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

doi:10.1016/j.jbc.2021.100412

Ang Li, Samantha L Yuen + Show 4 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100412

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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.

Highlights

Uptake function involving the transmembrane (TM) domain [4]
To study the SERCA2a–dwarf open reading frame (DWORF) interaction in living cells, we developed a fluorescence resonance energy transfer (FRET)-based biosensor system by fusing green fluorescent protein (GFP) and tagRFP to the N termini of SERCA2a and DWORF, respectively
We show that transient transfection of unlabeled DWORF or mutant DWORF into a stable cell line expressing GFP–SERCA2a does not alter the fluorescence lifetime (FLT) of the donor (Fig. S1)

Summary

Introduction

Uptake function involving the transmembrane (TM) domain [4]. Of the 12 known SERCA isoforms expressed in muscle and nonmuscle cells [5], SERCA2a is the principal isoform expressed in cardiac myocytes. We have investigated this mechanism by using fluorescence lifetime (FLT) detection of FRET, to determine quantitatively the interactions of PLB and DWORF (separately and in combination) with SERCA2a and correlate with functional assays of SERCA activity on the same samples [10, 17]. With this approach, we find that DWORF increases the Ca-ATPase. Using site-directed mutagenesis, we identified two DWORF residues, P15 and W22 (in the human isoform), as essential for activation of SERCA2a These findings provide key insights needed to develop effective therapies for heart failure

Results

Discussion

Conclusion