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Proteins must fold into three-dimensional structures to execute their biological functions. Therefore, maintenance of protein homeostasis, proteostasis, including prevention of protein misfolding is essential for cellular activity and health. Molecular chaperones are key actors in proteostasis. BRICHOS domain is an intramolecular chaperone that also interferes with several aggregation-prone proteins including amyloid β (Aβ), involved in Alzheimer's disease (AD). To extend the knowledge about Bri2 BRICHOS interactome we here used recombinant human (rh) Bri2 BRICHOS-mCherry fusion protein to probe for potential binding partners. Firstly, exogenously added Bri2 BRICHOS-mCherry was used to stain brain sections of wildtype and amyloid precursor protein (App) knock-in AD mice exhibiting robust pathology. Unexpectedly, we found that rh Bri2 BRICHOS-mCherry stained the cytoplasm of neurons which are devoid of deposits. To identify these intraneuronal proteins that bind to the rh Bri2 BRICHOS domain, we performed co-immunoprecipitation (co-IP) of mouse brain hippocampi homogenates using the Bri2 BRICHOS-mCherry probe and analyzed co-IP proteins by LC-MS/MS. This identified several cytoskeletal proteins including spectrin alpha and beta chain, drebrin, tubulin β3, and β-actin as binding partners. The interactions were confirmed by a second round of pulldown experiments using rh Bri2 BRICHOS linked to magnetic beads. The interaction of rh Bri2 BRICHOS and tubulin β3 was further investigated by staining both mouse brain sections and SH-SY5Y neuroblastoma cells with rh Bri2 BRICHOS-mCherry and tubulin β3 immunostaining, which revealed partial co-localization. These data suggest a possible interplay of extracellular chaperone Bri2 BRICHOS domain in the intracellular space including the cytoskeleton. Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.


Makoto Shimozawa, Helene Tigro, Henrik Biverstål, Ganna Shevchenko, Jonas Bergquist, Ruin Moaddel, Jan Johansson, Per Nilsson. Identification of cytoskeletal proteins as binding partners of Bri2 BRICHOS domain. Molecular and cellular neurosciences. 2023 Jun;125:103843

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PMID: 36935047

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