Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.

Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca2+-ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca2+ sensitivity of active SERCA, increasing the Ca2+ concentration required for pump cycling. However, PLB does not decrease Ca2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca2+ binding, altering transport Ca2+ dependence without actually affecting the equilibrium binding affinity of the Ca2+-coordinating sites. Here, we consider another hypothesis, that measurements of Ca2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca2+-binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca2+ affinity of SERCA by interrupting allosteric activation of the pump by ATP.

Micropeptide hetero-oligomerization adds complexity to the calcium pump regulatory network.

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) is an ion transporter that creates and maintains intracellular calcium stores. SERCA is inhibited or stimulated by several membrane micropeptides including another-regulin, dwarf open reading frame, endoregulin, phospholamban (PLB), and sarcolipin. We previously showed that these micropeptides assemble into homo-oligomeric complexes with varying affinity. Here, we tested whether different micropeptides can interact with each other, hypothesizing that coassembly into hetero-oligomers may affect micropeptide bioavailability to regulate SERCA. We quantified the relative binding affinity of each combination of candidates using automated fluorescence resonance energy transfer microscopy. All pairs were capable of interacting with good affinity, similar to the affinity of micropeptide self-binding (homo-oligomerization). Testing each pair at a 1:5 ratio and a reciprocal 5:1 ratio, we noted that the affinity of hetero-oligomerization of some micropeptides depended on whether they were the minority or majority species. In particular, sarcolipin was able to join oligomers when it was the minority species but did not readily accommodate other micropeptides in the reciprocal experiment when it was expressed in fivefold excess. The opposite was observed for endoregulin. PLB was a universal partner for all other micropeptides tested, forming avid hetero-oligomers whether it was the minority or majority species. Increasing expression of SERCA decreased PLB-dwarf open reading frame hetero-oligomerization, suggesting that SERCA-micropeptide interactions compete with micropeptide-micropeptide interactions. Thus, micropeptides populate a regulatory network of diverse protein assemblies. The data suggest that the complexity of this interactome increases exponentially with the number of micropeptides that are coexpressed in a particular tissue.

Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump.

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, the activity of this transporter is tightly controlled via direct interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, the micropeptide phospholamban (PLB) inhibits SERCA, while dwarf open reading frame (DWORF) stimulates SERCA. These competing interactions determine cardiac performance by modulating the amplitude of Ca2+ signals that drive the contraction/relaxation cycle. We hypothesized that the functions of these peptides may relate to their reciprocal preferences for SERCA binding; SERCA binds PLB more avidly at low cytoplasmic [Ca2+] but binds DWORF better when [Ca2+] is high. In the present study, we demonstrated this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca2+ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca2+]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca2+ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. A computational model showed that DWORF exaggerates changes in PLB-SERCA binding during the cardiac cycle. These results suggest a mechanistic basis for inhibitory versus stimulatory micropeptide function, as well as a new role for DWORF as a modulator of dynamic oscillations of PLB-SERCA regulatory interactions.

Reversible optogenetic control of kinase activity during differentiation and embryonic development.

A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of optogenetics in multicellular organisms, however, have not been widely reported. Here, we create an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proof-of-principle study, we develop an optogenetic Raf kinase that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves light-induced cell differentiation compared with co-transfection. When applied to Xenopus embryos, this system enables blue light-dependent reversible Raf activation at any desired developmental stage in specific cell lineages. Our system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings.

Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling.

The sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca 2+ stores. In the heart, SERCA is regulated by an inhibitory interaction with the monomeric form of the transmembrane micropeptide phospholamban (PLB). PLB also forms avid homo-pentamers, and dynamic exchange of PLB between pentamers and the regulatory complex with SERCA is an important determinant of cardiac responsiveness to exercise. Here, we investigated two naturally occurring pathogenic mutations of PLB, a cysteine substitution of arginine 9 (R9C) and an in-frame deletion of arginine 14 (R14del). Both mutations are associated with dilated cardiomyopathy. We previously showed that the R9C mutation causes disulfide crosslinking and hyperstabilization of pentamers. While the pathogenic mechanism of R14del is unclear, we hypothesized that this mutation may also alter PLB homo-oligomerization and disrupt the PLB-SERCA regulatory interaction. SDS-PAGE revealed a significantly increased pentamer:monomer ratio for R14del-PLB when compared to WT-PLB. In addition, we quantified homo-oligomerization and SERCA-binding in live cells using fluorescence resonance energy transfer (FRET) microscopy. R14del-PLB showed an increased affinity for homo-oligomerization and decreased binding affinity for SERCA compared to WT, suggesting that, like R9C, the R14del mutation stabilizes PLB in its pentameric form, decreasing its ability to regulate SERCA. Moreover, the R14del mutation reduces the rate of PLB unbinding from the pentamer after a transient Ca 2+ elevation, limiting the rate of re-binding to SERCA. A computational model predicted that hyperstabilization of PLB pentamers by R14del impairs the ability of cardiac Ca 2+ handling to respond to changing heart rates between rest and exercise. We postulate that impaired responsiveness to physiological stress contributes to arrhythmogenesis in human carriers of the R14del mutation.

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

The sarco-plasmic reticulum calcium pump (SERCA) plays a critical role in the contraction-relaxation cycle of muscle. In cardiac muscle, SERCA is regulated by the inhibitor phospholamban. A new regulator, dwarf open reading frame (DWORF), has been reported to displace phospholamban from SERCA. Here, we show that DWORF is a direct activator of SERCA, increasing its turnover rate in the absence of phospholamban. Measurement of in-cell calcium dynamics supports this observation and demonstrates that DWORF increases SERCA-dependent calcium reuptake. These functional observations reveal opposing effects of DWORF activation and phospholamban inhibition of SERCA. To gain mechanistic insight into SERCA activation, fluorescence resonance energy transfer experiments revealed that DWORF has a higher affinity for SERCA in the presence of calcium. Molecular modeling and molecular dynamics simulations provide a model for DWORF activation of SERCA, where DWORF modulates the membrane bilayer and stabilizes the conformations of SERCA that predominate during elevated cytosolic calcium.

Newly Discovered Micropeptide Regulators of SERCA Form Oligomers but Bind to the Pump as Monomers.

The recently-discovered single-span transmembrane proteins endoregulin (ELN), dwarf open reading frame (DWORF), myoregulin (MLN), and another-regulin (ALN) are reported to bind to the SERCA calcium pump in a manner similar to that of known regulators of SERCA activity, phospholamban (PLB) and sarcolipin (SLN). To determine how micropeptide assembly into oligomers affects the availability of the micropeptide to bind to SERCA in a regulatory complex, we used co-immunoprecipitation and fluorescence resonance energy transfer (FRET) to quantify micropeptide oligomerization and SERCA-binding. Micropeptides formed avid homo-oligomers with high-order stoichiometry (n > 2 protomers per homo-oligomer), but it was the monomeric form of all micropeptides that interacted with SERCA. In view of these two alternative binding interactions, we evaluated the possibility that oligomerization occurs at the expense of SERCA-binding. However, even the most avidly oligomeric micropeptide species still showed robust FRET with SERCA, and there was a surprising positive correlation between oligomerization affinity and SERCA-binding. This comparison of micropeptide family members suggests that the same structural determinants that support oligomerization are also important for binding to SERCA. Moreover, the unique oligomerization/SERCA-binding profile of DWORF is in harmony with its distinct role as a PLB-competing SERCA activator, in contrast to the inhibitory function of the other SERCA-binding micropeptides.

The DWORF micropeptide enhances contractility and prevents heart failure in a mouse model of dilated cardiomyopathy.

Calcium (Ca2+) dysregulation is a hallmark of heart failure and is characterized by impaired Ca2+ sequestration into the sarcoplasmic reticulum (SR) by the SR-Ca2+-ATPase (SERCA). We recently discovered a micropeptide named DWORF (DWarf Open Reading Frame) that enhances SERCA activity by displacing phospholamban (PLN), a potent SERCA inhibitor. Here we show that DWORF has a higher apparent binding affinity for SERCA than PLN and that DWORF overexpression mitigates the contractile dysfunction associated with PLN overexpression, substantiating its role as a potent activator of SERCA. Additionally, using a well-characterized mouse model of dilated cardiomyopathy (DCM) due to genetic deletion of the muscle-specific LIM domain protein (MLP), we show that DWORF overexpression restores cardiac function and prevents the pathological remodeling and Ca2+ dysregulation classically exhibited by MLP knockout mice. Our results establish DWORF as a potent activator of SERCA within the heart and as an attractive candidate for a heart failure therapeutic.