Site-Specific C-Terminal Fluorescent Labeling of Tau Protein.
Site-Specific C-Terminal Fluorescent Labeling of Tau Protein.
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DOI：
10.1021/acsomega.2c06139
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发表时间：
2022-12-20
期刊：
影响因子：
4.1
通讯作者：
Mayer, Michael
中科院分区：
文献类型：
--
作者：
Bryan, Louise;Awasthi, Saurabh;Li, Yuanjie;Nirmalraj, Peter Niraj;Balog, Sandor;Yang, Jerry;Mayer, Michael
神经元中Tau蛋白聚集体的形成是包括阿尔茨海默病在内的几种神经退行性疾病的病理学标志。因此，双标记的Tau蛋白可用于研究这些病理性蛋白质的聚集并鉴定潜在的治疗靶点。通常，半胱氨酸残基用于标记Tau蛋白;然而，全长Tau同种型在微管结合区中含有两个半胱氨酸残基，其通过形成分子间二硫键而参与Tau聚集。为了防止荧光标记干扰微管结合区，我们开发了一种策略，在其C-末端荧光标记Tau，同时保留半胱氨酸残基不受干扰。我们利用分选酶A介导的转肽方法将具有His标签和共价连接的Alexa 647荧光团的短肽（GGGH 6-Alexa 647）结合到Tau的C末端。该反应依赖于分选酶识别基序（LPXTG）的存在，我们将其连接到重组表达的Tau的C末端。我们证明，C-末端修饰的Tau蛋白的结果在天然和C-末端标记的Tau单体之间没有显着差异，关于聚集动力学，二级结构，和原纤维形态。
Formation of Tau protein aggregates in neurons is a pathological hallmark of several neurodegenerative diseases, including Alzheimer’s disease. Fluorescently labeled Tau protein is therefore useful to study the aggregation of these pathological proteins and to identify potential therapeutic targets. Conventionally, cysteine residues are used for labeling Tau proteins; however, the full-length Tau isoform contains two cysteine residues in the microtubule-binding region, which are implicated in Tau aggregation by forming intermolecular disulfide bonds. To prevent the fluorescent label from disturbing the microtubule binding region, we developed a strategy to fluorescently label Tau at its C-terminus while leaving cysteine residues unperturbed. We took advantage of a Sortase A-mediated transpeptidation approach to bind a short peptide (GGGH6-Alexa647) with a His-tag and a covalently attached Alexa 647 fluorophore to the C-terminus of Tau. This reaction relies on the presence of a Sortase recognition motif (LPXTG), which we attached to the C-terminus of recombinantly expressed Tau. We demonstrate that C-terminal modification of Tau protein results in no significant differences between the native and C-terminally labeled Tau monomer with regard to aggregation kinetics, secondary structure, and fibril morphology.
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Tau组装：PHF6（VQIVYK）在微管结合区域重复R3中的主要作用。
影响因子：
3.3
作者：
Ganguly, Pritam;Do, Thanh D.;Larini, Luca;LaPointe, Nichole E.;Sercel, Alexander J.;Shade, Madeleine F.;Feinstein, Stuart C.;Bowers, Michael T.;Shea, Joan-Emma
通讯作者：
Shea, Joan-Emma
影响因子：
2.9
作者：
Karikari TK;Nagel DA;Grainger A;Clarke-Bland C;Hill EJ;Moffat KG
通讯作者：
Moffat KG
影响因子：
2.9
作者：
Jeganathan, Sadasivam;von Bergen, Martin;Mandelkow, Eckhard
通讯作者：
Mandelkow, Eckhard
DOI：
10.1073/pnas.1101046108
发表时间：
2011-07-12
影响因子：
11.1
作者：
Chen, Irwin;Dorr, Brent M.;Liu, David R.
通讯作者：
Liu, David R.
影响因子：
4.8
作者：
Di Primio, Cristina;Quercioli, Valentina;Cattaneo, Antonino
通讯作者：
Cattaneo, Antonino