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Title: Phospholamban mutants compete with wild type for SERCA binding in living cells

Abstract

Highlights: Black-Right-Pointing-Pointer PLB phosphorylation in HEK cells increased FRET between YFP-PLB and CFP-SERCA. Black-Right-Pointing-Pointer Competition: Expressing loss-of-function PLB mutants in the system decreased FRET. Black-Right-Pointing-Pointer The FRET assay could screen potential therapeutic PLB mutants to activate SERCA. -- Abstract: We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca{sup 2+} cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCA activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLB{sub M}) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bindmore » to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLB{sub M} in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLB{sub M} and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLB{sub M} for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.« less

Authors:
;  [1]
  1. Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, MN 55455 (United States)
Publication Date:
OSTI Identifier:
22207791
Resource Type:
Journal Article
Journal Name:
Biochemical and Biophysical Research Communications
Additional Journal Information:
Journal Volume: 420; Journal Issue: 2; Other Information: Copyright (c) 2012 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA); Journal ID: ISSN 0006-291X
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; CALCIUM IONS; ENERGY TRANSFER; FLUORESCENCE; GENE THERAPY; HEART FAILURE; HYDROFLUORIC ACID; INHIBITION; MUSCLES; MUTANTS; OPTIMIZATION; PHOSPHORYLATION; PROTEINS; SARCOPLASMIC RETICULUM; SODIUM; SULFATES

Citation Formats

Gruber, Simon J., Haydon, Suzanne, and Thomas, David D., E-mail: ddt@umn.edu. Phospholamban mutants compete with wild type for SERCA binding in living cells. United States: N. p., 2012. Web. doi:10.1016/J.BBRC.2012.02.125.
Gruber, Simon J., Haydon, Suzanne, & Thomas, David D., E-mail: ddt@umn.edu. Phospholamban mutants compete with wild type for SERCA binding in living cells. United States. https://doi.org/10.1016/J.BBRC.2012.02.125
Gruber, Simon J., Haydon, Suzanne, and Thomas, David D., E-mail: ddt@umn.edu. 2012. "Phospholamban mutants compete with wild type for SERCA binding in living cells". United States. https://doi.org/10.1016/J.BBRC.2012.02.125.
@article{osti_22207791,
title = {Phospholamban mutants compete with wild type for SERCA binding in living cells},
author = {Gruber, Simon J. and Haydon, Suzanne and Thomas, David D., E-mail: ddt@umn.edu},
abstractNote = {Highlights: Black-Right-Pointing-Pointer PLB phosphorylation in HEK cells increased FRET between YFP-PLB and CFP-SERCA. Black-Right-Pointing-Pointer Competition: Expressing loss-of-function PLB mutants in the system decreased FRET. Black-Right-Pointing-Pointer The FRET assay could screen potential therapeutic PLB mutants to activate SERCA. -- Abstract: We have used fluorescent fusion proteins stably expressed in HEK cells to detect directly the interaction between the sarcoplasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLB) in living cells, in order to design PLB mutants for gene therapy. Ca{sup 2+} cycling in muscle cells depends strongly on SERCA. Heart failure (HF), which contributes to 12% of US deaths, typically exhibits decreased SERCA activity, and several potential therapies for HF aim to increase SERCA activity. We are investigating the use of LOF-PLB mutants (PLB{sub M}) as gene therapy vectors to increase SERCA activity. Active SERCA1a and WT-PLB, tagged at their N termini with fluorescent proteins (CFP and YFP), were coexpressed in stable HEK cell lines, and fluorescence resonance energy transfer (FRET) was used to detect their interaction directly. Phosphorylation of PLB, induced by forskolin, caused an increase in FRET from CFP-SERCA to YFP-PLB, indicating that SERCA inhibition can be relieved without dissociation of the complex. This suggests that a LOF mutant might bind to SERCA with sufficient affinity to complete effectively with WT-PLB, thus relieving SERCA inhibition. Therefore, we transiently expressed a series of PLB{sub M} in the CFP-SERCA/YFP-PLB cell line, and found decreased FRET, implying competition between PLB{sub M} and WT-PLB for binding to SERCA. These results establish this FRET assay as a rapid and quantitative means of screening PLB{sub M} for optimization of gene therapy to activate SERCA, as needed for gene therapy in HF.},
doi = {10.1016/J.BBRC.2012.02.125},
url = {https://www.osti.gov/biblio/22207791}, journal = {Biochemical and Biophysical Research Communications},
issn = {0006-291X},
number = 2,
volume = 420,
place = {United States},
year = {Fri Apr 06 00:00:00 EDT 2012},
month = {Fri Apr 06 00:00:00 EDT 2012}
}