65 results on '"Robia SL"'
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2. Pathological mutations in the phospholamban cytoplasmic region affect its topology and dynamics modulating the extent of SERCA inhibition.
- Author
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Weber DK, Reddy UV, Robia SL, and Veglia G
- Subjects
- Humans, Phosphorylation genetics, Mutation genetics, Calcium metabolism, Cytoplasm metabolism, Cytoplasm genetics, Animals, Binding Sites genetics, Protein Binding, Calcium-Binding Proteins genetics, Calcium-Binding Proteins chemistry, Calcium-Binding Proteins metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics
- Abstract
Phospholamban (PLN) is a 52 amino acid regulin that allosterically modulates the activity of the sarco(endo)plasmic reticulum Ca
2+ -ATPase (SERCA) in the heart muscle. In its unphosphorylated form, PLN binds SERCA within its transmembrane (TM) domains, approximately 20 Å away from the Ca2+ binding site, reducing SERCA's apparent Ca2+ affinity (pKCa ) and decreasing cardiac contractility. During the enzymatic cycle, the inhibitory TM domain of PLN remains anchored to SERCA, whereas its cytoplasmic region transiently binds the ATPase's headpiece. Phosphorylation of PLN at Ser16 by protein kinase A increases the affinity of its cytoplasmic domain to SERCA, weakening the TM interactions with the ATPase, reversing its inhibitory function, and augmenting muscle contractility. How the structural changes caused by pathological mutations in the PLN cytoplasmic region are transmitted to its inhibitory TM domain is still unclear. Using solid-state NMR spectroscopy and activity assays, we analyzed the structural and functional effects of a series of mutations and their phosphorylated forms located in the PLN cytoplasmic region and linked to dilated cardiomyopathy. We found that these missense mutations affect the overall topology and dynamics of PLN and ultimately modulate its inhibitory potency. Also, the changes in the TM tilt angle and cytoplasmic dynamics of PLN caused by these mutations correlate well with the extent of SERCA inhibition. Our study unveils new molecular determinants for designing variants of PLN that outcompete endogenous PLN to regulate SERCA in a tunable manner., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial funding, personal interests or relationships that may appear to influence the work reported in this article., (Copyright © 2024 Elsevier B.V. All rights reserved.)- Published
- 2024
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3. Protein carbonylation causes sarcoplasmic reticulum Ca 2+ overload by increasing intracellular Na + level in ventricular myocytes.
- Author
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Bovo E, Seflova J, Robia SL, and Zima AV
- Subjects
- Animals, Mice, Sodium-Calcium Exchanger metabolism, Heart Ventricles metabolism, Heart Ventricles cytology, Pyruvaldehyde pharmacology, Pyruvaldehyde metabolism, Calcium Signaling physiology, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Action Potentials drug effects, Mice, Inbred C57BL, Cells, Cultured, Male, Myocytes, Cardiac metabolism, Myocytes, Cardiac drug effects, Sarcoplasmic Reticulum metabolism, Sarcoplasmic Reticulum drug effects, Calcium metabolism, Sodium metabolism, Protein Carbonylation drug effects
- Abstract
Diabetes is commonly associated with an elevated level of reactive carbonyl species due to alteration of glucose and fatty acid metabolism. These metabolic changes cause an abnormality in cardiac Ca
2+ regulation that can lead to cardiomyopathies. In this study, we explored how the reactive α-dicarbonyl methylglyoxal (MGO) affects Ca2+ regulation in mouse ventricular myocytes. Analysis of intracellular Ca2+ dynamics revealed that MGO (200 μM) increases action potential (AP)-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load, with a limited effect on L-type Ca2+ channel-mediated Ca2+ transients and SERCA-mediated Ca2+ uptake. At the same time, MGO significantly slowed down cytosolic Ca2+ extrusion by Na+ /Ca2+ exchanger (NCX). MGO also increased the frequency of Ca2+ waves during rest and these Ca2+ release events were abolished by an external solution with zero [Na+ ] and [Ca2+ ]. Adrenergic receptor activation with isoproterenol (10 nM) increased Ca2+ transients and SR Ca2+ load, but it also triggered spontaneous Ca2+ waves in 27% of studied cells. Pretreatment of myocytes with MGO increased the fraction of cells with Ca2+ waves during adrenergic receptor stimulation by 163%. Measurements of intracellular [Na+ ] revealed that MGO increases cytosolic [Na+ ] by 57% from the maximal effect produced by the Na+ -K+ ATPase inhibitor ouabain (20 μM). This increase in cytosolic [Na+ ] was a result of activation of a tetrodotoxin-sensitive Na+ influx, but not an inhibition of Na+ -K+ ATPase. An increase in cytosolic [Na+ ] after treating cells with ouabain produced similar effects on Ca2+ regulation as MGO. These results suggest that protein carbonylation can affect cardiac Ca2+ regulation by increasing cytosolic [Na+ ] via a tetrodotoxin-sensitive pathway. This, in turn, reduces Ca2+ extrusion by NCX, causing SR Ca2+ overload and spontaneous Ca2+ waves., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)- Published
- 2024
- Full Text
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4. Phosphorylation of phospholamban promotes SERCA2a activation by dwarf open reading frame (DWORF).
- Author
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Bovo E, Jamrozik T, Kahn D, Karkut P, Robia SL, and Zima AV
- Subjects
- Humans, Phosphorylation, HEK293 Cells, Open Reading Frames genetics, Calcium metabolism, Enzyme Activation, Calcium-Calmodulin-Dependent Protein Kinase Type 2 metabolism, Cyclic AMP-Dependent Protein Kinases metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Calcium-Binding Proteins metabolism
- Abstract
In cardiac myocytes, the type 2a sarco/endoplasmic reticulum Ca-ATPase (SERCA2a) plays a key role in intracellular Ca regulation. Due to its critical role in heart function, SERCA2a activity is tightly regulated by different mechanisms, including micropeptides. While phospholamban (PLB) is a well-known SERCA2a inhibitor, dwarf open reading frame (DWORF) is a recently identified SERCA2a activator. Since PLB phosphorylation is the most recognized mechanism of SERCA2a activation during adrenergic stress, we studied whether PLB phosphorylation also affects SERCA2a regulation by DWORF. By using confocal Ca imaging in a HEK293 expressing cell system, we analyzed the effect of the co-expression of PLB and DWORF using a bicistronic construct on SERCA2a-mediated Ca uptake. Under these conditions of matched expression of PLB and DWORF, we found that SERCA2a inhibition by non-phosphorylated PLB prevails over DWORF activating effect. However, when PLB is phosphorylated at PKA and CaMKII sites, not only PLB's inhibitory effect was relieved, but SERCA2a was effectively activated by DWORF. Förster resonance energy transfer (FRET) analysis between SERCA2a and DWORF showed that DWORF has a higher relative affinity for SERCA2a when PLB is phosphorylated. Thus, SERCA2a regulation by DWORF responds to the PLB phosphorylation status, suggesting that DWORF might contribute to SERCA2a activation during conditions of adrenergic stress., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
5. Phospholamban inhibits the cardiac calcium pump by interrupting an allosteric activation pathway.
- Author
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Cleary SR, Seflova J, Cho EE, Bisht K, Khandelia H, Espinoza-Fonseca LM, and Robia SL
- Subjects
- Animals, Humans, Adenosine Triphosphate metabolism, Allosteric Regulation, Myocardium metabolism, Dogs, HEK293 Cells, Models, Molecular, Protein Structure, Tertiary, Calcium metabolism, Calcium-Binding Proteins metabolism, Calcium-Binding Proteins chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry
- Abstract
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 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., 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.)- Published
- 2024
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6. Protein carbonylation causes sarcoplasmic reticulum Ca2+ overload by increasing intracellular Na+ level in ventricular myocytes.
- Author
-
Bovo E, Seflova J, Robia SL, and Zima AV
- Abstract
Diabetes is commonly associated with an elevated level of reactive carbonyl species due to alteration of glucose and fatty acid metabolism. These metabolic changes cause an abnormality in cardiac Ca
2+ regulation that can lead to cardiomyopathies. In this study, we explored how the reactive α-dicarbonyl methylglyoxal (MGO) affects Ca2+ regulation in mouse ventricular myocytes. Analysis of intracellular Ca2+ dynamics revealed that MGO (200 μM) increases action potential (AP)-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load, with a limited effect on L-type Ca2+ channel-mediated Ca2+ transients and SERCA-mediated Ca2+ uptake. At the same time, MGO significantly slowed down cytosolic Ca2+ extrusion by Na+ /Ca2+ exchanger (NCX). MGO also increased the frequency of Ca2+ waves during rest and these Ca2+ release events were abolished by an external solution with zero [Na+ ] and [Ca2+ ]. Adrenergic receptor activation with isoproterenol (10 nM) increased Ca2+ transients and SR Ca2+ load, but it also triggered spontaneous Ca2+ waves in 27% of studied cells. Pretreatment of myocytes with MGO increased the fraction of cells with Ca2+ waves during adrenergic receptor stimulation by 163%. Measurements of intracellular [Na+ ] revealed that MGO increases cytosolic [Na+ ] by 57% from the maximal effect produced by the Na+ -K+ ATPase inhibitor ouabain (20 μM). This increase in cytosolic [Na+ ] was a result of activation of a tetrodotoxin-sensitive Na+ influx, but not an inhibition of Na+ -K+ ATPase. An increase in cytosolic [Na+ ] after treating cells with ouabain produced similar effects on Ca2+ regulation as MGO. These results suggest that protein carbonylation can affect cardiac Ca2+ regulation by increasing cytosolic [Na+ ] via a tetrodotoxin-sensitive pathway. This, in turn, reduces Ca2+ extrusion by NCX, causing SR Ca2+ overload and spontaneous Ca2+ waves., Competing Interests: Additional Declarations: No competing interests reported.- Published
- 2024
- Full Text
- View/download PDF
7. Mechanisms for cardiac calcium pump activation by its substrate and a synthetic allosteric modulator using fluorescence lifetime imaging.
- Author
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Šeflová J, Cruz-Cortés C, Guerrero-Serna G, Robia SL, and Espinoza-Fonseca LM
- Abstract
The discovery of allosteric modulators is an emerging paradigm in drug discovery, and signal transduction is a subtle and dynamic process that is challenging to characterize. We developed a time-correlated single photon-counting imaging approach to investigate the structural mechanisms for small-molecule activation of the cardiac sarcoplasmic reticulum Ca
2+ -ATPase, a pharmacologically important pump that transports Ca2+ at the expense of adenosine triphosphate (ATP) hydrolysis. We first tested whether the dissociation of sarcoplasmic reticulum Ca2+ -ATPase from its regulatory protein phospholamban is required for small-molecule activation. We found that CDN1163, a validated sarcoplasmic reticulum Ca2+ -ATPase activator, does not have significant effects on the stability of the sarcoplasmic reticulum Ca2+ -ATPase-phospholamban complex. Time-correlated single photon-counting imaging experiments using the nonhydrolyzable ATP analog β,γ-Methyleneadenosine 5'-triphosphate (AMP-PCP) showed ATP is an allosteric modulator of sarcoplasmic reticulum Ca2+ -ATPase, increasing the fraction of catalytically competent structures at physiologically relevant Ca2+ concentrations. Unlike ATP, CDN1163 alone has no significant effects on the Ca2+ -dependent shifts in the structural populations of sarcoplasmic reticulum Ca2+ -ATPase, and it does not increase the pump's affinity for Ca2+ ions. However, we found that CDN1163 enhances the ATP-mediated modulatory effects to increase the population of catalytically competent sarcoplasmic reticulum Ca2+ -ATPase structures. Importantly, this structural shift occurs within the physiological window of Ca2+ concentrations at which sarcoplasmic reticulum Ca2+ -ATPase operates. We demonstrated that ATP is both a substrate and modulator of sarcoplasmic reticulum Ca2+ -ATPase and showed that CDN1163 and ATP act synergistically to populate sarcoplasmic reticulum Ca2+ -ATPase structures that are primed for phosphorylation. This study provides novel insights into the structural mechanisms for sarcoplasmic reticulum Ca2+ -ATPase activation by its substrate and a synthetic allosteric modulator., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)- Published
- 2023
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8. RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET.
- Author
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Šeflová J, Schwarz JA, Smith AN, Svensson B, Blackwell DJ, Phillips TA, Nikolaienko R, Bovo E, Rebbeck RT, Zima AV, Thomas DD, Van Petegem F, Knollmann BC, Johnston JN, Robia SL, and Cornea RL
- Subjects
- Mice, Swine, Humans, Animals, Ryanodine chemistry, Ryanodine metabolism, Ryanodine therapeutic use, Fluorescence Resonance Energy Transfer methods, HEK293 Cells, Arrhythmias, Cardiac drug therapy, Arrhythmias, Cardiac metabolism, Calcium metabolism, Myocytes, Cardiac metabolism, Ryanodine Receptor Calcium Release Channel chemistry, Ryanodine Receptor Calcium Release Channel metabolism, Depsipeptides metabolism
- Abstract
Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca
2+ -release channels contributes to heart failure and arrhythmias. Reducing the RyR2 activity, particularly during cardiac relaxation (diastole), is a desirable therapeutic goal. We previously reported that the unnatural enantiomer ( ent ) of an insect-RyR activator, verticilide, inhibits porcine and mouse RyR2 at diastolic (nanomolar) Ca2+ and has in vivo efficacy against atrial and ventricular arrhythmia. To determine the ent -verticilide structural mode of action on RyR2 and guide its further development via medicinal chemistry structure-activity relationship studies, here, we used fluorescence lifetime (FLT)-measurements of Förster resonance energy transfer (FRET) in HEK293 cells expressing human RyR2. For these studies, we used an RyR-specific FRET molecular-toolkit and computational methods for trilateration (i.e., using distances to locate a point of interest). Multiexponential analysis of FLT-FRET measurements between four donor-labeled FKBP12.6 variants and acceptor-labeled ent -verticilide yielded distance relationships placing the acceptor probe at two candidate loci within the RyR2 cryo-EM map. One locus is within the Ry12 domain (at the corner periphery of the RyR2 tetrameric complex). The other locus is sandwiched at the interface between helical domain 1 and the SPRY3 domain. These findings document RyR2-target engagement by ent -verticilide, reveal new insight into the mechanism of action of this new class of RyR2-targeting drug candidate, and can serve as input in future computational determinations of the ent -verticilide binding site on RyR2 that will inform structure-activity studies for lead optimization.- Published
- 2023
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9. Dilated cardiomyopathy variant R14del increases phospholamban pentamer stability, blunting dynamic regulation of cardiac calcium handling.
- Author
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Cleary SR, Teng ACT, Kongmeneck AD, Fang X, Phillips TA, Cho EE, Kekenes-Huskey P, Gramolini AO, and Robia SL
- Abstract
The sarco(endo)plasmic reticulum Ca
2+ ATPase (SERCA) is a membrane transporter that creates and maintains intracellular Ca2+ 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 Ca2+ 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 Ca2+ 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.- Published
- 2023
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10. Micropeptide hetero-oligomerization adds complexity to the calcium pump regulatory network.
- Author
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Phillips TA, Hauck GT, Pribadi MP, Cho EE, Cleary SR, and Robia SL
- Subjects
- Ion Transport, Calcium-Binding Proteins chemistry, Micropeptides, Calcium metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
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.)
- Published
- 2023
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11. Regulation of the regulator: Endopeptidase cleavage controls the expression of a micropeptide that regulates SERCA.
- Author
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Phillips TA and Robia SL
- Subjects
- Muscle Proteins chemistry, Muscle Proteins metabolism, Calcium-Binding Proteins metabolism, Neprilysin metabolism, Endopeptidases metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Calcium metabolism
- Abstract
Micropeptides regulate cellular calcium handling by modulating the function of the calcium transporter SERCA. In a recent Nature Communications paper [4] authors Schiemann et al. describe regulation of an invertebrate SERCA-active micropeptide, sarcolamban, by an endopeptidase called neprilysin 4 (NEP4). NEP4 activity limits sarcolamban expression by cleavage of luminal residues near the micropeptide's c-terminus. This cleavage event liberates sarcolamban from the membrane, reduces its oligomerization, and prevents it from inhibiting SERCA. The study reveals a novel mechanism for "regulation of the regulator" that may be a general feature of micropeptide/SERCA physiology., (Copyright © 2022 Elsevier Ltd. All rights reserved.)
- Published
- 2022
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12. Primitive Phospholamban- and Sarcolipin-like Peptides Inhibit the Sarcoplasmic Reticulum Calcium Pump SERCA.
- Author
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Bak JJ, Aguayo-Ortiz R, Rathod N, Primeau JO, Khan MB, Robia SL, Lemieux MJ, Espinoza-Fonseca LM, and Young HS
- Subjects
- Animals, Calcium Signaling, Calcium-Binding Proteins chemistry, Humans, Mammals metabolism, Molecular Dynamics Simulation, Muscle Proteins, Peptides metabolism, Peptides pharmacology, Proteolipids chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry, Calcium metabolism, Sarcoplasmic Reticulum metabolism
- Abstract
Intracellular calcium signaling is essential for all kingdoms of life. An important part of this process is the sarco-endoplasmic reticulum Ca
2+ -ATPase (SERCA), which maintains the low cytosolic calcium levels required for intracellular calcium homeostasis. In higher organisms, SERCA is regulated by a series of tissue-specific transmembrane subunits such as phospholamban in cardiac muscles and sarcolipin in skeletal muscles. These regulatory axes are so important for muscle contractility that SERCA, phospholamban, and sarcolipin are practically invariant across mammalian species. With the recent discovery of the arthropod sarcolambans, the family of calcium pump regulatory subunits appears to span more than 550 million years of evolutionary divergence from arthropods to humans. This evolutionary divergence is reflected in the peptide sequences, which vary enormously from one another and only vaguely resemble phospholamban and sarcolipin. The discovery of the sarcolambans allowed us to address two questions. How much sequence variation is tolerated in the regulation of mammalian SERCA activity by the transmembrane peptides? Do divergent peptide sequences mimic phospholamban or sarcolipin in their regulatory activities despite limited sequence similarity? We expressed and purified recombinant sarcolamban peptides from three different arthropods. The peptides were coreconstituted into proteoliposomes with mammalian SERCA1a and the effect of each peptide on the apparent calcium affinity and maximal activity of SERCA was measured. All three peptides were superinhibitors of SERCA, exhibiting either phospholamban-like or sarcolipin-like characteristics. Molecular modeling, protein-protein docking, and molecular dynamics simulations revealed novel features of the divergent peptides and their SERCA regulatory properties.- Published
- 2022
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13. Inhibitory and stimulatory micropeptides preferentially bind to different conformations of the cardiac calcium pump.
- Author
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Cleary SR, Fang X, Cho EE, Pribadi MP, Seflova J, Beach JR, Kekenes-Huskey PM, and Robia SL
- Subjects
- Adenosine Triphosphate metabolism, Humans, Ion Transport, Peptides metabolism, Protein Binding, Calcium metabolism, Calcium-Binding Proteins metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
The ATP-dependent ion pump sarco/endoplasmic reticulum Ca
2+ -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., 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.)- Published
- 2022
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14. Fluorescence lifetime imaging microscopy reveals sodium pump dimers in live cells.
- Author
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Seflova J, Habibi NR, Yap JQ, Cleary SR, Fang X, Kekenes-Huskey PM, Espinoza-Fonseca LM, Bossuyt JB, and Robia SL
- Subjects
- Cell Membrane metabolism, Phosphoproteins metabolism, Phosphorylation, Sodium metabolism, Microscopy, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
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.)- Published
- 2022
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15. The Ile191Val is a partial loss-of-function variant of the TAS1R2 sweet-taste receptor and is associated with reduced glucose excursions in humans.
- Author
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Serrano J, Seflova J, Park J, Pribadi M, Sanematsu K, Shigemura N, Serna V, Yi F, Mari A, Procko E, Pratley RE, Robia SL, and Kyriazis GA
- Subjects
- Adult, Female, HEK293 Cells, Humans, Male, Glucose metabolism, Receptors, G-Protein-Coupled genetics, Receptors, G-Protein-Coupled metabolism, Taste genetics
- Abstract
Objective: Sweet taste receptors (STR) are expressed in the gut and other extra-oral tissues, suggesting that STR-mediated nutrient sensing may contribute to human physiology beyond taste. A common variant (Ile191Val) in the TAS1R2 gene of STR is associated with nutritional and metabolic outcomes independent of changes in taste perception. It is unclear whether this polymorphism directly alters STR function and how it may contribute to metabolic regulation., Methods: We implemented a combination of in vitro biochemical approaches to decipher the effects of TAS1R2 polymorphism on STR function. Then, as proof-of-concept, we assessed its effects on glucose homeostasis in apparently healthy lean participants., Results: The Ile191Val variant causes a partial loss of function of TAS1R2 through reduced receptor availability in the plasma membrane. Val minor allele carriers have reduced glucose excursions during an OGTT, mirroring effects previously seen in mice with genetic loss of function of TAS1R2. These effects were not due to differences in beta-cell function or insulin sensitivity., Conclusions: Our pilot studies on a common TAS1R2 polymorphism suggest that STR sensory function in peripheral tissues, such as the intestine, may contribute to the regulation of metabolic control in humans., (Copyright © 2021 The Author(s). Published by Elsevier GmbH.. All rights reserved.)
- Published
- 2021
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16. Presenilin 1 is a direct regulator of the cardiac sarco/endoplasmic reticulum calcium pump.
- Author
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Bovo E, Nikolaienko R, Kahn D, Cho E, Robia SL, and Zima AV
- Subjects
- HEK293 Cells, Humans, Presenilin-1 genetics, Presenilin-1 metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Calcium metabolism, Endoplasmic Reticulum metabolism
- Abstract
The gamma secretase catalytic subunit presenilin 1 (PS1) is expressed in the endoplasmic reticulum (ER) of neurons, where it regulates Ca
2+ signaling. PS1 is also expressed in heart, but its role in regulation of cardiac Ca2+ transport remains unknown. Since the type 2 sarco/endoplasmic reticulum Ca2+ ATPase (SERCA2a) plays a central role in cardiac Ca2+ homeostasis, we studied whether PS1 regulates the cardiac SERCA2a function. The experiments were conducted in an inducible human SERCA2a stable T-Rex-293 cell line transfected with fluorescently labeled PS1 and the ER Ca2+ sensor R-CEPIA1er. Confocal imaging showed that that PS1 is localized predominantly in the ER membrane. Fluorescent resonance energy transfer (FRET) experiments in HEK293 cells transfected with fluorescently labeled SERCA2a and PS1 revealed that the two proteins directly interact with a 1:1 stoichiometry. The functional significance of this interaction was investigated in a heterologous cellular environment using a novel approach to directly measure ER Ca2+ dynamics. Measurements of SERCA2a-mediated Ca2+ transport showed that PS1 enhanced Ca2+ uptake at low ER Ca2+ loads (<0.15 mM) and reduced uptake at high loads (>0.35 mM). The results of this study revealed that PS1 could act as an important regulator of the cardiac Ca2+ pump function with a complex stimulatory/inhibitory profile., (Published by Elsevier Ltd.)- Published
- 2021
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17. Dwarf open reading frame (DWORF) is a direct activator of the sarcoplasmic reticulum calcium pump SERCA.
- Author
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Fisher ME, Bovo E, Aguayo-Ortiz R, Cho EE, Pribadi MP, Dalton MP, Rathod N, Lemieux MJ, Espinoza-Fonseca LM, Robia SL, Zima AV, and Young HS
- Subjects
- Calcium-Binding Proteins metabolism, Enzyme Activation, HEK293 Cells, Humans, Molecular Dynamics Simulation, Peptides chemistry, Peptides genetics, Protein Conformation, Sarcoplasmic Reticulum genetics, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics, Structure-Activity Relationship, Time Factors, Calcium metabolism, Calcium Signaling, Peptides metabolism, Sarcoplasmic Reticulum enzymology, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
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., Competing Interests: MF, EB, RA, EC, MP, MD, NR, LE, SR, AZ, HY No competing interests declared, ML Reviewing editor, eLife, (© 2021, Fisher et al.)
- Published
- 2021
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18. FXYD proteins and sodium pump regulatory mechanisms.
- Author
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Yap JQ, Seflova J, Sweazey R, Artigas P, and Robia SL
- Subjects
- Cell Membrane metabolism, Humans, Ion Transport, Potassium metabolism, Sodium metabolism, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
The sodium/potassium-ATPase (NKA) is the enzyme that establishes gradients of sodium and potassium across the plasma membrane. NKA activity is tightly regulated for different physiological contexts through interactions with single-span transmembrane peptides, the FXYD proteins. This diverse family of regulators has in common a domain containing a Phe-X-Tyr-Asp (FXYD) motif, two conserved glycines, and one serine residue. In humans, there are seven tissue-specific FXYD proteins that differentially modulate NKA kinetics as appropriate for each system, providing dynamic responsiveness to changing physiological conditions. Our understanding of how FXYD proteins contribute to homeostasis has benefitted from recent advances described in this review: biochemical and biophysical studies have provided insight into regulatory mechanisms, genetic models have uncovered remarkable complexity of FXYD function in integrated physiological systems, new posttranslational modifications have been identified, high-resolution structural studies have revealed new details of the regulatory interaction with NKA, and new clinical correlations have been uncovered. In this review, we address the structural determinants of diverse FXYD functions and the special roles of FXYDs in various physiological systems. We also discuss the possible roles of FXYDs in protein trafficking and regulation of non-NKA targets., (© 2021 Yap et al.)
- Published
- 2021
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19. Dynamics-Driven Allostery Underlies Ca 2+ -Mediated Release of SERCA Inhibition by Phospholamban.
- Author
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Raguimova ON, Aguayo-Ortiz R, Robia SL, and Espinoza-Fonseca LM
- Subjects
- Calcium metabolism, Phosphorylation, Protein Binding, Sarcoplasmic Reticulum metabolism, Calcium-Binding Proteins metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
Sarcoplasmic reticulum (SR) Ca
2+ -ATPase (SERCA) and phospholamban (PLB) are essential for intracellular Ca2+ transport in myocytes. Ca2+ -dependent activation of SERCA-PLB provides a control function that regulates cytosolic and SR Ca2+ levels. Although experimental and computational studies alone have led to a greater insight into SERCA-PLB regulation, the structural mechanisms for Ca2+ binding reversing inhibition of the complex remain poorly understood. Therefore, we have performed atomistic simulations totaling 32.7 μs and cell-based intramolecular fluorescence resonance energy transfer (FRET) experiments to determine structural changes of PLB-bound SERCA in response to binding of a single Ca2+ ion. Complementary MD simulations and FRET experiments showed that open-to-closed transitions in the structure of the headpiece underlie PLB inhibition of SERCA, and binding of a single Ca2+ ion is sufficient to shift the protein population toward a structurally closed structure of the complex. Closure is accompanied by functional interactions between the N-domain β5-β6 loop and the A-domain and the displacement of the catalytic phosphorylation domain toward a competent structure. We propose that reversal of SERCA-PLB inhibition is achieved by stringing together its controlling modules (A-domain and loop Nβ5-β6) with catalytic elements (P-domain) to regulate function during intracellular Ca2+ signaling. We conclude that binding of a single Ca2+ is a critical mediator of allosteric signaling that dictates structural changes and motions that relieve SERCA inhibition by PLB. Understanding allosteric regulation is of paramount importance to guide therapeutic modulation of SERCA and other evolutionarily related ion-motive ATPases., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)- Published
- 2020
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20. Dimerization of SERCA2a Enhances Transport Rate and Improves Energetic Efficiency in Living Cells.
- Author
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Bovo E, Nikolaienko R, Cleary SR, Seflova J, Kahn D, Robia SL, and Zima AV
- Subjects
- Dimerization, HEK293 Cells, Humans, Ryanodine Receptor Calcium Release Channel metabolism, Sarcoplasmic Reticulum metabolism, Calcium metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
The type 2a sarco/endoplasmic reticulum (ER) Ca
2+ -ATPase (SERCA2a) plays a key role in intracellular Ca2+ 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 Ca2+ transport. Fluorescence resonance energy transfer experiments in HEK293 cells transfected with fluorescently labeled SERCA2a revealed increasing dimerization of Ca2+ pumps with increasing expression level. This concentration-dependent dimerization provided means of comparison of the functional characteristics of monomeric and dimeric pumps. SERCA-mediated Ca2+ uptake was measured with the ER-targeted Ca2+ sensor R-CEPIA1er in cells cotransfected with SERCA2a and ryanodine receptor. For each individual cell, the maximal ER Ca2+ uptake rate and the maximal Ca2+ load, together with the pump expression level, were analyzed. This analysis revealed that the ER Ca2+ uptake rate increased as a function of SERCA2a expression, with a particularly steep, nonlinear increase at high expression levels. Interestingly, the maximal ER Ca2+ 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 Ca2+ uptake rate but also the maximal Ca2+ load. These data suggest that SERCA2a dimerization regulates Ca2+ transport by improving both the SERCA2a turnover rate and catalytic efficacy. Analysis of ER Ca2+ uptake in cells cotransfected with human wild-type SERCA2a (SERCA2aWT ) and SERCA2a mutants with different catalytic activity revealed that an intact catalytic cycle in both protomers is required for enhancing the efficacy of Ca2+ 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 Ca2+ transport., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)- Published
- 2020
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21. Protein docking and steered molecular dynamics suggest alternative phospholamban-binding sites on the SERCA calcium transporter.
- Author
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Alford RF, Smolin N, Young HS, Gray JJ, and Robia SL
- Subjects
- Animals, Binding Sites, Humans, Molecular Dynamics Simulation, Calcium-Binding Proteins metabolism, Proteins metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
The transport activity of the sarco(endo)plasmic reticulum calcium ATPase (SERCA) in cardiac myocytes is modulated by an inhibitory interaction with a transmembrane peptide, phospholamban (PLB). Previous biochemical studies have revealed that PLB interacts with a specific inhibitory site on SERCA, and low-resolution structural evidence suggests that PLB interacts with distinct alternative sites on SERCA. High-resolution details of the structural determinants of SERCA regulation have been elusive because of the dynamic nature of the regulatory complex. In this study, we used computational approaches to develop a structural model of SERCA-PLB interactions to gain a mechanistic understanding of PLB-mediated SERCA transport regulation. We combined steered molecular dynamics and membrane protein-protein docking experiments to achieve both a global search and all-atom force calculations to determine the relative affinities of PLB for candidate sites on SERCA. We modeled the binding of PLB to several SERCA conformations, representing different enzymatic states sampled during the calcium transport catalytic cycle. The results of the steered molecular dynamics and docking experiments indicated that the canonical PLB-binding site (comprising transmembrane helices M2, M4, and M9) is the preferred site. This preference was even more stringent for a superinhibitory PLB variant. Interestingly, PLB-binding specificity became more ambivalent for other SERCA conformers. These results provide evidence for polymorphic PLB interactions with novel sites on M3 and with the outside of the SERCA helix M9. Our findings are compatible with previous physical measurements that suggest that PLB interacts with multiple binding sites, conferring dynamic responsiveness to changing physiological conditions., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Alford et al.)
- Published
- 2020
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22. Intrinsically disordered HAX-1 regulates Ca 2+ cycling by interacting with lipid membranes and the phospholamban cytoplasmic region.
- Author
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Larsen EK, Weber DK, Wang S, Gopinath T, Blackwell DJ, Dalton MP, Robia SL, Gao J, and Veglia G
- Subjects
- Adaptor Proteins, Signal Transducing chemistry, Animals, Calcium-Binding Proteins ultrastructure, Humans, Intrinsically Disordered Proteins, Magnetic Resonance Spectroscopy, Protein Structure, Secondary, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Adaptor Proteins, Signal Transducing physiology, Calcium metabolism, Calcium-Binding Proteins metabolism, Cytoplasm metabolism, Membrane Lipids metabolism, Myocardium metabolism
- Abstract
Hematopoietic-substrate-1 associated protein X-1 (HAX-1) is a 279 amino acid protein expressed ubiquitously. In cardiac muscle, HAX-1 was found to modulate the sarcoendoplasmic reticulum calcium ATPase (SERCA) by shifting its apparent Ca
2+ affinity (pCa). It has been hypothesized that HAX-1 binds phospholamban (PLN), enhancing its inhibitory function on SERCA. HAX-1 effects are reversed by cAMP-dependent protein kinase A that phosphorylates PLN at Ser16. To date, the molecular mechanisms for HAX-1 regulation of the SERCA/PLN complex are still unknown. Using enzymatic, in cell assays, circular dichroism, and NMR spectroscopy, we found that in the absence of a binding partner HAX-1 is essentially disordered and adopts a partial secondary structure upon interaction with lipid membranes. Also, HAX-1 interacts with the cytoplasmic region of monomeric and pentameric PLN as detected by NMR and in cell FRET assays, respectively. We propose that the regulation of the SERCA/PLN complex by HAX-1 is mediated by its interactions with lipid membranes, adding another layer of control in Ca2+ homeostatic balance in the heart muscle., (Copyright © 2019 Elsevier B.V. All rights reserved.)- Published
- 2020
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23. Newly Discovered Micropeptide Regulators of SERCA Form Oligomers but Bind to the Pump as Monomers.
- Author
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Singh DR, Dalton MP, Cho EE, Pribadi MP, Zak TJ, Šeflová J, Makarewich CA, Olson EN, and Robia SL
- Subjects
- Calcium Signaling genetics, Calcium Signaling physiology, Calcium-Binding Proteins metabolism, Cell Line, Fluorescence Resonance Energy Transfer, Humans, Muscle Proteins genetics, Muscle Proteins metabolism, Open Reading Frames genetics, Protein Binding, Protein Multimerization genetics, Protein Multimerization physiology, Proteolipids metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics, Peptides chemistry, Peptides metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
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., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2019
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24. Novel approach for quantification of endoplasmic reticulum Ca 2+ transport.
- Author
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Bovo E, Nikolaienko R, Bhayani S, Kahn D, Cao Q, Martin JL, Kuo IY, Robia SL, and Zima AV
- Subjects
- Adenosine Diphosphate metabolism, Adenosine Triphosphate metabolism, Animals, Biological Transport, Biosensing Techniques, Calcium-Binding Proteins metabolism, Endoplasmic Reticulum drug effects, Enzyme Inhibitors pharmacology, HEK293 Cells, Humans, Mice, Inbred C57BL, Mutation, Myocytes, Cardiac drug effects, Oxidative Stress, Ryanodine Receptor Calcium Release Channel genetics, Sarcoplasmic Reticulum drug effects, Sarcoplasmic Reticulum Calcium-Transporting ATPases antagonists & inhibitors, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics, Time Factors, Calcium metabolism, Endoplasmic Reticulum enzymology, Myocytes, Cardiac enzymology, Ryanodine Receptor Calcium Release Channel metabolism, Sarcoplasmic Reticulum enzymology, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
The type 2a sarco-/endoplasmic reticulum Ca
2+ -ATPase (SERCA2a) plays a key role in Ca2+ regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca2+ sensor R-CEPIA1er was used to measure the concentration of Ca2+ in the lumen of the endoplasmic reticulum (ER) ([Ca2+ ]ER ) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca2+ release channel ryanodine receptor (RyR2) created a Ca2+ release/reuptake system that mimicked aspects of cardiac myocyte Ca2+ handling. SERCA2a function was quantified from the rate of [Ca2+ ]ER refilling after ER Ca2+ depletion; then, ER Ca2+ leak was measured after SERCA inhibition. ER Ca2+ uptake and leak were analyzed as a function of [Ca2+ ]ER to determine maximum ER Ca2+ uptake rate and maximum ER Ca2+ load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/[ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function. NEW & NOTEWORTHY The aim of this study was to develop a sensitive approach to selectively measure sarco-/endoplasmic reticulum Ca2+ -ATPase (SERCA) function in the cellular environment. The newly developed Ca2+ sensor R-CEPIA1er was used to successfully analyze Ca2+ uptake mediated by recombinant and native cardiac SERCA. These results demonstrate that this new approach can be used as a powerful tool to study new mechanisms of Ca2+ pump regulation.- Published
- 2019
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25. Defects in assembly explain reduced antiviral activity of the G249D polymorphism in human TRIM5α.
- Author
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Kömürlü S, Bradley M, Smolin N, Imam S, Pauszek RF 3rd, Robia SL, Millar D, Nakayama EE, Shioda T, and Campbell EM
- Subjects
- Animals, Antiviral Restriction Factors, Capsid Proteins immunology, Capsid Proteins metabolism, Carrier Proteins immunology, Carrier Proteins metabolism, Cats, Genetic Predisposition to Disease, HEK293 Cells, HIV Infections immunology, HIV Infections virology, HIV-1 metabolism, Human Immunodeficiency Virus Proteins immunology, Human Immunodeficiency Virus Proteins metabolism, Humans, Molecular Dynamics Simulation, Polymorphism, Single Nucleotide, Protein Conformation, alpha-Helical genetics, Protein Domains genetics, Protein Structure, Quaternary genetics, Recombinant Proteins genetics, Recombinant Proteins immunology, Recombinant Proteins metabolism, Tripartite Motif Proteins, Ubiquitin-Protein Ligases, Carrier Proteins genetics, HIV Infections genetics, HIV-1 immunology
- Abstract
TRIM5α is an interferon inducible restriction factor which contributes to intrinsic defense against HIV infection by targeting the HIV capsid protein CA. Although human TRIM5α (huTRIM5α) does not potently inhibit HIV-1 infection, the ability of huTRIM5α to exhibit some control of HIV-1 infection is evidenced by a single nucleotide polymorphism in huTRIM5α which substitutes aspartic acid to glycine at position 249 (G249D) in the L2 region and is associated with higher susceptibility to HIV-1 infection. To understand the mechanistic basis for the reduced antiviral activity, we employed biophysical and cell biological methods coupled with molecular dynamics simulations to compare WT and the G249D polymorphism of huTRIM5α. We investigated the differences in conformational dynamics of rhesus and huTRIM5α Coiled Coil-Linker 2 (CC-L2) dimers utilizing circular dichroism and single molecule-Fluorescence Energy Transfer (sm-FRET). These methods revealed that the G249D dimer exhibits secondary structure and conformational dynamics similar to WT huTRIM5α. Homology modelling revealed that G249 was present on the hairpin of the antiparallel dimer, in a position which may act to stabilize the adjacent BBox2 domain which mediates the inter-dimeric contacts required for the formation of TRIM5 assemblies. We therefore asked if the G249D mutant forms assemblies in cells with the same efficiency as WT protein by expressing these proteins as YFP fusions and quantifying the number of assemblies in cells. In cells expressing comparable amounts of protein, the G249D mutant formed fewer assemblies than WT protein, in agreement with our homology modeling predictions and molecular dynamics simulations of dimers and higher oligomers of TRIM5α, providing a mechanistic explanation of the reduced antiviral activity of the G249D polymorphism., Competing Interests: The authors’ declare that they have no competing interests.
- Published
- 2019
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26. The DWORF micropeptide enhances contractility and prevents heart failure in a mouse model of dilated cardiomyopathy.
- Author
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Makarewich CA, Munir AZ, Schiattarella GG, Bezprozvannaya S, Raguimova ON, Cho EE, Vidal AH, Robia SL, Bassel-Duby R, and Olson EN
- Subjects
- Animals, Disease Models, Animal, Gene Knockout Techniques, Heart Failure prevention & control, LIM Domain Proteins deficiency, Mice, Knockout, Muscle Proteins deficiency, Calcium-Binding Proteins metabolism, Cardiomyopathy, Dilated physiopathology, Myocardial Contraction drug effects, Peptides metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
Calcium (Ca
2+ ) 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 ( DW arf O pen R eading F rame) 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., Competing Interests: CM CAM has filed a provisional patent application to use DWORF for treatment of heart failure (Application #62/324,706), AM, GS, SB, OR, EC, AV, SR No competing interests declared, RB RB-D has filed a provisional patent application to use DWORF for treatment of heart failure (Application #62/324,706), EO ENO has filed a provisional patent application to use DWORF for treatment of heart failure (Application #62/324,706), (© 2018, Makarewich et al.)- Published
- 2018
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27. Redistribution of SERCA calcium pump conformers during intracellular calcium signaling.
- Author
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Raguimova ON, Smolin N, Bovo E, Bhayani S, Autry JM, Zima AV, and Robia SL
- Subjects
- Crystallography, X-Ray, Humans, Models, Molecular, Molecular Dynamics Simulation, Protein Binding, Protein Conformation, RNA, Small Interfering genetics, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Calcium metabolism, Calcium Signaling, Endoplasmic Reticulum metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry
- Abstract
The conformational changes of a calcium transport ATPase were investigated with molecular dynamics (MD) simulations as well as fluorescence resonance energy transfer (FRET) measurements to determine the significance of a discrete structural element for regulation of the conformational dynamics of the transport cycle. Previous MD simulations indicated that a loop in the cytosolic domain of the SERCA calcium transporter facilitates an open-to-closed structural transition. To investigate the significance of this structural element, we performed additional MD simulations and new biophysical measurements of SERCA structure and function. Rationally designed in silico mutations of three acidic residues of the loop decreased SERCA domain-domain contacts and increased domain-domain separation distances. Principal component analysis of MD simulations suggested decreased sampling of compact conformations upon N-loop mutagenesis. Deficits in headpiece structural dynamics were also detected by measuring intramolecular FRET of a Cer-YFP-SERCA construct (2-color SERCA). Compared with WT, the mutated 2-color SERCA shows a partial FRET response to calcium, whereas retaining full responsiveness to the inhibitor thapsigargin. Functional measurements showed that the mutated transporter still hydrolyzes ATP and transports calcium, but that maximal enzyme activity is reduced while maintaining similar calcium affinity. In live cells, calcium elevations resulted in concomitant FRET changes as the population of WT 2-color SERCA molecules redistributed among intermediates of the transport cycle. Our results provide novel insights on how the population of SERCA pumps responds to dynamic changes in intracellular calcium., (© 2018 Raguimova et al.)
- Published
- 2018
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28. Skin cells prefer a slower calcium pump.
- Author
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Robia SL and Young HS
- Subjects
- Humans, Kinetics, Protein Conformation, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics, Calcium metabolism, Calcium Signaling, Darier Disease genetics, Darier Disease pathology, Mutation, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
Naturally occurring mutations of a calcium ion transporter can cause a skin condition known as Darier's disease. In this issue of JBC, Mikkelsen et al. describe a particularly interesting Darier's mutation that alters calcium transport by disrupting a kinetic braking mechanism that is unique to the SERCA2b calcium pump isoform. The study provides new insight into the intrinsic regulation of this transporter and reveals how disruption of regulation can lead to disease in Darier's patients., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2018
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29. Regulation of Focal Adhesion Kinase through a Direct Interaction with an Endogenous Inhibitor.
- Author
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Zak TJ, Koshman YE, Samarel AM, and Robia SL
- Subjects
- Animals, Focal Adhesion Kinase 1, Muscle, Smooth, Vascular cytology, Phosphorylation, Rats, Signal Transduction physiology, Focal Adhesion Protein-Tyrosine Kinases metabolism, Gene Expression Regulation, Enzymologic physiology, Myocytes, Smooth Muscle enzymology, Protein Kinase Inhibitors metabolism
- Abstract
Focal adhesion kinase (FAK) plays a key role in integrin and growth factor signaling pathways. FAK-related non-kinase (FRNK) is an endogenous inhibitor of FAK that shares its primary structure with the C-terminal third of FAK. FAK S910 phosphorylation is known to regulate FAK protein-protein interactions, but the role of the equivalent site on FRNK (S217) is unknown. Here we determined that S217 is highly phosphorylated by ERK in cultured rat aortic smooth muscle cells. Blocking phosphorylation by mutation (S217A) greatly increased FRNK inhibitory potency, resulting in strong inhibition of FAK autophosphorylation at Y397 and induction of smooth muscle cell apoptosis. FRNK has been proposed to compete for FAK anchoring sites in focal adhesions, but we did not detect displacement of FAK by WT-FRNK or superinhibitory S217A-FRNK. Instead, we found FRNK interacted directly with FAK, and this interaction is markedly strengthened for the superinhibitory S217A-FRNK. The results suggest that FRNK limits growth and survival signaling pathways by binding directly to FAK in an inhibitory complex, and this inhibition is relieved by phosphorylation of FRNK at S217.
- Published
- 2017
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30. Dynamic conformational changes in the rhesus TRIM5α dimer dictate the potency of HIV-1 restriction.
- Author
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Lamichhane R, Mukherjee S, Smolin N, Pauszek RF 3rd, Bradley M, Sastri J, Robia SL, Millar D, and Campbell EM
- Subjects
- Amino Acid Sequence, Animals, Carrier Proteins genetics, Cell Line, Dimerization, Disease Models, Animal, HIV Infections genetics, HIV Infections virology, HIV-1 genetics, Humans, Macaca mulatta genetics, Macaca mulatta virology, Mutation, Protein Conformation, Carrier Proteins chemistry, Carrier Proteins immunology, HIV Infections immunology, HIV-1 physiology, Macaca mulatta immunology
- Abstract
The TRIM5α protein from rhesus macaques (rhTRIM5α) mediates a potent inhibition of HIV-1 infection via a mechanism that involves the abortive disassembly of the viral core. We have demonstrated that alpha-helical elements within the Linker 2 (L2) region, which lies between the SPRY domain and the Coiled-Coil domain, influence the potency of restriction. Here, we utilize single-molecule FRET analysis to reveal that the L2 region of the TRIM5α dimer undergoes dynamic conformational changes, which results in the displacement of L2 regions by 25 angstroms relative to each other. Analysis of restriction enhancing or abrogating mutations in the L2 region reveal that restriction defective mutants are unable to undergo dynamic conformational changes and do not assume compact, alpha-helical conformations in the L2 region. These data suggest a model in which conformational changes in the L2 region mediate displacement of CA bound SPRY domains to induce the destabilization of assembled capsid during restriction., (Copyright © 2016 Elsevier Inc. All rights reserved.)
- Published
- 2017
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31. L30A Mutation of Phospholemman Mimics Effects of Cardiac Glycosides in Isolated Cardiomyocytes.
- Author
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Himes RD, Smolin N, Kukol A, Bossuyt J, Bers DM, and Robia SL
- Subjects
- Animals, Cell Line, Fluorescence Resonance Energy Transfer, Molecular Dynamics Simulation, Rabbits, Cardiac Glycosides pharmacology, Membrane Proteins genetics, Mutation, Myocytes, Cardiac drug effects, Phosphoproteins genetics
- Abstract
To determine if mutations introduced into phospholemman (PLM) could increase the level of PLM-Na,K-ATPase (NKA) binding, we performed scanning mutagenesis of the transmembrane domain of PLM and measured Förster resonance energy transfer (FRET) between each mutant and NKA. We observed an increased level of binding to NKA for several PLM mutants compared to that of the wild type (WT), including L27A, L30A, and I32A. In isolated cardiomyocytes, overexpression of WT PLM increased the amplitude of the Ca
2+ transient compared to the GFP control. The Ca2+ transient amplitude was further increased by L30A PLM overexpression. The L30A mutation also delayed Ca2+ extrusion and increased the duration of cardiomyocyte contraction. This mimics aspects of the effect of cardiac glycosides, which are known to increase contractility through inhibition of NKA. No significant differences between WT and L30A PLM-expressing myocytes were observed after treatment with isoproterenol, suggesting that the superinhibitory effects of L30A are reversible with β-adrenergic stimulation. We also observed a decrease in the extent of PLM tetramerization with L30A compared to WT using FRET, suggesting that L30 is an important residue for mediating PLM-PLM binding. Molecular dynamics simulations revealed that the potential energy of the L30A tetramer is greater than that of the WT, and that the transmembrane α helix is distorted by the mutation. The results identify PLM residue L30 as an important determinant of PLM tetramerization and of functional inhibition of NKA by PLM.- Published
- 2016
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32. Restrictive Cardiomyopathy Troponin I R145W Mutation Does Not Perturb Myofilament Length-dependent Activation in Human Cardiac Sarcomeres.
- Author
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Dvornikov AV, Smolin N, Zhang M, Martin JL, Robia SL, and de Tombe PP
- Subjects
- Amino Acid Substitution, Cyclic AMP-Dependent Protein Kinases chemistry, Cyclic AMP-Dependent Protein Kinases genetics, Cyclic AMP-Dependent Protein Kinases metabolism, Female, Humans, Male, Protein Kinase C chemistry, Protein Kinase C genetics, Protein Kinase C metabolism, Structure-Activity Relationship, Cardiomyopathy, Restrictive genetics, Cardiomyopathy, Restrictive metabolism, Molecular Dynamics Simulation, Mutation, Missense, Sarcomeres chemistry, Sarcomeres genetics, Sarcomeres metabolism, Troponin I chemistry, Troponin I genetics, Troponin I metabolism
- Abstract
The cardiac troponin I (cTnI) R145W mutation is associated with restrictive cardiomyopathy (RCM). Recent evidence suggests that this mutation induces perturbed myofilament length-dependent activation (LDA) under conditions of maximal protein kinase A (PKA) stimulation. Some cardiac disease-causing mutations, however, have been associated with a blunted response to PKA-mediated phosphorylation; whether this includes LDA is unknown. Endogenous troponin was exchanged in isolated skinned human myocardium for recombinant troponin containing either cTnI R145W, PKA/PKC phosphomimetic charge mutations (S23D/S24D and T143E), or various combinations thereof. Myofilament Ca
2+ sensitivity of force, tension cost, LDA, and single myofibril activation/relaxation parameters were measured. Our results show that both R145W and T143E uncouple the impact of S23D/S24D phosphomimetic on myofilament function, including LDA. Molecular dynamics simulations revealed a marked reduction in interactions between helix C of cTnC (residues 56, 59, and 63), and cTnI (residue 145) in the presence of either cTnI RCM mutation or cTnI PKC phosphomimetic. These results suggest that the RCM-associated cTnI R145W mutation induces a permanent structural state that is similar to, but more extensive than, that induced by PKC-mediated phosphorylation of cTnI Thr-143. We suggest that this structural conformational change induces an increase in myofilament Ca2+ sensitivity and, moreover, uncoupling from the impact of phosphorylation of cTnI mediated by PKA at the Ser-23/Ser-24 target sites. The R145W RCM mutation by itself, however, does not impact LDA. These perturbed biophysical and biochemical myofilament properties are likely to significantly contribute to the diastolic cardiac pump dysfunction that is seen in patients suffering from a restrictive cardiomyopathy that is associated with the cTnI R145W mutation., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2016
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33. Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer.
- Author
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Blackwell DJ, Zak TJ, and Robia SL
- Subjects
- Adenosine Triphosphate chemistry, Adenosine Triphosphate metabolism, Animals, Blotting, Western, Calcium chemistry, Calcium metabolism, Calcium-Binding Proteins chemistry, Calcium-Binding Proteins metabolism, Dogs, Fluorescence Resonance Energy Transfer, HEK293 Cells, Heart Ventricles chemistry, Heart Ventricles enzymology, Humans, Immunoprecipitation, Luminescent Proteins chemistry, Luminescent Proteins genetics, Luminescent Proteins metabolism, Models, Molecular, Muscle Cells chemistry, Muscle Cells enzymology, Mutation, Phosphorylation, Photobleaching, Protein Multimerization, Rabbits, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry
- Abstract
The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation., (Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
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34. Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides.
- Author
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Soller KJ, Verardi R, Jing M, Abrol N, Yang J, Walsh N, Vostrikov VV, Robia SL, Bowser MT, and Veglia G
- Subjects
- Amino Acids metabolism, Animals, Cell Survival, Epitopes metabolism, Fluorescence Resonance Energy Transfer, Kinetics, Magnetic Resonance Spectroscopy, Protein Binding, Rabbits, Sarcoplasmic Reticulum metabolism, Sus scrofa, Calcium-Binding Proteins metabolism, DNA, Single-Stranded metabolism, Membrane Proteins metabolism, Oligonucleotides metabolism, RNA, Untranslated metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
The membrane protein complex between sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLN) is a prime therapeutic target for reversing cardiac contractile dysfunctions caused by calcium mishandling. So far, however, efforts to develop drugs specific for this protein complex have failed. Here, we show that non-coding RNAs and single-stranded DNAs (ssDNAs) interact with and regulate the function of the SERCA/PLN complex in a tunable manner. Both in HEK cells expressing the SERCA/PLN complex, as well as in cardiac sarcoplasmic reticulum preparations, these short oligonucleotides bind and reverse PLN's inhibitory effects on SERCA, increasing the ATPase's apparent Ca(2+) affinity. Solid-state NMR experiments revealed that ssDNA interacts with PLN specifically, shifting the conformational equilibrium of the SERCA/PLN complex from an inhibitory to a non-inhibitory state. Importantly, we achieved rheostatic control of SERCA function by modulating the length of ssDNAs. Since restoration of Ca(2+) flux to physiological levels represents a viable therapeutic avenue for cardiomyopathies, our results suggest that oligonucleotide-based drugs could be used to fine-tune SERCA function to counterbalance the extent of the pathological insults.
- Published
- 2015
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35. ATP-Binding Cassette Transporter Structure Changes Detected by Intramolecular Fluorescence Energy Transfer for High-Throughput Screening.
- Author
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Iram SH, Gruber SJ, Raguimova ON, Thomas DD, and Robia SL
- Subjects
- ATP Binding Cassette Transporter, Subfamily B chemistry, ATP Binding Cassette Transporter, Subfamily B metabolism, Binding Sites, Cell Membrane metabolism, Drug Discovery, Green Fluorescent Proteins chemistry, Green Fluorescent Proteins metabolism, HEK293 Cells, High-Throughput Screening Assays methods, Humans, Models, Molecular, Protein Structure, Secondary, Small Molecule Libraries pharmacology, Adenosine Triphosphate metabolism, Fluorescence Resonance Energy Transfer methods, Small Molecule Libraries metabolism
- Abstract
Multidrug resistance protein 1 (MRP1) actively transports a wide variety of drugs out of cells. To quantify MRP1 structural dynamics, we engineered a "two-color MRP1" construct by fusing green fluorescent protein (GFP) and TagRFP to MRP1 nucleotide-binding domains NBD1 and NBD2, respectively. The recombinant MRP1 protein expressed and trafficked normally to the plasma membrane. Two-color MRP1 transport activity was normal, as shown by vesicular transport of [(3)H]17β-estradiol-17-β-(D-glucuronide) and doxorubicin efflux in AAV-293 cells. We quantified fluorescence resonance energy transfer (FRET) from GFP to TagRFP as an index of NBD conformational changes. Our results show that ATP binding induces a large-amplitude conformational change that brings the NBDs into closer proximity. FRET was further increased by substrate in the presence of ATP but not by substrate alone. The data suggest that substrate binding is required to achieve a fully closed and compact structure. ATP analogs bind MRP1 with reduced apparent affinity, inducing a partially closed conformation. The results demonstrate the utility of the two-color MRP1 construct for investigating ATP-binding cassette transporter structural dynamics, and it holds great promise for high-throughput screening of chemical libraries for unknown activators, inhibitors, or transportable substrates of MRP1., (Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.)
- Published
- 2015
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36. Acute inotropic and lusitropic effects of cardiomyopathic R9C mutation of phospholamban.
- Author
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Abrol N, de Tombe PP, and Robia SL
- Subjects
- Amino Acid Sequence, Animals, Calcium-Binding Proteins chemistry, Calcium-Binding Proteins metabolism, Cardiomyopathies metabolism, Cells, Cultured, Dogs, Fluorescence Resonance Energy Transfer, Humans, Molecular Sequence Data, Oxidative Stress, Point Mutation, Protein Multimerization, Rabbits, Calcium-Binding Proteins genetics, Cardiomyopathies genetics, Myocytes, Cardiac metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
A naturally occurring R9C mutation of phospholamban (PLB) triggers cardiomyopathy and premature death by altering regulation of sarco/endoplasmic reticulum calcium-ATPase (SERCA). The goal of this study was to investigate the acute physiological consequences of the R9C-PLB mutation on cardiomyocyte calcium kinetics and contractility. We measured the physiological consequences of R9C-PLB mutation on calcium transients and sarcomere shortening in adult cardiomyocytes. In contrast to studies of chronic R9C-PLB expression in transgenic mice, we found that acute expression of R9C-PLB exerts a positively inotropic and lusitropic effect in cardiomyocytes. Importantly, R9C-PLB exhibited blunted sensitivity to frequency potentiation and β-adrenergic stimulation, two major physiological mechanisms for the regulation of cardiac performance. To identify the molecular mechanism of R9C pathology, we quantified the effect of R9C on PLB oligomerization and PLB-SERCA binding. FRET measurements in live cells revealed that R9C-PLB exhibited an increased propensity for oligomerization, and this was further increased by oxidative stress. The R9C also decreased PLB binding to SERCA and altered the structure of the PLB-SERCA regulatory complex. The structural change after oxidative modification of R9C-PLB was similar to that observed after PLB phosphorylation. We conclude that R9C mutation of PLB decreases SERCA inhibition by decreasing the amount of the regulatory complex and altering its conformation. This has an acute inotropic/lusitropic effect but yields negative consequences of impaired frequency potentiation and blunted β-adrenergic responsiveness. We envision a self-reinforcing mechanism beginning with phosphomimetic R9C-PLB oxidation and loss of SERCA inhibition, leading to impaired calcium regulation and heart failure., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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37. A structural mechanism for calcium transporter headpiece closure.
- Author
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Smolin N and Robia SL
- Subjects
- Amino Acid Sequence, Animals, Conserved Sequence, Sequence Alignment, Models, Molecular, Molecular Dynamics Simulation, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry
- Abstract
To characterize the conformational dynamics of sarcoplasmic reticulum (SR) calcium pump (SERCA) we performed molecular dynamics simulations beginning with several different high-resolution structures. We quantified differences in structural disorder and dynamics for an open conformation of SERCA versus closed structures and observed that dynamic motions of SERCA cytoplasmic domains decreased with decreasing domain-domain separation distance. The results are useful for interpretation of recent intramolecular Förster resonance energy transfer (FRET) distance measurements obtained for SERCA fused to fluorescent protein tags. Those previous physical measurements revealed several discrete structural substates and suggested open conformations of SERCA are more dynamic than compact conformations. The present simulations support this hypothesis and provide additional details of SERCA molecular mechanisms. Specifically, all-atoms simulations revealed large-scale translational and rotational motions of the SERCA N-domain relative to the A- and P-domains during the transition from an open to a closed headpiece conformation over the course of a 400 ns trajectory. The open-to-closed structural transition was accompanied by a disorder-to-order transition mediated by an initial interaction of an N-domain loop (Nβ5-β6, residues 426-436) with residues 133-139 of the A-domain. Mutation of three negatively charged N-domain loop residues abolished the disorder-to-order transition and prevented the initial domain-domain interaction and subsequent closure of the cytoplasmic headpiece. Coarse-grained molecular dynamics simulations were in harmony with all-atoms simulations and physical measurements and revealed a close communication between fluorescent protein tags and the domain to which they were fused. The data indicate that previous intramolecular FRET distance measurements report SERCA structure changes with high fidelity and suggest a structural mechanism that facilitates the closure of the SERCA cytoplasmic headpiece.
- Published
- 2015
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38. Phospholamban C-terminal residues are critical determinants of the structure and function of the calcium ATPase regulatory complex.
- Author
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Abrol N, Smolin N, Armanious G, Ceholski DK, Trieber CA, Young HS, and Robia SL
- Subjects
- Animals, Biophysical Phenomena, Calcium chemistry, Calcium metabolism, Calcium-Binding Proteins chemistry, Calcium-Binding Proteins genetics, Cell Membrane chemistry, Cell Membrane metabolism, Cytoplasm chemistry, Cytoplasm metabolism, Fluorescence Resonance Energy Transfer, Green Fluorescent Proteins chemistry, Heart Failure pathology, Humans, Multiprotein Complexes, Protein Binding, Protein Structure, Quaternary genetics, Recombinant Fusion Proteins genetics, Sarcoplasmic Reticulum metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases genetics, Calcium-Binding Proteins metabolism, Heart Failure metabolism, Recombinant Fusion Proteins metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
To determine the structural and regulatory role of the C-terminal residues of phospholamban (PLB) in the membranes of living cells, we fused fluorescent protein tags to PLB and sarco/endoplasmic reticulum calcium ATPase (SERCA). Alanine substitution of PLB C-terminal residues significantly altered fluorescence resonance energy transfer (FRET) from PLB to PLB and SERCA to PLB, suggesting a change in quaternary conformation of PLB pentamer and SERCA-PLB regulatory complex. Val to Ala substitution at position 49 (V49A) had particularly large effects on PLB pentamer structure and PLB-SERCA regulatory complex conformation, increasing and decreasing probe separation distance, respectively. We also quantified a decrease in oligomerization affinity, an increase in binding affinity of V49A-PLB for SERCA, and a gain of inhibitory function as quantified by calcium-dependent ATPase activity. Notably, deletion of only a few C-terminal residues resulted in significant loss of PLB membrane anchoring and mislocalization to the cytoplasm and nucleus. C-terminal truncations also resulted in progressive loss of PLB-PLB FRET due to a decrease in the apparent affinity of PLB oligomerization. We quantified a similar decrease in the binding affinity of truncated PLB for SERCA and loss of inhibitory potency. However, despite decreased SERCA-PLB binding, intermolecular FRET for Val(49)-stop (V49X) truncation mutant was paradoxically increased as a result of an 11.3-Å decrease in the distance between donor and acceptor fluorophores. We conclude that PLB C-terminal residues are critical for localization, oligomerization, and regulatory function. In particular, the PLB C terminus is an important determinant of the quaternary structure of the SERCA regulatory complex., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
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39. Restriction of HIV-1 by rhesus TRIM5α is governed by alpha helices in the Linker2 region.
- Author
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Sastri J, Johnsen L, Smolin N, Imam S, Mukherjee S, Lukic Z, Brandariz-Nuñez A, Robia SL, Diaz-Griffero F, Wiethoff C, and Campbell EM
- Subjects
- Animals, Cell Line, Cell Line, Tumor, Chemokine CCL2 genetics, Chemokine CCL2 metabolism, Cytoplasm genetics, Cytoplasm metabolism, HEK293 Cells, HeLa Cells, Humans, Macaca mulatta genetics, Macaca mulatta microbiology, Macaca mulatta virology, Mutation genetics, Carrier Proteins genetics, Carrier Proteins metabolism, HIV-1 genetics, HIV-1 metabolism, Protein Structure, Secondary genetics
- Abstract
Unlabelled: TRIM5α proteins are a potent barrier to the cross-species transmission of retroviruses. TRIM5α proteins exhibit an ability to self-associate at many levels, ultimately leading to the formation of protein assemblies with hexagonal symmetry in vitro and cytoplasmic assemblies when expressed in cells. However, the role of these assemblies in restriction, the determinants that mediate their formation, and the organization of TRIM5α molecules within these assemblies have remained unclear. Here we show that α-helical elements within the Linker2 region of rhesus macaque TRIM5α govern the ability to form cytoplasmic assemblies in cells and restrict HIV-1 infection. Mutations that reduce α-helix formation by the Linker2 region disrupt assembly and restriction. More importantly, mutations that enhance the α-helical content of the Linker2 region, relative to the wild-type protein, also exhibit an increased ability to form cytoplasmic assemblies and restrict HIV-1 infection. Molecular modeling of the TRIM5α dimer suggests a model in which α-helical elements within the Linker2 region dock to α-helices of the coiled-coil domain, likely establishing proper orientation and spacing of protein domains necessary for assembly and restriction. Collectively, these studies provide critical insight into the determinants governing TRIM5α assembly and restriction and demonstrate that the antiviral potency of TRIM5α proteins can be significantly increased without altering the affinity of SPRY/capsid binding., Importance: Many members of the tripartite motif (TRIM) family of proteins act as restriction factors that directly inhibit viral infection and activate innate immune signaling pathways. Another common feature of TRIM proteins is the ability to form protein assemblies in the nucleus or the cytoplasm. However, the determinants in TRIM proteins required for assembly and the degree to which assembly affects TRIM protein function have been poorly understood. Here we show that alpha helices in the Linker2 (L2) region of rhesus TRIM5α govern assembly and restriction of HIV-1 infection. Helix-disrupting mutations disrupt the assembly and restriction of HIV-1, while helix-stabilizing mutations enhance assembly and restriction relative to the wild-type protein. Circular dichroism analysis suggests that that the formation of this helical structure is supported by intermolecular interactions with the coiled-coil (CC) domain in the CCL2 dimer. These studies reveal a novel mechanism by which the antiviral activity of TRIM5α proteins can be regulated and provide detailed insight into the assembly determinants of TRIM family proteins., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)
- Published
- 2014
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40. Discovery of enzyme modulators via high-throughput time-resolved FRET in living cells.
- Author
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Gruber SJ, Cornea RL, Li J, Peterson KC, Schaaf TM, Gillispie GD, Dahl R, Zsebo KM, Robia SL, and Thomas DD
- Subjects
- Animals, Green Fluorescent Proteins chemistry, HEK293 Cells, Hepatocytes metabolism, Humans, Luminescent Proteins chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases antagonists & inhibitors, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism, Small Molecule Libraries, Red Fluorescent Protein, Biosensing Techniques methods, Fluorescence Resonance Energy Transfer, High-Throughput Screening Assays, Sarcoplasmic Reticulum Calcium-Transporting ATPases isolation & purification
- Abstract
We have used a "two-color" SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified 10 compounds that significantly affected two-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns and is adaptable to many other targets.
- Published
- 2014
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41. Phosphorylated phospholamban stabilizes a compact conformation of the cardiac calcium-ATPase.
- Author
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Pallikkuth S, Blackwell DJ, Hu Z, Hou Z, Zieman DT, Svensson B, Thomas DD, and Robia SL
- Subjects
- Animals, Dependovirus metabolism, Detergents pharmacology, Enzyme Stability drug effects, Fluorescence Resonance Energy Transfer, Models, Molecular, Myocardium cytology, Myocytes, Cardiac enzymology, Phosphorylation drug effects, Protein Conformation, Rabbits, Solubility, Transfection, Calcium-Binding Proteins metabolism, Myocardium enzymology, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with β-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture., (Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2013
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42. 2-Color calcium pump reveals closure of the cytoplasmic headpiece with calcium binding.
- Author
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Hou Z, Hu Z, Blackwell DJ, Miller TD, Thomas DD, and Robia SL
- Subjects
- Animals, Cell Line, Cell Survival drug effects, Color, Cytoplasm drug effects, Cytosol drug effects, Cytosol metabolism, Dependovirus metabolism, Dogs, Fluorescence Resonance Energy Transfer, Humans, Microscopy, Fluorescence, Protein Structure, Secondary, Protein Structure, Tertiary, Protein Transport drug effects, Thapsigargin pharmacology, Calcium metabolism, Cytoplasm metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases chemistry, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The "2-color" SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.
- Published
- 2012
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43. Serine-910 phosphorylation of focal adhesion kinase is critical for sarcomere reorganization in cardiomyocyte hypertrophy.
- Author
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Chu M, Iyengar R, Koshman YE, Kim T, Russell B, Martin JL, Heroux AL, Robia SL, and Samarel AM
- Subjects
- Angiotensin II pharmacology, Animals, Animals, Newborn, Blotting, Western, Cardiomyopathy, Dilated pathology, Cells, Cultured, Dose-Response Relationship, Drug, Endothelin-1 pharmacology, Enzyme Activation, Fluorescence Recovery After Photobleaching, Focal Adhesion Kinase 1 genetics, Heart Failure pathology, Humans, Immunoprecipitation, Insulin-Like Growth Factor I pharmacology, Microscopy, Fluorescence, Mutation, Myocytes, Cardiac drug effects, Myocytes, Cardiac pathology, Paxillin genetics, Paxillin metabolism, Phenylephrine pharmacology, Phosphorylation, Protein Kinase C-delta metabolism, Rats, Rats, Sprague-Dawley, Recombinant Fusion Proteins metabolism, Sarcomeres drug effects, Sarcomeres pathology, Serine, Signal Transduction, Time Factors, Transfection, src-Family Kinases metabolism, Cardiomyopathy, Dilated enzymology, Focal Adhesion Kinase 1 metabolism, Heart Failure enzymology, Myocytes, Cardiac enzymology, Sarcomeres enzymology
- Abstract
Aims: Tyrosine-phosphorylated focal adhesion kinase (FAK) is required for the hypertrophic response of cardiomyocytes to growth factors and mechanical load, but the role of FAK serine phosphorylation in this process is unknown. The aims of the present study were to characterize FAK serine phosphorylation in cultured neonatal rat ventricular myocytes (NRVM), analyse its functional significance during hypertrophic signalling, and examine its potential role in the pathogenesis of human dilated cardiomyopathy (DCM)., Methods and Results: Endothelin-1 (ET-1) and other hypertrophic factors induced a time- and dose-dependent increase in FAK-S910 phosphorylation. ET-1-induced FAK-S910 phosphorylation required ET(A)R-dependent activation of PKCδ and Src via parallel Raf-1 → MEK1/2 → ERK1/2 and MEK5 → ERK5 signalling pathways. Replication-deficient adenoviruses expressing wild-type (WT) FAK and a non-phosphorylatable, S910A-FAK mutant were then used to examine the functional significance of FAK-S910 phosphorylation. Unlike WT-FAK, S910A-FAK increased the half-life of GFP-tagged paxillin within costameres (as determined by total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching) and increased the steady-state FAK-paxillin interaction (as determined by co-immunoprecipitation and western blotting). These alterations resulted in reduced NRVM sarcomere reorganization and cell spreading. Finally, we found that FAK was serine-phosphorylated at multiple sites in non-failing, human left ventricular tissue. FAK-S910 phosphorylation and ERK5 expression were dramatically reduced in patients undergoing heart transplantation for end-stage DCM., Conclusion: FAK undergoes S910 phosphorylation via PKCδ and Src-dependent pathways that are important for cell spreading and sarcomere reorganization. Reduced FAK-S910 phosphorylation may contribute to sarcomere disorganization in DCM.
- Published
- 2011
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44. TRIM5α associates with proteasomal subunits in cells while in complex with HIV-1 virions.
- Author
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Lukic Z, Hausmann S, Sebastian S, Rucci J, Sastri J, Robia SL, Luban J, and Campbell EM
- Subjects
- ATPases Associated with Diverse Cellular Activities, Antiviral Restriction Factors, Carrier Proteins genetics, Cytoplasm genetics, Cytoplasm metabolism, Cytoplasm virology, DNA, Recombinant genetics, DNA, Recombinant metabolism, Fluorescence Resonance Energy Transfer, Gene Library, HEK293 Cells, HIV Infections metabolism, HIV Infections virology, HIV-1 pathogenicity, Humans, Immunoprecipitation, Microscopy, Fluorescence, Proteasome Endopeptidase Complex genetics, Protein Binding, Protein Interaction Mapping methods, Species Specificity, Transfection, Tripartite Motif Proteins, Two-Hybrid System Techniques, Ubiquitin genetics, Ubiquitin metabolism, Ubiquitin-Protein Ligases, Ubiquitination, Carrier Proteins metabolism, HIV-1 metabolism, Proteasome Endopeptidase Complex metabolism
- Abstract
Background: The TRIM5 proteins are cellular restriction factors that prevent retroviral infection in a species-specific manner. Multiple experiments indicate that restriction activity requires accessory host factors, including E2-enzymes. To better understand the mechanism of restriction, we conducted yeast-two hybrid screens to identify proteins that bind to two TRIM5 orthologues., Results: The only cDNAs that scored on repeat testing with both TRIM5 orthologues were the proteasome subunit PSMC2 and ubiquitin. Using co-immunoprecipitation assays, we demonstrated an interaction between TRIM5α and PSMC2, as well as numerous other proteasome subunits. Fluorescence microscopy revealed co-localization of proteasomes and TRIM5α cytoplasmic bodies. Forster resonance energy transfer (FRET) analysis indicated that the interaction between TRIM5 and PSMC2 was direct. Previous imaging experiments demonstrated that, when cells are challenged with fluorescently-labeled HIV-1 virions, restrictive TRIM5α orthologues assemble cytoplasmic bodies around incoming virion particles. Following virus challenge, we observed localization of proteasome subunits to rhTRIM5α cytoplasmic bodies that contained fluorescently labeled HIV-1 virions., Conclusions: Taken together, the results presented here suggest that localization of the proteasome to TRIM5α cytoplasmic bodies makes an important contribution to TRIM5α-mediated restriction.
- Published
- 2011
- Full Text
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45. Focal adhesion kinase-related nonkinase inhibits vascular smooth muscle cell invasion by focal adhesion targeting, tyrosine 168 phosphorylation, and competition for p130(Cas) binding.
- Author
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Koshman YE, Chu M, Engman SJ, Kim T, Iyengar R, Robia SL, and Samarel AM
- Subjects
- Adenoviridae genetics, Animals, Carotid Arteries metabolism, Carotid Arteries pathology, Carotid Artery Injuries etiology, Carotid Artery Injuries metabolism, Carotid Artery Injuries pathology, Catheterization adverse effects, Cells, Cultured, Focal Adhesion Kinase 2 genetics, Focal Adhesion Protein-Tyrosine Kinases genetics, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Models, Animal, Phosphorylation physiology, Protein Binding physiology, RNA, Small Interfering pharmacology, Rats, Cell Movement physiology, Crk-Associated Substrate Protein metabolism, Focal Adhesion Kinase 2 metabolism, Focal Adhesion Protein-Tyrosine Kinases metabolism, Muscle, Smooth, Vascular metabolism, Muscle, Smooth, Vascular pathology, Protein-Tyrosine Kinases metabolism
- Abstract
Objective: Focal adhesion kinase-related nonkinase (FRNK), the C-terminal domain of focal adhesion kinase (FAK), is a tyrosine-phosphorylated, vascular smooth muscle cell (VSMC)-specific inhibitor of cell migration. FRNK inhibits both FAK and proline-rich tyrosine kinase 2 (PYK2) in cultured VSMCs, and both kinases may be involved in VSMC invasion during vascular remodeling., Methods and Results: Adenovirally mediated gene transfer of green fluorescent protein-tagged, wild-type (wt) FRNK into balloon-injured rat carotid arteries confirmed that FRNK overexpression inhibited both FAK and PYK2 phosphorylation and downstream signaling in vivo. To identify which kinase was involved in regulating VSMC invasion, adenovirally mediated expression of specific short hairpin RNAs was used to knock down FAK versus PYK2 in cultured VSMCs, but only FAK short hairpin RNA was effective in reducing VSMC invasion. The role of FRNK tyrosine phosphorylation was then examined using adenoviruses expressing nonphosphorylatable (Tyr168Phe-, Tyr232Phe-, and Tyr168,232Phe-) green fluorescent protein-FRNK mutants. wtFRNK and all FRNK mutants localized to FAs, but only Tyr168 phosphorylation was required for FRNK to inhibit invasion. Preventing Tyr168 phosphorylation also increased FRNK-paxillin interaction, as determined by coimmunoprecipitation, total internal reflection fluorescence microscopy, and fluorescence recovery after photobleaching. Furthermore, wtFRNK competed with FAK for binding to p130(Cas) (a critically important regulator of cell migration) and prevented its phosphorylation. However, Tyr168Phe-FRNK was unable to bind p130(Cas)., Conclusion: We propose a 3-stage mechanism for FRNK inhibition: focal adhesion targeting, Tyr168 phosphorylation, and competition with FAK for p130 binding and phosphorylation, which are all required for FRNK to inhibit VSMC invasion.
- Published
- 2011
- Full Text
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46. Phospholamban binds with differential affinity to calcium pump conformers.
- Author
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Bidwell P, Blackwell DJ, Hou Z, Zima AV, and Robia SL
- Subjects
- Animals, Bacterial Proteins metabolism, Binding Sites, Calcium-Transporting ATPases metabolism, Cytosol metabolism, Dogs, Fluorescence Resonance Energy Transfer methods, Heart Ventricles cytology, Luminescent Proteins metabolism, Membrane Proteins metabolism, Microscopy, Confocal methods, Models, Biological, Muscle Cells metabolism, Protein Binding, Protein Interaction Mapping, Rabbits, Calcium chemistry, Calcium-Binding Proteins metabolism
- Abstract
To investigate the mechanism of regulation of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) by phospholamban (PLB), we expressed Cerulean-SERCA and yellow fluorescent protein (YFP)-PLB in adult rabbit ventricular myocytes using adenovirus vectors. SERCA and PLB were localized in the sarcoplasmic reticulum and were mobile over multiple sarcomeres on a timescale of tens of seconds. We also observed robust fluorescence resonance energy transfer (FRET) from Cerulean-SERCA to YFP-PLB. Electrical pacing of cardiac myocytes elicited cytoplasmic Ca(2+) elevations, but these increases in Ca(2+) produced only modest changes in SERCA-PLB FRET. The data suggest that the regulatory complex is not disrupted by elevations of cytosolic calcium during cardiac contraction (systole). This conclusion was also supported by parallel experiments in heterologous cells, which showed that FRET was reduced but not abolished by calcium. Thapsigargin also elicited a small decrease in PLB-SERCA binding affinity. We propose that PLB is not displaced from SERCA by high calcium during systole, and relief of functional inhibition does not require dissociation of the regulatory complex. The observed modest reduction in the affinity of the PLB-SERCA complex with Ca(2+) or thapsigargin suggests that the binding interface is altered by SERCA conformational changes. The results are consistent with multiple modes of PLB binding or alternative binding sites.
- Published
- 2011
- Full Text
- View/download PDF
47. Spatiotemporally distinct protein kinase D activation in adult cardiomyocytes in response to phenylephrine and endothelin.
- Author
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Bossuyt J, Chang CW, Helmstadter K, Kunkel MT, Newton AC, Campbell KS, Martin JL, Bossuyt S, Robia SL, and Bers DM
- Subjects
- Aging drug effects, Animals, Cell Membrane drug effects, Cell Membrane metabolism, Enzyme Activation drug effects, Fluorescence Recovery After Photobleaching, Green Fluorescent Proteins, Histone Deacetylases metabolism, Models, Biological, Myocytes, Cardiac cytology, Myocytes, Cardiac drug effects, Protein Binding drug effects, Protein Transport drug effects, Rabbits, Recombinant Fusion Proteins metabolism, Sarcolemma drug effects, Sarcolemma enzymology, Signal Transduction drug effects, Time Factors, Aging metabolism, Endothelin-1 pharmacology, Myocytes, Cardiac enzymology, Phenylephrine pharmacology, Protein Kinase C metabolism
- Abstract
Protein kinase D (PKD) is a nodal point in cardiac hypertrophic signaling. It triggers nuclear export of class II histone deacetylase (HDAC) and regulates transcription. Although this pathway is thought to be critical in cardiac hypertrophy and heart failure, little is known about spatiotemporal aspects of PKD activation at the myocyte level. Here, we demonstrate that in adult cardiomyocytes two important neurohumoral stimuli that induce hypertrophy, endothelin-1 (ET1) and phenylephrine (PE), trigger comparable global PKD activation and HDAC5 nuclear export, but via divergent spatiotemporal PKD signals. PE-induced HDAC5 export is entirely PKD-dependent, involving fleeting sarcolemmal PKD translocation (for activation) and very rapid subsequent nuclear import. In contrast, ET1 recruits and activates PKD that remains predominantly sarcolemmal. This explains why PE-induced nuclear HDAC5 export in myocytes is totally PKD-dependent, whereas ET1-induced HDAC5 export depends more prominently on InsP(3) and CaMKII signaling. Thus α-adrenergic and ET-1 receptor signaling via PKD in adult myocytes feature dramatic differences in cellular localization and translocation in mediating hypertrophic signaling. This raises new opportunities for targeted therapeutic intervention into distinct limbs of this hypertrophic signaling pathway.
- Published
- 2011
- Full Text
- View/download PDF
48. Oligomeric interactions of sarcolipin and the Ca-ATPase.
- Author
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Autry JM, Rubin JE, Pietrini SD, Winters DL, Robia SL, and Thomas DD
- Subjects
- Animals, Bacterial Proteins, Baculoviridae genetics, Cloning, Molecular, Dogs, Fluorescence Resonance Energy Transfer, Green Fluorescent Proteins, Heart Atria cytology, Insecta cytology, Luminescent Proteins, Muscle Fibers, Fast-Twitch, Mutagenesis, Site-Directed, Protein Binding, Rabbits, Muscle Proteins metabolism, Protein Multimerization, Proteolipids metabolism, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
We have detected directly the interactions of sarcolipin (SLN) and the sarcoplasmic reticulum Ca-ATPase (SERCA) by measuring fluorescence resonance energy transfer (FRET) between fusion proteins labeled with cyan fluorescent protein (donor) and yellow fluorescent protein (acceptor). SLN is a membrane protein that helps control contractility by regulating SERCA activity in fast-twitch and atrial muscle. Here we used FRET microscopy and spectroscopy with baculovirus expression in insect cells to provide direct evidence for: 1) oligomerization of SLN and 2) regulatory complex formation between SLN and the fast-twitch muscle Ca-ATPase (SERCA1a isoform). FRET experiments demonstrated that SLN monomers self-associate into dimers and higher order oligomers in the absence of SERCA, and that SLN monomers also bind to SERCA monomers in a 1:1 binary complex when the two proteins are coexpressed. FRET experiments further demonstrated that the binding affinity of SLN for itself is similar to that for SERCA. Mutating SLN residue isoleucine-17 to alanine (I17A) decreased the binding affinity of SLN self-association and converted higher order oligomers into monomers and dimers. The I17A mutation also decreased SLN binding affinity for SERCA but maintained 1:1 stoichiometry in the regulatory complex. Thus, isoleucine-17 plays dual roles in determining the distribution of SLN homo-oligomers and stabilizing the formation of SERCA-SLN heterodimers. FRET results for SLN self-association were supported by the effects of SLN expression in bacterial cells. We propose that SLN exists as multiple molecular species in muscle, including SERCA-free (monomer, dimer, oligomer) and SERCA-bound (heterodimer), with transmembrane zipper residues of SLN serving to stabilize oligomeric interactions.
- Published
- 2011
- Full Text
- View/download PDF
49. Phosphomimetic mutations enhance oligomerization of phospholemman and modulate its interaction with the Na/K-ATPase.
- Author
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Song Q, Pallikkuth S, Bossuyt J, Bers DM, and Robia SL
- Subjects
- Amino Acid Substitution, Cell Line, Humans, Membrane Proteins genetics, Mutation, Missense, Phosphoproteins genetics, Protein Binding, Protein Structure, Quaternary, Sodium-Potassium-Exchanging ATPase genetics, Membrane Proteins metabolism, Phosphoproteins metabolism, Protein Multimerization physiology, Sodium-Potassium-Exchanging ATPase metabolism
- Abstract
Na/K-ATPase (NKA) activity is dynamically regulated by an inhibitory interaction with a small transmembrane protein, phospholemman (PLM). Inhibition is relieved upon PLM phosphorylation. Phosphorylation may alter how PLM interacts with NKA and/or itself, but details of these interactions are unknown. To address this, we quantified FRET between PLM and its regulatory target NKA in live cells. Phosphorylation of PLM was mimicked by mutation S63E (PKC site), S68E (PKA/PKC site), or S63E/S68E. The dependence of FRET on protein expression in live cells yielded information about the structure and binding affinity of the PLM-NKA regulatory complex. PLM phosphomimetic mutations altered the quaternary structure of the regulatory complex and reduced the apparent affinity of the PLM-NKA interaction. The latter effect was likely due to increased oligomerization of PLM phosphomimetic mutants, as suggested by PLM-PLM FRET measurements. Distance constraints obtained by FRET suggest that phosphomimetic mutations slightly alter the oligomer quaternary conformation. Photon-counting histogram measurements revealed that the major PLM oligomeric species is a tetramer. We conclude that phosphorylation of PLM increases its oligomerization into tetramers, decreases its binding to NKA, and alters the structures of both the tetramer and NKA regulatory complex.
- Published
- 2011
- Full Text
- View/download PDF
50. Lethal Arg9Cys phospholamban mutation hinders Ca2+-ATPase regulation and phosphorylation by protein kinase A.
- Author
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Ha KN, Masterson LR, Hou Z, Verardi R, Walsh N, Veglia G, and Robia SL
- Subjects
- Calcium metabolism, Cardiomyopathy, Dilated metabolism, Fluorescence Resonance Energy Transfer, Humans, Myocardial Contraction physiology, Oxidative Stress genetics, Phosphorylation, Calcium-Binding Proteins genetics, Calcium-Binding Proteins metabolism, Cardiomyopathy, Dilated genetics, Cyclic AMP-Dependent Protein Kinases metabolism, Mutation, Missense genetics, Myocardial Contraction genetics, Sarcoplasmic Reticulum Calcium-Transporting ATPases metabolism
- Abstract
The regulatory interaction of phospholamban (PLN) with Ca(2+)-ATPase controls the uptake of calcium into the sarcoplasmic reticulum, modulating heart muscle contractility. A missense mutation in PLN cytoplasmic domain (R9C) triggers dilated cardiomyopathy in humans, leading to premature death. Using a combination of biochemical and biophysical techniques both in vitro and in live cells, we show that the R9C mutation increases the stability of the PLN pentameric assembly via disulfide bridge formation, preventing its binding to Ca(2+)-ATPase as well as phosphorylation by protein kinase A. These effects are enhanced under oxidizing conditions, suggesting that oxidative stress may exacerbate the cardiotoxic effects of the PLN(R9C) mutant. These results reveal a regulatory role of the PLN pentamer in calcium homeostasis, going beyond the previously hypothesized role of passive storage for active monomers.
- Published
- 2011
- Full Text
- View/download PDF
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