Structural mapping of the surfaces of fibrillar 1N3R and 1N4R human tau

The structural characterization of pathogenic tau filaments that accumulate in tauopathies, including Alzheimer disease, is key for understanding their pathogenic function and identify the structural determinants of distinct tau filaments involved in distinct tauopathies. Fibrillar tau surfaces play a crucial role in tau fibrils interaction with various protein partners and binding to neurons, a key step for their prion-like propagation propensity. In order to identify the amino acid residues stretches that are exposed and those that are not exposed to the solvent within tau fibrils, we used structural proteomic approaches: proteolysis and molecular covalent surface painting using NHS-Biotin followed by MS-based analysis of the proteolytic products. We compared the solvent accessible amino acid residues on the surface of htau proteins upon assembly of 1N3R and 1N4R human htau monomers into fibrils in vitro. We mapped the amino acid segments exposed or unexposed on the surface of htau fibrils by determining the accessibility GluC enzyme and NHS-biotin followed by proteolysis. The proteolytic peptides were analyzed by nanoLC-MSMS and processed using Mascot for their identification and PeakView for intensity extraction of labeled and unlabeled proteolytic peptides.

在包括阿尔茨海默病（Alzheimer disease）在内的tau蛋白病（tauopathies）中蓄积的致病性tau丝（tau filaments）的结构表征，是解析其致病功能、明确不同tau蛋白病相关特异性tau丝结构决定因素的关键。tau原纤维的表面在其与多种蛋白质伴侣相互作用、结合神经元的过程中发挥关键作用，而这一环节正是其类朊病毒传播倾向的核心基础。为了明确tau原纤维内部暴露于溶剂与未暴露于溶剂的氨基酸残基区段，我们采用了结构蛋白质组学研究策略：蛋白水解法与基于NHS-生物素（NHS-Biotin）的分子共价表面标记法，随后对蛋白水解产物进行质谱（MS）分析。我们在体外将1N3R与1N4R型人源htau单体组装为原纤维后，对比了htau蛋白表面可被溶剂接触的氨基酸残基分布情况。我们通过利用GluC酶与NHS-生物素分析可及性、并结合蛋白水解的方式，绘制了htau原纤维表面暴露与未暴露的氨基酸区段图谱。本研究采用纳流液相色谱-串联质谱（nanoLC-MSMS）对蛋白水解肽段进行分析，并分别借助Mascot完成肽段鉴定、利用PeakView提取标记与未标记蛋白水解肽段的信号强度。