Delineation of functional subdomains of Huntingtin protein and their interaction with HAP40.

Autor:	Alteen MG; Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada; POINT Biopharma, 22 St Clair Avenue E Suite 1201, Toronto, ON M4T 2S3, Canada., Deme JC; Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA., Alvarez CP; Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada; SCIEX, 71 Four Valley Dr, Vaughan, ON L4K 4V8, Canada., Loppnau P; Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada., Hutchinson A; Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada., Seitova A; Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada., Chandrasekaran R; Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada., Silva Ramos E; Neuroproteomics, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125 Berlin, Germany., Secker C; Neuroproteomics, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125 Berlin, Germany., Alqazzaz M; Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada., Wanker EE; Neuroproteomics, Max Delbrück Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125 Berlin, Germany., Lea SM; Center for Structural Biology, Center for Cancer Research, National Cancer Institute, Frederick, MD 21702, USA., Arrowsmith CH; Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada; Princess Margaret Cancer Centre and Department of Medical Biophysics, University of Toronto, Toronto, ON M5G 1L7, Canada. Electronic address: cheryl.arrowsmith@uhnresearch.ca., Harding RJ; Structural Genomics Consortium, University of Toronto, Toronto, ON M5G 1L7, Canada; Department of Pharmacology and Toxicology, University of Toronto, Toronto, ON M5S 1A8, Canada. Electronic address: rachel.harding@utoronto.ca.
Jazyk:	angličtina
Zdroj:	Structure (London, England : 1993) [Structure] 2023 Sep 07; Vol. 31 (9), pp. 1121-1131.e6. Date of Electronic Publication: 2023 Jun 29.
DOI:	10.1016/j.str.2023.06.002
Abstrakt:	The huntingtin (HTT) protein plays critical roles in numerous cellular pathways by functioning as a scaffold for its many interaction partners and HTT knock out is embryonic lethal. Interrogation of HTT function is complicated by the large size of this protein so we studied a suite of structure-rationalized subdomains to investigate the structure-function relationships within the HTT-HAP40 complex. Protein samples derived from the subdomain constructs were validated using biophysical methods and cryo-electron microscopy, revealing they are natively folded and can complex with validated binding partner, HAP40. Derivatized versions of these constructs enable protein-protein interaction assays in vitro, with biotin tags, and in cells, with luciferase two-hybrid assay-based tags, which we use in proof-of-principle analyses to further interrogate the HTT-HAP40 interaction. These open-source biochemical tools enable studies of fundamental HTT biochemistry and biology, will aid the discovery of macromolecular or small-molecule binding partners and help map interaction sites across this large protein. Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. (Copyright © 2023 Elsevier Ltd. All rights reserved.)
Databáze:	MEDLINE
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