Abstract 282: Phospholamban Truncation Mutations Including Heart Failure Mutant L39stop Disrupt Membrane Localization.

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Neha Abrol ◽  
Nikolai Smolin ◽  
Chris Stefonowicz ◽  
Seth L Robia
Keyword(s):  
Heart Failure ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Full Length ◽  
Membrane Localization ◽  
Minimum Length ◽  
Human Heart Failure ◽  
C Terminus ◽  
Dynamics Simulations ◽  
And Function

Introduction: Phospholamban (PLB) is an integral sarcoplasmic reticulum (SR) membrane protein, which directly regulates cardiac Ca 2+ handling and contractility by reversibly inhibiting SR Ca 2+ ATPase (SERCA). Our previous studies have suggested that the naturally occurring human heart failure mutation of PLB, L39X disrupts membrane localization. Hypothesis: We hypothesize that the membrane localization of PLB is a prerequisite for PLB oligomerization and interaction with SERCA. The truncation mutations in C-terminus of PLB will disrupt membrane localization, PLB oligomerization, and SERCA regulation. Results and Methods: To identify the minimum length of PLB required for membrane localization and function, we generated a series of C-terminal transmembrane truncation mutants of PLB (tagged N-terminally with Cer or YFP) including L51X, M50X, V49X, I48X, I38X, I33X, and the heart-failure mutant L39X. Confocal microscopy revealed that progressive truncation of the C-terminal residues of PLB resulted in escalating increase in mislocalization of PLB to the cytoplasm and nucleus. In addition, we observed an increased solubilization of PLB as indicated by loss of YFP fluorescence after selective permeabilization of the plasma membrane by saponin. As expected, there was no change in localization of Cer-SERCA upon saponin permeabilization. Next, western blot analysis exhibited a decrease in molecular weight corresponding to the relative sizes of truncation mutants compared to full length PLB, indicating that protein degradation is not the cause of membrane mislocalization. Fluorescence resonance energy transfer analysis revealed that truncating the C-terminal residues of PLB results in a progressive decrease in apparent affinity of PLB oligomerization and interaction with SERCA. Finally, molecular dynamics simulations exhibited that the heart failure mutant L39X was unstable compared to full length PLB pentamer and started protruding out of the bilayer until complete solubilization. Conclusions: Truncating only two C-terminal residues of PLB resulted in significant mislocalization, while deleting five or more residues profoundly disrupted membrane localization, PLB oligomerization and SERCA regulation.

scholarly journals Heart failure leads to altered β2-adrenoceptor/cyclic adenosine monophosphate dynamics in the sarcolemmal phospholemman/Na,K ATPase microdomain

2018 ◽  
Vol 115 (3) ◽  
pp. 546-555 ◽  
Author(s):  
Zeynep Bastug-Özel ◽  
Peter T Wright ◽  
Axel E Kraft ◽  
Davor Pavlovic ◽  
Jacqueline Howie ◽  
...  
Keyword(s):  
Heart Failure ◽  
Ventricular Myocytes ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Adult Rat ◽  
C Terminus ◽  
Complex Forms ◽  
Cardiac Excitation

Abstract Aims Cyclic adenosine monophosphate (cAMP) regulates cardiac excitation–contraction coupling by acting in microdomains associated with sarcolemmal ion channels. However, local real time cAMP dynamics in such microdomains has not been visualized before. We sought to directly monitor cAMP in a microdomain formed around sodium–potassium ATPase (NKA) in healthy and failing cardiomyocytes and to better understand alterations of cAMP compartmentation in heart failure. Methods and results A novel Förster resonance energy transfer (FRET)-based biosensor termed phospholemman (PLM)-Epac1 was developed by fusing a highly sensitive cAMP sensor Epac1-camps to the C-terminus of PLM. Live cell imaging in PLM-Epac1 and Epac1-camps expressing adult rat ventricular myocytes revealed extensive regulation of NKA/PLM microdomain-associated cAMP levels by β2-adrenoceptors (β2-ARs). Local cAMP pools stimulated by these receptors were tightly controlled by phosphodiesterase (PDE) type 3. In chronic heart failure following myocardial infarction, dramatic reduction of the microdomain-specific β2-AR/cAMP signals and β2-AR dependent PLM phosphorylation was accompanied by a pronounced loss of local PDE3 and an increase in PDE2 effects. Conclusions NKA/PLM complex forms a distinct cAMP microdomain which is directly regulated by β2-ARs and is under predominant control by PDE3. In heart failure, local changes in PDE repertoire result in blunted β2-AR signalling to cAMP in the vicinity of PLM.

Features of MOG required for recognition by patients with MOG antibody-associated disorders

Brain ◽  
2021 ◽  
Author(s):  
Caterina Macrini ◽  
Ramona Gerhards ◽  
Stephan Winklmeier ◽  
Lena Bergmann ◽  
Simone Mader ◽  
...  
Keyword(s):  
Complement Activation ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Structural Features ◽  
Full Length ◽  
Target Antigen ◽  
Terminal Part ◽  
Hydrophobic Domain ◽  
C Terminus ◽  
Better Than

Abstract Antibodies (Abs) to myelin oligodendrocyte glycoprotein (MOG) define a distinct disease entity. Here we aimed to understand essential structural features of MOG required for recognition by autoantibodies from patients. We produced the N-terminal part of MOG in a conformationally correct form; this domain was insufficient to identify patients with MOG-Abs by ELISA even after site-directed binding. This was neither due to a lack of lipid embedding nor to a missing putative epitope at the C-terminus, which we confirmed to be an intracellular domain. When MOG was displayed on transfected cells, patients with MOG-Abs recognized full-length MOG much better than its N-terminal part with the first hydrophobic domain (p < 0.0001). Even antibodies affinity-purified with the extracellular part of MOG recognized full-length MOG better than the extracellular part of MOG after transfection. The second hydrophobic domain of MOG enhanced the recognition of the extracellular part of MOG by antibodies from patients as seen with truncated variants of MOG. We confirmed the pivotal role of the second hydrophobic domain by fusing the intracellular part of MOG from the evolutionary distant opossum to the human extracellular part; the chimeric construct restored the antibody-binding completely. Further, we found that in contrast to 8-18C5, MOG-Abs from patients bound preferentially as F(ab’)2 rather than Fab. It was previously found that bivalent binding of human IgG1, the prominent isotype of MOG-Abs, requires that its target antigen is displayed at a distance of 13-16 nm. We found that, upon transfection, molecules of MOG did not interact so closely to induce a Förster resonance energy transfer (FRET) signal, indicating that they are more than 6 nm apart. We propose that the intracellular part of MOG holds the monomers apart at a suitable distance for bivalent binding; this could explain why a cell-based assay is needed to identify MOG-Abs. Our finding that MOG-Abs from most patients require bivalent binding has implications for understanding the pathogenesis of MOG-antibody-associated-disorders. Since bivalently bound antibodies have been reported to only poorly bind C1q, we speculate that the pathogenicity of MOG-Abs is mostly mediated by other mechanisms than complement activation. Therefore, therapeutic inhibition of complement activation should be less efficient in MOG-Ab associated disorders than in patients with Abs to aquaporin-4.

Structural dynamics of P-type ATPase ion pumps

2019 ◽  
Vol 47 (5) ◽  
pp. 1247-1257 ◽  
Author(s):  
Mateusz Dyla ◽  
Sara Basse Hansen ◽  
Poul Nissen ◽  
Magnus Kjaergaard
Keyword(s):  
Structural Dynamics ◽  
Single Molecule ◽  
New Technologies ◽  
Atp Hydrolysis ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Time Resolved ◽  
Dynamics Simulations ◽  
Types Of Information ◽  
P Type

Abstract P-type ATPases transport ions across biological membranes against concentration gradients and are essential for all cells. They use the energy from ATP hydrolysis to propel large intramolecular movements, which drive vectorial transport of ions. Tight coordination of the motions of the pump is required to couple the two spatially distant processes of ion binding and ATP hydrolysis. Here, we review our current understanding of the structural dynamics of P-type ATPases, focusing primarily on Ca2+ pumps. We integrate different types of information that report on structural dynamics, primarily time-resolved fluorescence experiments including single-molecule Förster resonance energy transfer and molecular dynamics simulations, and interpret them in the framework provided by the numerous crystal structures of sarco/endoplasmic reticulum Ca2+-ATPase. We discuss the challenges in characterizing the dynamics of membrane pumps, and the likely impact of new technologies on the field.

scholarly journals S ‐glutathiolation impairs phosphoregulation and function of cardiac myosin‐binding protein C in human heart failure

2016 ◽  
Vol 30 (5) ◽  
pp. 1849-1864 ◽  
Author(s):  
Konstantina Stathopoulou ◽  
Ilka Wittig ◽  
Juliana Heidler ◽  
Angelika Piasecki ◽  
Florian Richter ◽  
...  
Keyword(s):  
Heart Failure ◽  
Protein C ◽  
Human Heart ◽  
Binding Protein ◽  
Cardiac Myosin ◽  
Human Heart Failure ◽  
Myosin Binding Protein ◽  
Myosin Binding Protein C ◽  
Myosin Binding ◽  
And Function

scholarly journals Lipid Bilayer Composition Affects Transmembrane Protein Orientation and Function

2011 ◽  
Vol 2011 ◽  
pp. 1-9 ◽  
Author(s):  
Katie D. Hickey ◽  
Mary M. Buhr
Keyword(s):  
Lipid Bilayer ◽  
Lipid Composition ◽  
Protein Function ◽  
Transmembrane Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Outer Leaflet ◽  
Lipid Environment ◽  
Protein Incorporation ◽  
And Function

Sperm membranes change in structure and composition upon ejaculation to undergo capacitation, a molecular transformation which enables spermatozoa to undergo the acrosome reaction and be capable of fertilization. Changes to the membrane environment including lipid composition, specifically lipid microdomains, may be responsible for enabling capacitation. To study the effect of lipid environment on proteins, liposomes were created using lipids extracted from bull sperm membranes, with or without a protein (Na+K+-ATPase or -amylase). Protein incorporation, function, and orientation were determined. Fluorescence resonance energy transfer (FRET) confirmed protein inclusion in the lipid bilayer, and protein function was confirmed using a colourometric assay of phosphate production from ATP cleavage. In the native lipid liposomes, ATPase was oriented with the subunit facing the outer leaflet, while changing the lipid composition to 50% native lipids and 50% exogenous lipids significantly altered this orientation of Na+K+-ATPase within the membranes.

scholarly journals Rational design of protein-specific folding modifiers

2020 ◽  
Author(s):  
Anirban Das ◽  
Anju Yadav ◽  
Mona Gupta ◽  
R Purushotham ◽  
Vishram L. Terse ◽  
...  
Keyword(s):  
Single Molecule ◽  
Rational Design ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Force Measurements ◽  
Folding Nucleus ◽  
Dynamics Simulations ◽  
General Method

AbstractProtein folding can go wrong in vivo and in vitro, with significant consequences for the living cell and the pharmaceutical industry, respectively. Here we propose a general design principle for constructing small peptide-based protein-specific folding modifiers. We construct a ‘xenonucleus’, which is a pre-folded peptide that resembles the folding nucleus of a protein, and demonstrate its activity on the folding of ubiquitin. Using stopped-flow kinetics, NMR spectroscopy, Förster Resonance Energy transfer, single-molecule force measurements, and molecular dynamics simulations, we show that the ubiquitin xenonucleus can act as an effective decoy for the native folding nucleus. It can make the refolding faster by 33 ± 5% at 3 M GdnHCl. In principle, our approach provides a general method for constructing specific, genetically encodable, folding modifiers for any protein which has a well-defined contiguous folding nucleus.

scholarly journals Regulatory γ1 subunits defy symmetry in functional modulation of BK channels

2018 ◽  
Vol 115 (40) ◽  
pp. 9923-9928 ◽  
Author(s):  
Vivian Gonzalez-Perez ◽  
Manu Ben Johny ◽  
Xiao-Ming Xia ◽  
Christopher J. Lingle
Keyword(s):  
Single Channel ◽  
Regulatory Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Bk Channels ◽  
Bk Channel ◽  
Single Type ◽  
Regulatory Subunits ◽  
And Function

Structural symmetry is a hallmark of homomeric ion channels. Nonobligatory regulatory proteins can also critically define the precise functional role of such channels. For instance, the pore-forming subunit of the large conductance voltage and calcium-activated potassium (BK, Slo1, or KCa1.1) channels encoded by a single KCa1.1 gene assembles in a fourfold symmetric fashion. Functional diversity arises from two families of regulatory subunits, β and γ, which help define the range of voltages over which BK channels in a given cell are activated, thereby defining physiological roles. A BK channel can contain zero to four β subunits per channel, with each β subunit incrementally influencing channel gating behavior, consistent with symmetry expectations. In contrast, a γ1 subunit (or single type of γ1 subunit complex) produces a functionally all-or-none effect, but the underlying stoichiometry of γ1 assembly and function remains unknown. Here we utilize two distinct and independent methods, a Forster resonance energy transfer-based optical approach and a functional reporter in single-channel recordings, to reveal that a BK channel can contain up to four γ1 subunits, but a single γ1 subunit suffices to induce the full gating shift. This requires that the asymmetric association of a single regulatory protein can act in a highly concerted fashion to allosterically influence conformational equilibria in an otherwise symmetric K+channel.

Abstract 234: Transcriptional Profiling of Neuregulin-Induced Myocardial Recovery After Infarction in Rats and Swine

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Cristi L Galindo ◽  
Abigail Murphy ◽  
Michael Hill ◽  
John Cleator ◽  
Ehab Kasasbeh ◽  
...  
Keyword(s):  
Heart Failure ◽  
Therapeutic Potential ◽  
Transcriptional Profiling ◽  
Mapk Signaling ◽  
Left Ventricular ◽  
Experimental Myocardial Infarction ◽  
Human Heart Failure ◽  
Cardiovascular Tissue ◽  
And Function ◽  
Vehicle Treated Control

Neuregulin-1 (NRG-1) mediates cell-cell interactions and is a critical growth and developmental signaling molecule in the heart. We have been examining whether the recombinant NRG-1 isoform known as glial growth factor 2 (GGF2) has therapeutic potential for heart failure. In rats and swine with experimental myocardial infarction we have found that GGF2 treatment improves myocardial function and limits progressive myocardial remodeling. To understand potential mechanisms for this effect, we compared gene expression in these animals using microarrays. In rats we compared Sham operated, MI treated with vehicle, and MI treated with GGF2 at a single dose. We found that GGF2 treatment was associated with correction of mitochondrial and metabolic genes altered by MI compared to Sham-operated rats. When compared to 9 published datasets of ∼400 samples from rodents and human heart failure, we identified 563 genes associated with heart failure that were also reversed in expression in response to GGF2. Ingenuity pathway analysis demonstrated clusters of genes associated with energy production and cardiovascular tissue development as particularly enriched in GGF2-treated versus untreated MI rats. In swine our analysis was confined to animals with MI +/- GGF2 treatment at two doses. There were 527 genes altered by GGF2 at both doses compared to untreated controls, with a clear GGF2 dose response. Transcripts altered in response to GGF2 treatment were mainly those associated with extracellular matrix structure and function, MAPK signaling, and p53-mediated apoptosis. Electron microscopy of remote infarct left ventricular tissue from swine confirmed extreme morphological differences in mitochondria from GGF2-treated and vehicle-treated control pigs. Most striking was recovery of intercalated discs in response to GGF2, compared to severe disruption of intercalated disc structures in vehicle-treated control animals.

scholarly journals Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

2015 ◽  
Vol 112 (15) ◽  
pp. 4660-4665 ◽  
Author(s):  
Piyali Guhathakurta ◽  
Ewa Prochniewicz ◽  
David D. Thomas
Keyword(s):  
Light Chain ◽  
Contractile Function ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Catalytic Efficiency ◽  
Power Stroke ◽  
Time Resolved ◽  
Muscle Disorders ◽  
Essential Light Chain ◽  
C Terminus

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40–45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC’s location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATP-bound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.

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