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Evangelia G. Kranias - One of the best experts on this subject based on the ideXlab platform.
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Identification of a protein phosphatase-1/Phospholamban complex that is regulated by cAMP-dependent phosphorylation.
PLOS ONE, 2013Co-Authors: Elizabeth Vafiadaki, Despina Sanoudou, Demetrios A. Arvanitis, Evangelia G. KraniasAbstract:In human and experimental heart failure, the activity of the type 1 phosphatase is significantly increased, associated with dephosphorylation of Phospholamban, inhibition of the sarco(endo)plasmic reticulum Ca2+ transport ATPase (SERCA2a) and depressed function. In the current study, we investigated the molecular mechanisms controlling protein phosphatase-1 activity. Using recombinant proteins and complementary in vitro binding studies, we identified a multi-protein complex centered on protein phosphatase-1 that includes its muscle specific glycogen-targeting subunit GM and substrate Phospholamban. GM interacts directly with Phospholamban and this association is mediated by the cytosolic regions of the proteins. Our findings suggest the involvement of GM in mediating formation of the phosphatase-1/GM/Phospholamban complex through the direct and independent interactions of GM with both protein phosphatase-1 and Phospholamban. Importantly, the protein phosphatase-1/GM/Phospholamban complex dissociates upon protein kinase A phosphorylation, indicating its significance in the β-adrenergic signalling axis. Moreover, protein phosphatase-1 activity is regulated by two binding partners, inhibitor-1 and the small heat shock protein 20, Hsp20. Indeed, human genetic variants of inhibitor-1 (G147D) or Hsp20 (P20L) result in reduced binding and inhibition of protein phosphatase-1, suggesting aberrant enzymatic regulation in human carriers. These findings provide insights into the mechanisms underlying fine-tuned regulation of protein phosphatase-1 and its impact on the SERCA2/Phospholamban interactome in cardiac function.
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Phospholamban overexpression in rabbit ventricular myocytes does not alter sarcoplasmic reticulum Ca transport
American journal of physiology. Heart and circulatory physiology, 2008Co-Authors: Jason R. Waggoner, Kobra Haghighi, Donald M. Bers, Kenneth S. Ginsburg, Bryan Mitton, Jeffrey Robbins, Evangelia G. KraniasAbstract:Phospholamban has been suggested to be a key regulator of cardiac sarcoplasmic reticulum (SR) Ca cycling and contractility and a potential therapeutic target in restoring the depressed Ca cycling in failing hearts. Our understanding of the function of Phospholamban stems primarily from studies in genetically altered mouse models. To evaluate the significance of this protein in larger mammalian species, which exhibit Ca cycling properties similar to humans, we overexpressed Phospholamban in adult rabbit cardiomyocytes. Adenoviral-mediated gene transfer, at high multiplicities of infection, resulted in an insignificant 1.22-fold overexpression of Phospholamban. There were no effects on twitch Ca-transient amplitude or decay under basal or isoproterenol-stimulated conditions. Furthermore, the SR Ca load and Na/Ca exchanger function were not altered. These apparent differences between Phospholamban overexpression in rabbit compared with previous findings in the mouse may be due to a significantly higher (1.5-fold) endogenous Phospholamban-to-sarco(endo)plasmic reticulum Ca-ATPase (SERCA) 2a ratio and potential functional saturation of SERCA2a by Phospholamban in rabbit cardiomyocytes. The findings suggest that important species-dependent differences in Phospholamban regulation of SERCA2a occur. In larger mammals, a higher fraction of SERCA2a pumps are regulated by Phospholamban, and this may influence therapeutic strategies to enhance cardiac contractility and functional cardiac reserve.
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Phospholamban overexpression in transgenic rabbits
Transgenic Research, 2008Co-Authors: James Scott Pattison, Evangelia G. Kranias, Jason R. Waggoner, Jeanne James, Lisa Martin, James Gulick, Hanna Osinska, Raisa Klevitsky, Jeffrey RobbinsAbstract:There has been considerable interest in pursuing Phospholamban as a putative therapeutic target for overcoming depressed calcium handling in human heart failure. Studies predominantly done in mice have shown that Phospholamban is a key regulator of sarcoplasmic reticulum calcium cycling and cardiac function. However, mice differ significantly from humans in how they regulate calcium, whereas rabbits better recapitulate human cardiac function and calcium handling. To investigate Phospholamban’s role in the rabbit heart, transgenic rabbits that overexpressed wild-type Phospholamban in the ventricular cardiomyocytes and slow-twitch skeletal muscles were generated. Rabbits expressing high levels of Phospholamban were not viable due to severe skeletal muscle wasting, the onset of cardiac pathology and early death. A viable transgenic line exhibited a 30% increase in PLN protein levels in the heart. These animals showed isolated foci of cardiac pathology, but cardiac function as well as the response to β-adrenergic stimulation were normal. SR-calcium uptake measurements showed that the transgenic hearts had the expected reduced affinity for calcium. The data show that Phospholamban-overexpressing transgenic rabbits differ markedly in phenotype from analogous transgenic mice in that rabbits are quite sensitive to alterations in Phospholamban levels. Exceeding a relatively narrow window of Phospholamban expression results in significant morbidity and early death.
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altered calcium handling is critically involved in the cardiotoxic effects of chronic β adrenergic stimulation
Circulation, 2004Co-Authors: Stefan Engelhardt, Evangelia G. Kranias, Lutz Hein, Vitaly Dyachenkow, Gerrit Isenberg, Martin J LohseAbstract:Background— Chronic adrenergic stimulation leads to cardiac hypertrophy and heart failure in experimental models and contributes to the progression of heart failure in humans. The pathways mediating the detrimental effects of chronic β-adrenergic stimulation are only partly understood. We investigated whether genetic modification of calcium handling through deletion of Phospholamban in mice would affect the development of heart failure in mice with transgenic overexpression of the β1-adrenergic receptor. Methods and Results— We crossed β1-adrenergic receptor transgenic (β1TG) mice with mice homozygous for a targeted deletion of the Phospholamban gene (PLB−/−). Phospholamban ablation dramatically enhanced survival of β1TG mice. The decrease of left ventricular contractility typically observed in β1TG mice was reverted back to normal by Phospholamban ablation. Cardiac hypertrophy and fibrosis were significantly inhibited in β1TG/PLB−/− mice compared with β1TG mice, and the heart failure–specific gene expres...
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Phospholamban: a crucial regulator of cardiac contractility
Nature reviews. Molecular cell biology, 2003Co-Authors: David H Maclennan, Evangelia G. KraniasAbstract:Heart failure is a major cause of death and disability. Impairments in blood circulation that accompany heart failure can be traced, in part, to alterations in the activity of the sarcoplasmic reticulum Ca2+ pump that are induced by its interactions with Phospholamban, a reversible inhibitor. If Phospholamban becomes superinhibitory or chronically inhibitory, contractility is diminished, inducing dilated cardiomyopathy in mice and humans. In mice, Phospholamban seems to encumber an otherwise healthy heart, but humans with a Phospholamban-null genotype develop early-onset dilated cardiomyopathy.
Howard S. Young - One of the best experts on this subject based on the ideXlab platform.
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The Phospholamban pentamer interacts with the sarcoplasmic reticulum calcium pump SERCA
2018Co-Authors: Howard S. Young, John Paul Glaves, Joseph O. Primeau, M. Joanne Lemieux, L. Michel Espinoza-fonsecaAbstract:The interaction of Phospholamban with the sarcoplasmic reticulum calcium pump (SERCA) is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric Phospholamban and storage of Phospholamban as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that Phospholamban remains associated with SERCA and that the Phospholamban pentamer is required for cardiac contractility. We have previously used two-dimensional crystallization and electron microscopy to study the interaction between SERCA and Phospholamban. To further understand this interaction, we compared small helical crystals and large two-dimensional crystals of SERCA in the absence and presence of Phospholamban. In both crystal forms, SERCA molecules are organized into identical anti-parallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large two-dimensional crystals, which allow Phospholamban differential access to potential sites of interaction with SERCA. Nonetheless, we show that a Phospholamban oligomer interacts with SERCA in a similar manner in both crystal forms. In the two-dimensional crystals, a Phospholamban pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of Phospholamban also interacts with M3 of SERCA, though the Phospholamban oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of Phospholamban interacts with SERCA, and that it plays distinct structural and functional roles in SERCA regulation.
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Conformational memory in the association of the transmembrane protein Phospholamban with the sarcoplasmic reticulum calcium pump SERCA.
The Journal of biological chemistry, 2017Co-Authors: Serena Smeazzetto, Howard S. Young, M. Joanne Lemieux, Gareth P. Armanious, Marai Rosa Moncelli, Jessi J. Bak, Francesco Tadini-buoninsegniAbstract:Abstract The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by Phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how Phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of Phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady state current measurements, proteoliposomes containing SERCA and Phospholamban or sarcolipin were adsorbed to a solid-supported membranes and activated by substrate concentration jumps. We observed that Phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, while sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of Phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that Phospholamban can establish an inhibitory interaction with multiple SERCA conformational states, with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and Phospholamban, and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and Phospholamban, thus providing insights into the physiological role of Phospholamban and its regulatory effect on SERCA transport activity.
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conformational memory in the association of the transmembrane protein Phospholamban with the sarcoplasmic reticulum calcium pump serca
Journal of Biological Chemistry, 2017Co-Authors: Serena Smeazzetto, Howard S. Young, Gareth P. Armanious, Marai Rosa Moncelli, Jessi J. Bak, Joanne M Lemieux, Francesco TadinibuoninsegniAbstract:The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by Phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how Phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of Phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and Phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that Phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of Phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that Phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and Phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and Phospholamban, thus providing insights into the physiological role of Phospholamban and its regulatory effect on SERCA transport activity.
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Phospholamban and Sarcolipin Pentamers Naturally Associate with the Sarcoplasmic Reticulum Calcium Pump
Biophysical Journal, 2014Co-Authors: John Paul Glaves, Joseph O. Primeau, Catharine A. Trieber, Przemek A. Gorski, Howard S. YoungAbstract:Phospholamban and sarcolipin interact with the sarcoplasmic reticulum calcium pump (SERCA) and regulate contractility in smooth, cardiac and skeletal muscle. While both proteins can form oligomers, it is thought that only the monomers interact with and inhibit SERCA. To address the role of the Phospholamban and sarcolipin pentamers, we have studied their interaction with SERCA using electron cryo-microscopy of two-dimensional co-crystals. In our previous studies, Phospholamban oligomers were found interspersed between SERCA dimers and we constructed a three-dimensional model of the complex. We also addressed the molecular characteristics of Phospholamban that contribute to its interaction with SERCA and we examined the effects of phosphorylation and mutation of Phospholamban on the structure of the complex with SERCA. In our recent work, we compared two crystal forms of SERCA in the absence and presence of Phospholamban by electron cryo-microscopy - namely, small helical crystals and large two-dimensional crystals. The SERCA dimer ribbons that are found in both crystal forms consist of a rigid assembly of calcium-free SERCA molecules. While the lattice formed by the SERCA dimer ribbons is different in the helical and two-dimensional crystals, we show that a Phospholamban oligomer interacts with SERCA in a similar manner in both crystal types. With this information, we next undertook a structural investigation of SERCA and sarcolipin in the two-dimensional crystals. A projection map was determined for SERCA in the presence of sarcolipin to a resolution of 8.5 A and was consistent with a pentameric state for sarcolipin. While both Phospholamban and sarcolipin interacted with transmembrane segment M3 of SERCA, the interaction of the sarcolipin pentamer was mediated by an additional density consistent with a SLN monomer. We conclude that pentameric forms of both Phospholamban and sarcolipin naturally associate with SERCA.
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Lethal, hereditary mutants of Phospholamban elude phosphorylation by protein kinase A.
The Journal of biological chemistry, 2012Co-Authors: Delaine K. Ceholski, Catharine A. Trieber, Charles F. B. Holmes, Howard S. YoungAbstract:The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, Phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by β-adrenergic stimulation and phosphorylation of Phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of Phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14) deletion, alter regulation of SERCA. Deletion of Arg(14) disrupts the protein kinase A recognition motif, which abrogates Phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and Phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg(9) in Phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the Phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.
John Paul Glaves - One of the best experts on this subject based on the ideXlab platform.
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The Phospholamban pentamer interacts with the sarcoplasmic reticulum calcium pump SERCA
2018Co-Authors: Howard S. Young, John Paul Glaves, Joseph O. Primeau, M. Joanne Lemieux, L. Michel Espinoza-fonsecaAbstract:The interaction of Phospholamban with the sarcoplasmic reticulum calcium pump (SERCA) is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric Phospholamban and storage of Phospholamban as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that Phospholamban remains associated with SERCA and that the Phospholamban pentamer is required for cardiac contractility. We have previously used two-dimensional crystallization and electron microscopy to study the interaction between SERCA and Phospholamban. To further understand this interaction, we compared small helical crystals and large two-dimensional crystals of SERCA in the absence and presence of Phospholamban. In both crystal forms, SERCA molecules are organized into identical anti-parallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large two-dimensional crystals, which allow Phospholamban differential access to potential sites of interaction with SERCA. Nonetheless, we show that a Phospholamban oligomer interacts with SERCA in a similar manner in both crystal forms. In the two-dimensional crystals, a Phospholamban pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of Phospholamban also interacts with M3 of SERCA, though the Phospholamban oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of Phospholamban interacts with SERCA, and that it plays distinct structural and functional roles in SERCA regulation.
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Phospholamban and Sarcolipin Pentamers Naturally Associate with the Sarcoplasmic Reticulum Calcium Pump
Biophysical Journal, 2014Co-Authors: John Paul Glaves, Joseph O. Primeau, Catharine A. Trieber, Przemek A. Gorski, Howard S. YoungAbstract:Phospholamban and sarcolipin interact with the sarcoplasmic reticulum calcium pump (SERCA) and regulate contractility in smooth, cardiac and skeletal muscle. While both proteins can form oligomers, it is thought that only the monomers interact with and inhibit SERCA. To address the role of the Phospholamban and sarcolipin pentamers, we have studied their interaction with SERCA using electron cryo-microscopy of two-dimensional co-crystals. In our previous studies, Phospholamban oligomers were found interspersed between SERCA dimers and we constructed a three-dimensional model of the complex. We also addressed the molecular characteristics of Phospholamban that contribute to its interaction with SERCA and we examined the effects of phosphorylation and mutation of Phospholamban on the structure of the complex with SERCA. In our recent work, we compared two crystal forms of SERCA in the absence and presence of Phospholamban by electron cryo-microscopy - namely, small helical crystals and large two-dimensional crystals. The SERCA dimer ribbons that are found in both crystal forms consist of a rigid assembly of calcium-free SERCA molecules. While the lattice formed by the SERCA dimer ribbons is different in the helical and two-dimensional crystals, we show that a Phospholamban oligomer interacts with SERCA in a similar manner in both crystal types. With this information, we next undertook a structural investigation of SERCA and sarcolipin in the two-dimensional crystals. A projection map was determined for SERCA in the presence of sarcolipin to a resolution of 8.5 A and was consistent with a pentameric state for sarcolipin. While both Phospholamban and sarcolipin interacted with transmembrane segment M3 of SERCA, the interaction of the sarcolipin pentamer was mediated by an additional density consistent with a SLN monomer. We conclude that pentameric forms of both Phospholamban and sarcolipin naturally associate with SERCA.
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Phosphorylation and Mutation Induce an Order-To-Disorder Transition in the Cytoplasmic Domain of Phospholamban
Biophysical Journal, 2011Co-Authors: John Paul Glaves, Catharine A. Trieber, Delaine K. Ceholski, David L. Stokes, Howard S. YoungAbstract:Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We have studied this interaction using electron microscopy of two-dimensional crystals of SERCA in complex with Phospholamban. In previous studies, Phospholamban oligomers were found interspersed between SERCA dimer ribbons and a three-dimensional model was constructed to show interactions with SERCA. In the present study, we have examined the effects of phosphorylation and mutation of Phospholamban on the interaction with SERCA in the two-dimensional crystals. Based on projection maps from negatively-stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild-type Phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27-to-Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of Phospholamban (Arg14-to-Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34-to-Ala) was disordered. The functional state of Phospholamban correlated with an order-to-disorder transition of Phospholamban's cytoplasmic domain in the two-dimensional co-crystals. Furthermore, the residues studied (Ser16, Lys27 and Asn34) may implicate domain Ib of Phospholamban in the order-to-disorder transition. In summary, the two-dimensional co-crystals with SERCA require a functional pentameric form of Phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the Phospholamban monomer for the inhibitory association. Phosphorylation or mutation of Phospholamban alters the SERCA-pentamer interaction in a manner normally associated with inhibition by the monomer.
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Phosphorylation and mutation of Phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump.
Journal of molecular biology, 2010Co-Authors: John Paul Glaves, Catharine A. Trieber, Delaine K. Ceholski, David L. Stokes, Howard S. YoungAbstract:Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We studied this interaction using electron microscopy of 2D crystals of SERCA in complex with Phospholamban. In earlier studies, Phospholamban oligomers were found interspersed between SERCA dimer ribbons and a 3D model was constructed to show interactions with SERCA. In this study, we examined the oligomeric state of Phospholamban and the effects of phosphorylation and mutation of Phospholamban on the interaction with SERCA in the 2D crystals. On the basis of projection maps from negatively stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild type Phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of Phospholamban (Arg14Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34Ala) was also disordered. The functional state of Phospholamban was correlated with an order-to-disorder transition of the Phospholamban cytoplasmic domain in the 2D co-crystals. Furthermore, co-crystals of the gain-of-function mutant (Lys27Ala) facilitated data collection from frozen-hydrated crystals. An improved projection map was calculated to a resolution of 8 A, which supports the pentamer as the oligomeric state of Phospholamban in the crystals. The 2D co-crystals with SERCA require a functional pentameric form of Phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the Phospholamban monomer for the inhibitory association.
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A Functional, Pentameric Form of Phospholamban Is Required For Two-Dimensional Crystallization With the Sarcoplasmic Reticulum Calcium Pump
Biophysical Journal, 2010Co-Authors: John Paul Glaves, Catharine A. Trieber, David L. Stokes, Howard S. YoungAbstract:A Functional, Pentameric Form Of Phospholamban Is Required For Two-Dimensional Crystallization With The Sarcoplasmic Reticulum Calcium PumpPhospholamban physically interacts with the sarcoplasmic reticulum calcium pump (also known as SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We have studied this interaction using electron microscopy of large two-dimensional crystals of SERCA in complex with Phospholamban. In our original work, Phospholamban oligomers were found interspersed between dimer arrays of SERCA and a three-dimensional model was constructed to show potential interactions between the two proteins. In the present study, we have examined the effects of Phospholamban phosphorylation and mutation on the formation of two-dimensional co-crystals with SERCA. Phospholamban phosphorylation at Ser16 and a well-characterized loss-of-function mutation (Asn34-to-Ala) significantly reduced crystal formation. More importantly, projection maps calculated from these crystals revealed that the densities attributable to Phospholamban become disordered, suggesting a reduced interaction with SERCA. In contrast, a pentameric gain-of-function mutant (Lys27-to-Ala) significantly enhanced crystal formation. These latter crystals were used to calculate an improved projection map from frozen-hydrated crystals to a resolution of 8 A. We conclude that the oligomeric state of Phospholamban in the crystals is a pentamer, and that phosphorylation and mutation of Phospholamban alter physical interactions in the crystals in a manner that is consistent with a functional association with SERCA. Combined, the data suggest that the pentameric state of Phospholamban is not simply an inactive storage form.
Catharine A. Trieber - One of the best experts on this subject based on the ideXlab platform.
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Phospholamban and Sarcolipin Pentamers Naturally Associate with the Sarcoplasmic Reticulum Calcium Pump
Biophysical Journal, 2014Co-Authors: John Paul Glaves, Joseph O. Primeau, Catharine A. Trieber, Przemek A. Gorski, Howard S. YoungAbstract:Phospholamban and sarcolipin interact with the sarcoplasmic reticulum calcium pump (SERCA) and regulate contractility in smooth, cardiac and skeletal muscle. While both proteins can form oligomers, it is thought that only the monomers interact with and inhibit SERCA. To address the role of the Phospholamban and sarcolipin pentamers, we have studied their interaction with SERCA using electron cryo-microscopy of two-dimensional co-crystals. In our previous studies, Phospholamban oligomers were found interspersed between SERCA dimers and we constructed a three-dimensional model of the complex. We also addressed the molecular characteristics of Phospholamban that contribute to its interaction with SERCA and we examined the effects of phosphorylation and mutation of Phospholamban on the structure of the complex with SERCA. In our recent work, we compared two crystal forms of SERCA in the absence and presence of Phospholamban by electron cryo-microscopy - namely, small helical crystals and large two-dimensional crystals. The SERCA dimer ribbons that are found in both crystal forms consist of a rigid assembly of calcium-free SERCA molecules. While the lattice formed by the SERCA dimer ribbons is different in the helical and two-dimensional crystals, we show that a Phospholamban oligomer interacts with SERCA in a similar manner in both crystal types. With this information, we next undertook a structural investigation of SERCA and sarcolipin in the two-dimensional crystals. A projection map was determined for SERCA in the presence of sarcolipin to a resolution of 8.5 A and was consistent with a pentameric state for sarcolipin. While both Phospholamban and sarcolipin interacted with transmembrane segment M3 of SERCA, the interaction of the sarcolipin pentamer was mediated by an additional density consistent with a SLN monomer. We conclude that pentameric forms of both Phospholamban and sarcolipin naturally associate with SERCA.
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Lethal, hereditary mutants of Phospholamban elude phosphorylation by protein kinase A.
The Journal of biological chemistry, 2012Co-Authors: Delaine K. Ceholski, Catharine A. Trieber, Charles F. B. Holmes, Howard S. YoungAbstract:The sarcoplasmic reticulum calcium pump (SERCA) and its regulator, Phospholamban, are essential components of cardiac contractility. Phospholamban modulates contractility by inhibiting SERCA, and this process is dynamically regulated by β-adrenergic stimulation and phosphorylation of Phospholamban. Herein we reveal mechanistic insight into how four hereditary mutants of Phospholamban, Arg(9) to Cys, Arg(9) to Leu, Arg(9) to His, and Arg(14) deletion, alter regulation of SERCA. Deletion of Arg(14) disrupts the protein kinase A recognition motif, which abrogates Phospholamban phosphorylation and results in constitutive SERCA inhibition. Mutation of Arg(9) causes more complex changes in function, where hydrophobic substitutions such as cysteine and leucine eliminate both SERCA inhibition and Phospholamban phosphorylation, whereas an aromatic substitution such as histidine selectively disrupts phosphorylation. We demonstrate that the role of Arg(9) in Phospholamban function is multifaceted: it is important for inhibition of SERCA, it increases the efficiency of phosphorylation, and it is critical for protein kinase A recognition in the context of the Phospholamban pentamer. Given the synergistic consequences on contractility, it is not surprising that the mutants cause lethal, hereditary dilated cardiomyopathy.
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Phosphorylation and Mutation Induce an Order-To-Disorder Transition in the Cytoplasmic Domain of Phospholamban
Biophysical Journal, 2011Co-Authors: John Paul Glaves, Catharine A. Trieber, Delaine K. Ceholski, David L. Stokes, Howard S. YoungAbstract:Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We have studied this interaction using electron microscopy of two-dimensional crystals of SERCA in complex with Phospholamban. In previous studies, Phospholamban oligomers were found interspersed between SERCA dimer ribbons and a three-dimensional model was constructed to show interactions with SERCA. In the present study, we have examined the effects of phosphorylation and mutation of Phospholamban on the interaction with SERCA in the two-dimensional crystals. Based on projection maps from negatively-stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild-type Phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27-to-Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of Phospholamban (Arg14-to-Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34-to-Ala) was disordered. The functional state of Phospholamban correlated with an order-to-disorder transition of Phospholamban's cytoplasmic domain in the two-dimensional co-crystals. Furthermore, the residues studied (Ser16, Lys27 and Asn34) may implicate domain Ib of Phospholamban in the order-to-disorder transition. In summary, the two-dimensional co-crystals with SERCA require a functional pentameric form of Phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the Phospholamban monomer for the inhibitory association. Phosphorylation or mutation of Phospholamban alters the SERCA-pentamer interaction in a manner normally associated with inhibition by the monomer.
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Phosphorylation and mutation of Phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump.
Journal of molecular biology, 2010Co-Authors: John Paul Glaves, Catharine A. Trieber, Delaine K. Ceholski, David L. Stokes, Howard S. YoungAbstract:Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We studied this interaction using electron microscopy of 2D crystals of SERCA in complex with Phospholamban. In earlier studies, Phospholamban oligomers were found interspersed between SERCA dimer ribbons and a 3D model was constructed to show interactions with SERCA. In this study, we examined the oligomeric state of Phospholamban and the effects of phosphorylation and mutation of Phospholamban on the interaction with SERCA in the 2D crystals. On the basis of projection maps from negatively stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild type Phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of Phospholamban (Arg14Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34Ala) was also disordered. The functional state of Phospholamban was correlated with an order-to-disorder transition of the Phospholamban cytoplasmic domain in the 2D co-crystals. Furthermore, co-crystals of the gain-of-function mutant (Lys27Ala) facilitated data collection from frozen-hydrated crystals. An improved projection map was calculated to a resolution of 8 A, which supports the pentamer as the oligomeric state of Phospholamban in the crystals. The 2D co-crystals with SERCA require a functional pentameric form of Phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the Phospholamban monomer for the inhibitory association.
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A Functional, Pentameric Form of Phospholamban Is Required For Two-Dimensional Crystallization With the Sarcoplasmic Reticulum Calcium Pump
Biophysical Journal, 2010Co-Authors: John Paul Glaves, Catharine A. Trieber, David L. Stokes, Howard S. YoungAbstract:A Functional, Pentameric Form Of Phospholamban Is Required For Two-Dimensional Crystallization With The Sarcoplasmic Reticulum Calcium PumpPhospholamban physically interacts with the sarcoplasmic reticulum calcium pump (also known as SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We have studied this interaction using electron microscopy of large two-dimensional crystals of SERCA in complex with Phospholamban. In our original work, Phospholamban oligomers were found interspersed between dimer arrays of SERCA and a three-dimensional model was constructed to show potential interactions between the two proteins. In the present study, we have examined the effects of Phospholamban phosphorylation and mutation on the formation of two-dimensional co-crystals with SERCA. Phospholamban phosphorylation at Ser16 and a well-characterized loss-of-function mutation (Asn34-to-Ala) significantly reduced crystal formation. More importantly, projection maps calculated from these crystals revealed that the densities attributable to Phospholamban become disordered, suggesting a reduced interaction with SERCA. In contrast, a pentameric gain-of-function mutant (Lys27-to-Ala) significantly enhanced crystal formation. These latter crystals were used to calculate an improved projection map from frozen-hydrated crystals to a resolution of 8 A. We conclude that the oligomeric state of Phospholamban in the crystals is a pentamer, and that phosphorylation and mutation of Phospholamban alter physical interactions in the crystals in a manner that is consistent with a functional association with SERCA. Combined, the data suggest that the pentameric state of Phospholamban is not simply an inactive storage form.
Judy M. Harrer - One of the best experts on this subject based on the ideXlab platform.
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Pentameric assembly of Phospholamban facilitates inhibition of cardiac function in vivo
The Journal of biological chemistry, 1998Co-Authors: Guoxiang Chu, Judy M. Harrer, Yoji Sato, Vivek J. Kadambi, Brian D. Hoit, Donald M. Bers, Evangelia G. KraniasAbstract:Abstract Phospholamban has been proposed to coexist as pentamers and monomers in native sarcoplasmic reticulum membranes. To determine its functional unit in vivo, we reintroduced wild-type (pentameric) or monomeric mutant (C41F) Phospholamban in the hearts of Phospholamban knockout mice. Transgenic lines, expressing similar levels of mutant or wild-type Phospholamban, were identified, and their cardiac phenotypes were characterized in parallel. Sarcoplasmic reticulum Ca2+ transport assays indicated similar decreases in SERCA2 Ca2+ affinity by mutant or wild-type Phospholamban. However, the time constants of relaxation and Ca2+ transient decline in isolated cardiomyocytes were diminished to a greater extent by wild-type than mutant Phospholamban, even without significant differences in the amplitudes of myocyte contraction and Ca2+ transients between the two groups. Langendorff perfusion also indicated that mutant Phospholamban was not capable of depressing the enhanced relaxation parameters of the Phospholamban knockout hearts to the same extent as wild-type Phospholamban. Moreover, in vivo assessment of mouse hemodynamics revealed a greater depression of cardiac function in wild-type than mutant Phospholamban hearts. Thus, the mutant or monomeric form of Phospholamban was not as effective in slowing Ca2+ decline or relaxation in cardiomyocytes, hearts, or intact animals as wild-type or pentameric Phospholamban. These findings suggest that pentameric assembly of Phospholamban is necessary for optimal regulation of myocardial contractility in vivo.
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Monomeric Phospholamban Overexpression in Transgenic Mouse Hearts
Circulation research, 1997Co-Authors: Guoxiang Chu, Judy M. Harrer, Wusheng Luo, Vivek J. Kadambi, Gerald W. Dorn, Richard A. Walsh, Evangelia G. KraniasAbstract:Abstract Phospholamban, a prominent modulator of the sarcoplasmic reticulum (SR) Ca2+-ATPase activity and basal contractility in the mammalian heart, has been proposed to form pentamers in native SR membranes. However, the monomeric form of Phospholamban, which is associated with mutating Cys41 to Phe41, was shown to be as effective as pentameric Phospholamban in inhibiting Ca2+ transport in expression systems. To determine whether this monomeric form of Phospholamban is also functional in vivo, we generated transgenic mice with cardiac-specific overexpression of the mutant (Cys41→Phe41) Phospholamban. Quantitative immunoblotting indicated a 2-fold increase in the cardiac Phospholamban protein levels compared with wild-type controls, with ≈50% of Phospholamban migrating as monomers and ≈50% as pentamers upon SDS-PAGE. The mutant-Phospholamban transgenic hearts were analyzed in parallel with transgenic hearts overexpressing (2-fold) wild-type Phospholamban, which migrated as pentamers upon SDS-PAGE. SR Ca2+-uptake assays revealed that the EC50 values for Ca2+ were as follows: 0.32±0.01 μmol/L in hearts overexpressing monomeric Phospholamban, 0.49±0.05 μmol/L in hearts overexpressing wild-type Phospholamban, and 0.26±0.01 μmol/L in wild-type control mouse hearts. Analysis of cardiomyocyte mechanics and Ca2+ kinetics indicated that the inhibitory effects of mutant-Phospholamban overexpression (mt) were less pronounced than those of wild-type Phospholamban overexpression (ov) as assessed by depression of the following: (1) shortening fraction (25% mt versus 45% ov), (2) rates of shortening (27% mt versus 48% ov), (3) rates of relengthening (25% mt versus 50% ov), (4) amplitude of the Ca2+ signal (21% mt versus 40% ov), and (5) time for decay of the Ca2+ signal (25% mt versus 106% ov) compared with control (100%) myocytes. The differences in basal cardiac myocyte mechanics and Ca2+ transients among the animal groups overexpressing monomeric or wild-type Phospholamban and wild-type control mice were abolished upon isoproterenol stimulation. These findings suggest that pentameric assembly of Phospholamban is important for mediating its optimal regulatory effects on myocardial contractility in vivo.
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Phospholamban Gene Dosage Effects in the Mammalian Heart
Circulation research, 1996Co-Authors: Wusheng Luo, Judy M. Harrer, Donald G. Ferguson, Beata M. Wolska, Ingrid L. Grupp, Kobra Haghighi, Jay Patrick Slack, Gunter Grupp, Thomas Doetschman, R. John SolaroAbstract:Phospholamban ablation has been shown to result in significant increases in cardiac contractile parameters and loss of beta-adrenergic stimulation. To determine whether partial reduction in Phospholamban levels is also associated with enhancement of cardiac performance and to further examine the sensitivity of the contractile system to alterations in Phospholamban levels, hearts from wild-type, Phospholamban-heterozygous, and Phospholamban-deficient mice were studied in parallel at the subcellular, cellular, and organ levels. The Phospholamban-heterozygous mice expressed reduced cardiac Phospholamban mRNA and protein levels (40 +/- 5%) compared with wild type mice. The reduced Phospholamban levels were associated with significant decreases in the EC50 of the sarcoplasmic reticulum Ca2+ pump for CA2+ and increases in the contractile parameters of isolated myocytes and beating hearts. The relative Phospholamban levels among wild-type, Phospholamban-heterozygous, and Phospholamban-deficient mouse hearts correlated well with the (1) EC50 of the Ca(2+)-ATPase for Ca2+ in sarcoplasmic reticulum, (2) rates of relaxation and contraction in isolated cardiac myocytes, and (3) rates of relaxation and intact beating hearts. These findings suggest that physiological and pathological changes in the levels of Phospholamban will result in parallel changes in sarcoplasmic reticulum function and cardiac contraction.
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Expression of Phospholamban in C2C12 cells and regulation of endogenous SERCA1 activity.
Molecular and Cellular Biochemistry, 1995Co-Authors: Judy M. Harrer, Sathivel Ponniah, Donald G. Ferguson, Evangelia G. KraniasAbstract:Phospholamban (PLB) is a regulator of the sarcoplasmic reticulum Ca2+-ATPase (SERCA2) expressed in cardiac, slow-twitch skeletal, and smooth muscles. Phospholamban is not expressed in the sarcoplasmic reticulum of fast-twitch skeletal muscle, but it can regulate the sarcoplasmic reticulum Ca2+-ATPase activity (SERCA1) expressed in this muscle,in vitro. To determine whether Phospholamban can regulate SERCA1 activity in its native membrane environment, Phospholamban was stably transfected into a cell line (C2C12) derived from murine fast-twitch skeletal muscle. Differentiation of C2C12 myoblasts to myotubes was associated with induction of SERCA1 expression, assessed by Western blotting analysis using Ca2+-ATPase isoform specific antibodies. The expressed Phospholamban protein was localized in the microsomal fraction isolated from C2C12 myotubes. To determine the effect of Phospholamban expression on SERCA1 activity, microsomes were isolated from transfected and nontransfected C2C12 cell myotubes, and the initial rates of45Ca2+-uptake were determined over a wide range of Ca2+ concentrations (0.1–10 μM). Expression of Phospholamban was associated with inhibition of the initial rates of Ca2+-uptake at low [Ca2+] and this resulted in a decrease in the affinity of SERCA1 for Ca2+ (0.27±0.02 μM in nontransfected vs. 0.41±0.03 μM in PLB transfected C2C12 cells). These findings indicate that Phospholamban expression in C2C12 cells is associated within inhibition of the endogenous SERCA1 activity and provide evidence that Phospholamban is capable of regulating this Ca2+-ATPase isoform in its native membrane environment.
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Expression of Phospholamban in C_2C_12 cells and regulation of endogenous SERCA1 activity
Molecular and Cellular Biochemistry, 1995Co-Authors: Judy M. Harrer, Sathivel Ponniah, Donald G. Ferguson, Evangelia G. KraniasAbstract:Phospholamban (PLB) is a regulator of the sarcoplasmic reticulum Ca^2+-ATPase (SERCA2) expressed in cardiac, slow-twitch skeletal, and smooth muscles. Phospholamban is not expressed in the sarcoplasmic reticulum of fast-twitch skeletal muscle, but it can regulate the sarcoplasmic reticulum Ca^2+-ATPase activity (SERCA1) expressed in this muscle, in vitro . To determine whether Phospholamban can regulate SERCA1 activity in its native membrane environment, Phospholamban was stably transfected into a cell line (C_2C_12) derived from murine fast-twitch skeletal muscle. Differentiation of C_2C_12 myoblasts to myotubes was associated with induction of SERCA1 expression, assessed by Western blotting analysis using Ca^2+-ATPase isoform specific antibodies. The expressed Phospholamban protein was localized in the microsomal fraction isolated from C_2C_12 myotubes. To determine the effect of Phospholamban expression on SERCA1 activity, microsomes were isolated from transfected and nontransfected C_2C_12 cell myotubes, and the initial rates of^45Ca^2+-uptake were determined over a wide range of Ca^2+ concentrations (0.1–10 μM). Expression of Phospholamban was associated with inhibition of the initial rates of Ca^2+-uptake at low [Ca^2+] and this resulted in a decrease in the affinity of SERCA1 for Ca^2+ (0.27±0.02 μM in nontransfected vs. 0.41±0.03 μM in PLB transfected C_2C_12 cells). These findings indicate that Phospholamban expression in C_2C_12 cells is associated within inhibition of the endogenous SERCA1 activity and provide evidence that Phospholamban is capable of regulating this Ca^2+-ATPase isoform in its native membrane environment.