The transmembrane peptide DWORF activates SERCA2a via dual mechanisms

Ang Li, Samantha L. Yuen, Daniel R. Stroik, Evan Kleinboehl, Razvan L. Cornea, David D. Thomas

Research output: Contribution to journalArticlepeer-review

Abstract

The Ca-ATPase isoform 2a (SERCA2a) pumps cytosolic Ca2+ into the sarcoplasmic reticulum (SR) of cardiac myocytes, enabling muscle relaxation during diastole. Abnormally high cytosolic [Ca2+] is a central factor in heart failure, suggesting that augmentation of SERCA2a Ca2+ transport activity could be a promising therapeutic approach. SERCA2a is inhibited by the protein phospholamban (PLB), and a novel transmembrane peptide, dwarf open reading frame (DWORF), is proposed to enhance SR Ca2+ uptake and myocyte contractility by displacing PLB from binding to SERCA2a. However, establishing DWORF’s precise physiological role requires further investigation. In the present study, we developed cell-based FRET biosensor systems that can report on protein–protein interactions and structural changes in SERCA2a complexes with PLB and/or DWORF. To test the hypothesis that DWORF competes with PLB to occupy the SERCA2a-binding site, we transiently transfected DWORF into a stable HEK cell line expressing SERCA2a labeled with a FRET donor and PLB labeled with a FRET acceptor. We observed a significant decrease in FRET efficiency, consistent with a decrease in the fraction of SERCA2a bound to PLB. Surprisingly, we also found that DWORF also activates SERCA’s enzymatic activity directly in the absence of PLB at subsaturating calcium levels. Using site-directed mutagenesis, we generated DWORF variants that do not activate SERCA, thus identifying residues P15 and W22 as necessary for functional SERCA2a–DWORF interactions. This work advances our mechanistic understanding of the regulation of SERCA2a by small transmembrane proteins and sets the stage for future therapeutic development in heart failure research.

Original languageEnglish (US)
Article number100412
JournalJournal of Biological Chemistry
Volume296
DOIs
StatePublished - Feb 14 2021
Externally publishedYes

Bibliographical note

Funding Information:
Funding and additional information—This work was supported in part by NIH grants R01HL139065 (to D. D. T. and R. L. C.; formerly R01GM027906) and R37AG026160 (to D. D. T.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Publisher Copyright:
© 2021 American Society for Biochemistry and Molecular Biology Inc.. All rights reserved.

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