Leffers Karl-Werner, Wille Holger, Stöhr Jan, Junger Erika, Prusiner Stanley B, Riesner Detlev
Institut für Physikalische Biologie und Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität Düsseldorf, D-40225 Düsseldorf, Germany.
Biol Chem. 2005 Jun;386(6):569-80. doi: 10.1515/BC.2005.067.
The conversion of the alpha-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP Sc ) is the fundamental event in the prion diseases. The C-terminal fragment of PrP Sc (PrP 27-30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrP Sc , PrP 27-30 polymerizes into rod-shaped structures with the ultra-structural and tinctorial properties of amyloid. To study the folding of PrP, both with respect to the formation of PrP Sc from PrP C and the assembly of rods from PrP 27-30, we solubilized Syrian hamster (sol SHa) PrP 27-30 in low concentrations (0.2%) of sodium dodecyl sulfate (SDS) under conditions previously used to study the structural transitions of this protein. Sol SHaPrP 27-30 adopted a beta-sheet-rich structure at SDS concentrations between 0.02% and 0.04% and remained soluble. Here we report that NaCl stabilizes SHaPrP 27-30 in a soluble, beta-sheet-rich state that allows fibril assembly to proceed over several weeks. Under these conditions, fibril formation occurred not only with sol PrP 27-30, but also with native SHaPrP C . Addition of sphingolipids seems to increase fibril growth. When recombinant (rec) SHaPrP(90-231) was exposed to low concentrations of SDS, similar to those used to polymerize sol SHaPrP 27-30 in the presence of 250 mM NaCl, fibril formation occurred regularly. When fibrils formed from PrP 27-30 or PrP C were bioassayed in transgenic mice overexpressing full-length SHaPrP, no infectivity was obtained, whereas amyloid fibrils formed of rec mouse PrP(89-230) were infectious. At present, it cannot be determined whether the lack of infectivity is caused by a difference in the structure of the fibrils or in the bioassay conditions.
朊病毒蛋白的细胞型α-螺旋异构体（PrP C ）转变为不溶性、富含β-折叠、具有传染性的致病异构体（PrP Sc ）是朊病毒疾病的基本事件。PrP Sc 的C末端片段（PrP 27-30）通过有限的蛋白酶解形成，并保留传染性。与全长PrP Sc 不同，PrP 27-30聚合成具有淀粉样超微结构和染色特性的杆状结构。为了研究PrP的折叠，包括从PrP C 形成PrP Sc 以及从PrP 27-30组装成杆状结构，我们在先前用于研究该蛋白结构转变的条件下，将叙利亚仓鼠（sol SHa）PrP 27-30溶解在低浓度（0.2%）的十二烷基硫酸钠（SDS）中。在SDS浓度介于0.02%和0.04%之间时，Sol SHaPrP 27-30呈现富含β-折叠的结构并保持可溶。在此我们报告，NaCl能使SHaPrP 27-30稳定在一种可溶的、富含β-折叠的状态，使原纤维组装能持续数周进行。在这些条件下，不仅sol PrP 27-30能形成原纤维，天然的SHaPrP C 也能形成。添加鞘脂似乎能促进原纤维生长。当重组（rec）SHaPrP(90-231)暴露于低浓度SDS时，类似于在250 mM NaCl存在下使sol SHaPrP 27-30聚合所用的浓度，能有规律地形成原纤维。当在过表达全长SHaPrP的转基因小鼠中对由PrP 27-30或PrP C 形成的原纤维进行生物测定时，未获得传染性，而由rec小鼠PrP(89-230)形成的淀粉样原纤维具有传染性。目前，尚无法确定缺乏传染性是由原纤维结构差异还是生物测定条件差异所致。
