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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 . doi:10.1021/acschemneuro.0c00469 (In Press)
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WRAP-An-extensive-plasmid-library-prepare-tau-protein-variants-study-functional-Keeling-2020.pdf - Accepted Version Embargoed item. Restricted access to Repository staff only until 24 August 2021. Contact author directly, specifying your specific needs. - Requires a PDF viewer. Download (2962Kb) |
Official URL: http://dx.doi.org/10.1021/acschemneuro.0c00469
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 | ||||||||||||||||||||||||||||||||||||
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| 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: | 16 September 2020 | ||||||||||||||||||||||||||||||||||||
| Dates: |
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| Date of first compliant deposit: | 17 September 2020 | ||||||||||||||||||||||||||||||||||||
| DOI: | 10.1021/acschemneuro.0c00469 | ||||||||||||||||||||||||||||||||||||
| Status: | Peer Reviewed | ||||||||||||||||||||||||||||||||||||
| Publication Status: | In Press | ||||||||||||||||||||||||||||||||||||
| Publisher Statement: | “This document is the Accepted Manuscript version of a Published Work that appeared in final form in ACS Chemical Neuroscience, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see [insert ACS Articles on Request author-directed link to Published Work, see http://pubs.acs.org/page/policy/articlesonrequest/index.html].” | ||||||||||||||||||||||||||||||||||||
| Access rights to Published version: | Restricted or Subscription Access | ||||||||||||||||||||||||||||||||||||
| Copyright Holders: | Copyright © 2020 American Chemical Society | ||||||||||||||||||||||||||||||||||||
| RIOXX Funder/Project Grant: |
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