Bhattacharya K, Rank K B, Evans D B, Sharma S K
Pharmacia Corporation, Kalamazoo, Michigan 49007, USA.
Biochem Biophys Res Commun. 2001 Jul 6;285(1):20-6. doi: 10.1006/bbrc.2001.5116.
Filamentous tau pathology is central to a large number of dementing disorders, including Alzheimer's disease in which polymerized tau is hyperphosphorylated. Previous studies on heparin-dependent tau polymerization, using recombinant tau isoforms lacking Cys-291, suggest that tau dimerization via Cys-322 is critical for initiation of assembly of soluble tau into filaments. We report heparin-dependent in vitro polymerization of human recombinant tau (1-383 isoform), containing both Cys-291 and Cys-322, into paired helical filaments as characterized by electron microscopy. Tau polymerization, under physiological tau concentrations in the presence of dithiothreitol (DTT), was followed by a Thioflavine S fluorescence assay. To understand the molecular basis for heparin-induced tau polymerization, we expressed and purified C291A, C322A, and C291A/C322A tau mutants. The DTT requirement for tau polymerization was abolished using either the C291A or C322A tau mutant and polymerization was not observed with the C291A/C322A tau double mutant. Analysis by sodium dodecyl sulfate gel electrophoresis showed that, unlike wild type tau, a significant amount of the C291A mutant and the C322A mutant is present as a disulfide bonded dimer. Taken together these results suggest that, in isoforms containing both Cys-291 and Cys-322, a dimeric tau with an intermolecular disulfide bond through either Cys-291 or Cys-322 is presumably acting as a seed for initiation of tau polymerization.
丝状tau蛋白病理是许多痴呆症的核心，包括阿尔茨海默病，其中聚合的tau蛋白发生了过度磷酸化。先前关于肝素依赖性tau蛋白聚合的研究，使用缺乏Cys-291的重组tau蛋白异构体，表明通过Cys-322的tau蛋白二聚化对于可溶性tau蛋白组装成细丝的起始至关重要。我们报告了人重组tau蛋白（1-383异构体）在肝素依赖性体外聚合成双螺旋细丝，这通过电子显微镜进行了表征。在二硫苏糖醇（DTT）存在下，在生理tau蛋白浓度下的tau蛋白聚合，随后进行硫黄素S荧光测定。为了理解肝素诱导tau蛋白聚合的分子基础，我们表达并纯化了C291A、C322A和C291A/C322A tau蛋白突变体。使用C291A或C322A tau蛋白突变体消除了tau蛋白聚合对DTT的需求，并且在C291A/C322A tau蛋白双突变体中未观察到聚合。十二烷基硫酸钠凝胶电泳分析表明，与野生型tau蛋白不同，大量的C291A突变体和C322A突变体以二硫键结合的二聚体形式存在。综合这些结果表明，在同时含有Cys-291和Cys-322的异构体中，通过Cys-291或Cys-322形成分子间二硫键的二聚体tau蛋白可能作为tau蛋白聚合起始的种子。
