m
复制
发表时间：
2000-01-18
期刊：
影响因子：
2.9
通讯作者：
Surewicz, WK
中科院分区：
文献类型：
--
作者：
Swietnicki, W;Morillas, M;Surewicz, WK
根据“仅蛋白”假说，朊病毒疾病发病的关键步骤是朊病毒蛋白正常（PrPC）和病理（PrPSc）亚型之间的构象转变。为了深入了解这种转变的机制，我们对重组蛋白的生物物理性质进行了表征，该重组蛋白对应于人类朊病毒蛋白残基90-231 （huPrP90-231）。在1 M胍- hcl存在的酸性条件下（pH 3.6-5）孵育，导致huPrP90-231从α -螺旋结构到β -薄片结构的时间依赖性转变，并寡聚成大分子量的聚集体。在本实验中未检测到稳定的富含β -薄片的单体折叠中间体。数据的动力学分析表明，β -片结构的形成和蛋白质的低聚化可能同时发生。富含β -片的寡聚物具有明显增强的蛋白酶K消化抗性和纤维状形态（即，它们具有PrPSc的基本物理化学性质）。与之前的建议相反，重组朊病毒蛋白可以在非还原条件下转化为PrP（Sc-）样形式，而不需要破坏二硫键。尿素中的实验表明，除了酸性pH外，另一个控制huPrP90-231向低聚β -片结构转变的关键因素是盐的存在。
According to the "protein-only" hypothesis, the critical step in the pathogenesis of pi-ion diseases is the conformational transition between the normal (PrPC) and pathological (PrPSc) isoforms of prion protein. To gain insight into the mechanism of this transition, we have characterized the biophysical properties of the recombinant protein corresponding to residues 90-231 of the human prion protein (huPrP90-231). Incubation of the protein under acidic conditions (pH 3.6-5) in the presence of 1 M guanidine-HCl resulted in a time-dependent transition from an alpha-helical conformation to a beta-sheet structure and oligomerization of huPrP90-231 into large molecular weight aggregates, No stable monomeric beta-sheet-rich folding intermediate of the protein could be detected in the present experiments. Kinetic analysis of the data indicates that the formation of beta-sheet structure and protein oligomerization likely occur concomitantly. The beta-sheet-rich oligomers were characterized by a markedly increased resistance to proteinase K digestion and a fibrillar morphology (i.e., they had the essential physicochemical properties of PrPSc), Contrary to previous suggestions, the conversion of the recombinant prion protein into a PrP(Sc-)like form could be accomplished under nonreducing conditions, without the need to disrupt the disulfide bond. Experiments in urea indicate that, in addition to acidic pH, another critical factor controlling the transition of huPrP90-231 to an oligomeric beta-sheet structure is the presence of salt.