Extensive Plasmid Library to Prepare Tau Protein Variants and Study Their Functional Biochemistry

Abstract

Tau neurofibrillary tangles are key pathological features of Alzheimer’s disease and other tauopathies. Recombinant protein technology is vital for studying the structure and function of tau in physiology and aggregation in pathophysiology. However, open-source and well-characterized plasmids for efficiently expressing and purifying different tau variants are lacking. We generated 44 sequence-verified plasmids including those encoding full length (FL) tau-441, its four-repeat microtubule-binding (K18) fragment, and their respective selected familial pathological variants (N279K, V337M, P301L, C291R, and S356T). Moreover, plasmids for expressing single (C291A), double (C291A/C322A), and triple (C291A/C322A/I260C) cysteine-modified variants were generated to study alterations in cysteine content and locations. Furthermore, protocols for producing representative tau forms were developed. We produced and characterized the aggregation behavior of the triple cysteine-modified tau-K18, often used in real-time cell internalization and aggregation studies because it can be fluorescently labeled on a cysteine outside the microtubule-binding core. Similar to the wild type (WT), triple cysteine-modified tau-K18 aggregated by progressive β-sheet enrichment, albeit at a slower rate. On prolonged incubation, cysteine-modified K18 formed paired helical filaments similar to those in Alzheimer’s disease, sharing morphological phenotypes with WT tau-K18 filaments. Nonetheless, cysteine-modified tau-K18 filaments were significantly shorter (p = 0.002) and mostly wider than WT filaments, explainable by their different principal filament elongation pathways: vertical (end-to-end) and lateral growth for WT and cysteine-modified, respectively. Cysteine rearrangement may therefore induce filament polymorphism. Together, the plasmid library, the protein production methods, and the new insights into cysteine-dependent aggregation should facilitate further studies and the design of antiaggregation agents.

Publication DOI: https://doi.org/10.1021/acschemneuro.0c00469
Divisions: Life & Health Sciences > Biosciences
Life & Health Sciences > Chronic and Communicable Conditions
Life & Health Sciences
Life & Health Sciences > Clinical and Systems Neuroscience
Additional Information: This is an open access article published under a Creative Commons Attribution (CC-BY) License, which permits unrestricted use, distribution and reproduction in any medium, provided the author and source are cited. Funding: This study was partly funded by the UK NC3Rs through the CRACK IT: Untangle grant (#NC/C013101/1). T.K.K. was supported by the University of Warwick Chancellor’s Scholarship, the Biotechnology and Biological Sciences Research Council (#BB/J014532/1) through the Midlands Integrative Biosciences Training Partnership, the BrightFocus Foundation (#A2020812F), the Swedish Brain Foundation (Hjarnfonden; ̈ #FO2020-0240), the Swedish Alzheimer Foundation (#AF930627), the Swedish Dementia Foundation (Demensförbundet), the Agneta Prytz-Folkes & Gösta Folkes Foundation (#2020-00124), Gamla Tjanarinnor Foundation, the Aina ̈ (Ann) Wallströms and Mary-Ann Sjöbloms Foundation, the Gun & Bertil Stohnes Foundation, and the Anna Lisa and Brother Björnsson’s Foundation
Uncontrolled Keywords: Alzheimer’s disease,MAPT mutations,Tau,expression and purification,frontotemporal dementia,plasmid,Biochemistry,Physiology,Cognitive Neuroscience,Cell Biology
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Related URLs: http://www.scop ... tnerID=8YFLogxK (Scopus URL)
PURE Output Type: Article
Published Date: 2020-10-07
Published Online Date: 2020-09-16
Accepted Date: 2020-08-24
Authors: Karikari, Thomas K.
Keeling, Sophie
Hill, Emily
Lantero Rodrı́guez, Juan
Nagel, David A.
Becker, Bruno
Höglund, Kina
Zetterberg, Henrik
Blennow, Kaj
Hill, Eric J. ( 0000-0002-9419-1500)
Moffat, Kevin G.

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