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Phospholamban (PLB) is a transmembrane micropeptide that regulates the sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) in cardiac muscle, but the physical mechanism of this regulation remains poorly understood. PLB reduces the Ca 2+ sensitivity of active SERCA, increasing the Ca 2+ concentration required for pump cycling. However, PLB does not decrease Ca 2+ binding to SERCA when ATP is absent, suggesting PLB does not inhibit SERCA Ca 2+ affinity. The prevailing explanation for these seemingly conflicting results is that PLB slows transitions in the SERCA enzymatic cycle associated with Ca 2+ binding, altering transport Ca 2+ dependence without actually affecting the equilibrium binding affinity of the Ca 2+ -coordinating sites. Here, we consider another hypothesis, that measurements of Ca 2+ binding in the absence of ATP overlook important allosteric effects of nucleotide binding that increase SERCA Ca 2+ binding affinity. We speculated that PLB inhibits SERCA by reversing this allostery. To test this, we used a fluorescent SERCA biosensor to quantify the Ca 2+ affinity of non-cycling SERCA in the presence and absence of a non-hydrolyzable ATP-analog, AMPPCP. Nucleotide activation increased SERCA Ca 2+ affinity, and this effect was reversed by co-expression of PLB. Interestingly, PLB had no effect on Ca 2+ affinity in the absence of nucleotide. These results reconcile the previous conflicting observations from ATPase assays versus Ca 2+ binding assays. Moreover, structural analysis of SERCA revealed a novel allosteric pathway connecting the ATP- and Ca 2+ -binding sites. We propose this pathway is disrupted by PLB binding. Thus, PLB reduces the equilibrium Ca 2+ affinity of SERCA by interrupting allosteric activation of the pump by ATP., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Closed-loop recycling via an efficient chemical process can help alleviate the global plastic waste crisis. However, conventional depolymerization methods for polyolefins, which compose more than 50% of plastics, demand high temperatures and pressures, employ precious noble metals, and/or yield complex mixtures of products limited to single-use fuels or oils. Superacidic forms of sulfated zirconia (SZrO) with Hammet Acidity Functions ( H 0 ) ≤ - 12 (i.e., stronger than 100% H 2 SO 4 ) are industrially deployed heterogeneous catalysts capable of activating hydrocarbons under mild conditions and are shown to decompose polyolefins at temperatures near 200 °C and ambient pressure. Additionally, confinement of active sites in porous supports is known to radically increase selectivity, coking and sintering resistance, and acid site activity, presenting a possible approach to low-energy polyolefin depolymerization. However, a critical examination of the literature on SZrO led us to a surprising conclusion: despite 40 years of catalytic study, engineering, and industrial use, the surface chemistry of SZrO is poorly understood. Ostensibly spurred by SZrO's impressive catalytic activity, the application-driven study of SZrO has resulted in deleterious ambiguity in requisite synthetic conditions for superacidity and insufficient characterization of acidity, porosity, and active site structure. This ambiguity has produced significant knowledge gaps surrounding the synthesis, structure, and mechanisms of hydrocarbon activation for optimized SZrO, stunting the potential of this catalyst in olefin cracking and other industrially relevant reactions, such as isomerization, esterification, and alkylation. Toward mitigating these long extant issues, we herein identify and highlight these current shortcomings and knowledge gaps, propose explicit guidelines for characterization of and reporting on characterization of solid acidity, and discuss the potential of pore-confined superacids in the efficient and selective depolymerization of polyolefins.

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The ATP-dependent ion pump sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) sequesters Ca 2+ 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 Ca 2+ 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 [Ca 2+ ] but binds DWORF better when [Ca 2+ ] is high. In the present study, we demonstrated this opposing Ca 2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate states that SERCA samples during the Ca 2+ transport cycle. We show PLB binds best to the SERCA E1-ATP state, which prevails at low [Ca 2+ ]. In contrast, DWORF binds most avidly to E1P and E2P states that are more populated when Ca 2+ is elevated. Moreover, FRET microscopy revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca 2+ 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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The sodium-potassium ATPase (Na/K-ATPase, NKA) establishes ion gradients that facilitate many physiological functions including action potentials and secondary transport processes. NKA comprises a catalytic subunit (alpha) that interacts closely with an essential subunit (beta) and regulatory transmembrane micropeptides called FXYD proteins. In the heart, a key modulatory partner is the FXYD protein phospholemman (PLM, FXYD1), but the stoichiometry of the alpha-beta-PLM regulatory complex is unknown. Here, we used fluorescence lifetime imaging and spectroscopy to investigate the structure, stoichiometry, and affinity of the NKA-regulatory complex. We observed a concentration-dependent binding of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit as well as lower affinity alpha-alpha and alpha-PLM interactions. These data provide the first evidence that, in intact live cells, the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits. Docking and molecular dynamics (MD) simulations generated a structural model of the complex that is consistent with our experimental observations. We propose that alpha-alpha subunit interactions support conformational coupling of the catalytic subunits, which may enhance NKA turnover rate. These observations provide insight into the pathophysiology of heart failure, wherein low NKA expression may be insufficient to support formation of the complete regulatory complex with the stoichiometry (alpha-beta-PLM) 2 ., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The type 2a sarco/endoplasmic reticulum (ER) Ca 2+ -ATPase (SERCA2a) plays a key role in intracellular Ca 2+ regulation in the heart. We have previously shown evidence of stable homodimers of SERCA2a in heterologous cells and cardiomyocytes. However, the functional significance of the pump dimerization remains unclear. Here, we analyzed how SERCA2a dimerization affects ER Ca 2+ transport. Fluorescence resonance energy transfer experiments in HEK293 cells transfected with fluorescently labeled SERCA2a revealed increasing dimerization of Ca 2+ pumps with increasing expression level. This concentration-dependent dimerization provided means of comparison of the functional characteristics of monomeric and dimeric pumps. SERCA-mediated Ca 2+ uptake was measured with the ER-targeted Ca 2+ sensor R-CEPIA1er in cells cotransfected with SERCA2a and ryanodine receptor. For each individual cell, the maximal ER Ca 2+ uptake rate and the maximal Ca 2+ load, together with the pump expression level, were analyzed. This analysis revealed that the ER Ca 2+ uptake rate increased as a function of SERCA2a expression, with a particularly steep, nonlinear increase at high expression levels. Interestingly, the maximal ER Ca 2+ load also increased with an increase in the pump expression level, suggesting improved catalytic efficiency of the dimeric species. Reciprocally, thapsigargin inhibition of a fraction of the population of SERCA2a reduced not only the maximal ER Ca 2+ uptake rate but also the maximal Ca 2+ load. These data suggest that SERCA2a dimerization regulates Ca 2+ transport by improving both the SERCA2a turnover rate and catalytic efficacy. Analysis of ER Ca 2+ uptake in cells cotransfected with human wild-type SERCA2a (SERCA2a WT ) and SERCA2a mutants with different catalytic activity revealed that an intact catalytic cycle in both protomers is required for enhancing the efficacy of Ca 2+ transport by a dimer. The data are consistent with the hypothesis of functional coupling of two SERCA2a protomers in a dimer that reduces the energy barrier of rate-limiting steps of the catalytic cycle of Ca 2+ transport., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Whistleblowing by health professionals is an infrequent and extraordinary event and need not occur if internal voices are heard. Mannion and Davies' editorial on "Cultures of Silence and Cultures of Voice: The Role of Whistleblowing in Healthcare Organisations" asks the question whether whistleblowing ameliorates or exacerbates the 'deaf effect' prevalent in healthcare organisations. This commentary argues that the focus should remain on internal processes and hearer courage ., (© 2016 by Kerman University of Medical Sciences.)