1.
Assays to measure small molecule Hsp70 agonist activity in vitro and in vivo.
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Shapiro, O
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Woods, C
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Gleixner, AM
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Sannino, S
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Ngo, M
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McDaniels, MD
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Wipf, P
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Hukriede, NA
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Donnelly, CJ
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Brodsky, JL
Abstract:
Hsp70 prevents protein aggregation and is cytoprotective, but sustained Hsp70 overexpression is problematic. Therefore, we characterized small molecule agonists that augment Hsp70 activity. Because cumbersome assays were required to assay agonists, we developed cell-based and in vivo assays in which disease-associated consequences of Hsp70 activation can be quantified. One assay uses an optogenetic system in which the formation of TDP-43 inclusions can be controlled, and the second assay employs a zebrafish model for acute kidney injury (AKI). These complementary assays will facilitate future work to identify new Hsp70 agonists as well as optimized agonist derivatives.
2.
Quantitative real-time kinetics of optogenetic proteins CRY2 and CIB1/N using single-molecule tools.
Abstract:
In this work we evaluate the interaction of two optogenetic protein variants (CIB1, CIBN) with their complementary protein CRY2 by single-molecule tools in cell-free extracts. After validating the blue light induced co-localization of CRY2 and CIB1/N by Förster resonance energy transfer (FRET) in live cells, a fluorescence correlation spectroscopy (FCS) based method was developed to quantitatively determine the in vitro association of the extracted proteins. Our experiments suggest that CIB1, in comparison with CIBN, possesses a better coupling efficiency with CRY2 due to its intact protein structure and lower diffusion rate within 300s detection window.
