Dwarf open reading frame (DWORF) peptide is a direct activator of the sarcoplasmic reticulum calcium pump SERCA

Abstract

The cardiac sarcoplasmic reticulum calcium pump, SERCA, sequesters calcium in the sarco-endoplasmic reticulum (SR/ER) and plays a critical role in the contraction-relaxation cycle of the heart. A well-known regulator of SERCA in cardiac muscle is phospholamban (PLN), which interacts with the pump and reduces its apparent calcium affinity. A newly discovered SERCA regulatory subunit in cardiac muscle, dwarf open reading frame (DWORF), has added a new level of SERCA regulation. In this report, we modeled the structure of DWORF and evaluated it using molecular dynamics simulations. DWORF structure was modeled as a discontinuous helix with an unwound region at Pro15. This model orients an N-terminal amphipathic helix along the membrane surface and leaves a relatively short C-terminal transmembrane helix. We determined the functional regulation of SERCA by DWORF using a membrane reconstitution system. Surprisingly, we observed that DWORF directly activated SERCA by increasing its turnover rate. Furthermore, in-cell imaging of calcium dynamics demonstrated that DWORF increased SERCA-dependent ER calcium load, calcium reuptake rate, and spontaneous calcium release. Together, these functional assays suggest opposing effects of DWORF and PLN on SERCA function. The results agree with fluorescence resonance energy transfer experiments, which revealed changes in the affinity of DWORF for SERCA at low versus high cytosolic calcium concentrations. We found that DWORF has a higher affinity for SERCA in the presence of calcium, while PLN had the opposite behavior, a higher affinity for SERCA in low calcium. We propose a new mechanism for DWORF regulation of cardiac calcium handling in which DWORF directly enhances SERCA turnover by stabilizing the conformations of SERCA that predominate during elevated cytosolic calcium.

A Kink in DWORF Helical Structure Controls the Activation of the Sarco-plasmic Reticulum Ca2+-ATPase

“…Rather, PLN and DWORF produced an overall weighted functional effect on SERCA. A similar increase in V max was recently reported by Fisher et al ., also using a reconstituted assay system, but an inhibitory effect on pK Ca was not observed and competitive displacement of PLN was not determined (Fisher et al, 2020). These discrepancies are likely arising from variation in reconstitution protocols that affect PLN:SERCA and lipid:protein ratios.…”

SARS-CoV-2 ORF7b: is a bat virus protein homologue a major cause of COVID-19 symptoms?

“…However, transmembrane leucine-zipper interactions seem to be less abundant. They have been identified mainly in signaling and regulatory proteins, as PLN, SLN, MNL, but supposedly also DWarf Open Reading Frame (DWORF) ( 36 ), endoregulin (ENL) ( 37 ), and another-regulin (ALN) ( 37 ). Interestingly, they are also present in membrane glycoproteins, typically in E-cadherin, where they mediate dimer formation of the transmembrane helices.…”

“…Heart conditions related to SARS-CoV-2 are numerous and can arise through a variety of underlying putative mechanisms which have been put forward in the context of COVID-19 ( 39 ); a prominent cause includes diastolic dysfunction, which can occur due to impaired calcium transport. Defects in this activity are associated with cardiomyopathies and heart failure ( 36 ). Calcium traffic through sarco-endoplasmic reticulum Ca 2+ adenosine tri-phosphatase (SERCA) is regulated by PLN, and as well by at least one other small transmembrane protein, SLN, MLN, and DWORF.…”

The small molecule SERCA activator CDN1163 increases energy metabolism in human skeletal muscle cells

“…CDN1163 is a quinoline derivative that has been reported to be a SERCA activating compound ( Cornea et al., 2013 ; Fisher et al., 2020 ; Gruber et al., 2014 ) which directly binds SERCA and upturn the activity at saturating [Ca 2+ ] (V max ) in a concentration-dependent manner ( Gruber et al., 2014 ). The ability of CDN1163 to activate SERCA has been demonstrated in liver endoplasmic reticulum microsomes purified from ob/ob mice ( Kang et al., 2016 ), sarcoplasmic reticulum (SR) membrane vesicles from pig hearts, skeletal muscle SR obtained from New Zealand white rabbits ( Cornea et al., 2013 ), SERCA2 transfected HEK cells ( Gruber et al., 2014 ) and reconstituted vesicles system of SERCA1 from rabbit skeletal muscle ( Fisher et al., 2020 ). Furthermore, pharmacological activation of SERCA2 by CDN1163 has been shown to have a potential therapeutic effect in different animal models of oxidative stress-related diseases.…”