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Extensive plasmid library to prepare Tau protein variants and study their functional biochemistry

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Karikari, Thomas K., Keeling, Sophie, Hill, Emily, Lantero Rodrı́guez, Juan, Nagel, David A., Becker, Bruno, Höglund, Kina, Zetterberg, Henrik, Blennow, Kaj, Hill, Eric J. and Moffat, Kevin G. (2020) Extensive plasmid library to prepare Tau protein variants and study their functional biochemistry. ACS Chemical Neuroscience, 11 (19). pp. 3117-3129. doi:10.1021/acschemneuro.0c00469

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Official URL: http://dx.doi.org/10.1021/acschemneuro.0c00469

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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.

Item Type: Journal Article
Subjects: R Medicine > RC Internal medicine
Divisions: Faculty of Science > Life Sciences (2010- )
Library of Congress Subject Headings (LCSH): Alzheimer's disease, Alzheimer's disease -- Immunotherapy, Nerve tissue proteins, Tubulins, Alzheimer's disease -- Animal models
Journal or Publication Title: ACS Chemical Neuroscience
Publisher: American Chemical Society
ISSN: 1948-7193
Official Date: 7 October 2020
Dates:
DateEvent
7 October 2020Published
24 August 2020Available
24 August 2020Accepted
Volume: 11
Number: 19
Page Range: pp. 3117-3129
DOI: 10.1021/acschemneuro.0c00469
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Open Access
Copyright Holders: Copyright © 2020 American Chemical Society
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
NC/C013101/1National Centre for the Replacement, Refinement, and Reduction of Animals in Research http://viaf.org/viaf/156961849
BB/J014532/1[BBSRC] Biotechnology and Biological Sciences Research Councilhttp://dx.doi.org/10.13039/501100000268
A2020812FBrightFocus Foundationhttp://dx.doi.org/10.13039/100006312
FO2020-0240Hjärnfondenhttp://dx.doi.org/10.13039/501100003792
AF-930627Alzheimerfondenhttp://dx.doi.org/10.13039/501100008599
UNSPECIFIEDDemensförbundethttp://dx.doi.org/10.13039/100010773
2020-00124Agneta Prytz-Folkes och Gösta Folkes Stiftelsehttp://dx.doi.org/10.13039/501100007484
UNSPECIFIEDStiftelsen Gamla Tjänarinnorhttp://viaf.org/viaf/136756250
UNSPECIFIEDAina (Ann) Wallströms och Mary-Ann Sjöbloms stiftelseUNSPECIFIED
UNSPECIFIEDGun och Bertil Stohnes Stiftelsehttp://dx.doi.org/10.13039/100009673
UNSPECIFIEDAnna-Lisa och Bror Björnssons Stiftelsehttp://dx.doi.org/10.13039/100010107

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