Biology subjects

Seflova, J.

Publications and source records attributed to Seflova, J..

6 recordsLinked to original sources

bioRxiv2026

A Novel Mechanism of Cardiomyopathy: Toxic Peptides Dysregulate Calcium Transport

A hallmark of dilated cardiomyopathy (DCM) is calcium mishandling, including reduced transport activity of the SERCA calcium pump in cardiac muscle cells. This has focused attention on SERCA as mechanism of disease and potential therapeutic target. Previously, diminished SERCA activity has been attributed to decreased protein expression, but recent studies suggest SERCA levels are unchanged in DCM. Thus, another mechanism must be responsible for the deficit. Since proteolysis is increased and proteosome function is impaired in DCM, we reasoned that accumulation of toxic protein fragments may contribute to SERCA dysfunction. In particular, previous studies showed diverse species of hydrophobic -helices can inhibit SERCA, so we hypothesized that SERCA may become congested with transmembrane peptides that mimic endogenous regulatory partners. We purified cell membranes from non-failing and DCM human ventricles and subjected them to mass spectrometry to identify protein species upregulated in DCM. Select candidates were screened for binding and inhibition of SERCA. Several small membrane proteins and membrane protein fragments bound avidly to SERCA and significantly reduced cellular calcium stores. The data suggest a novel pathophysiological mechanism in which transmembrane protein debris obstructs SERCA function and regulation, contributing to cardiac muscle dysfunction in heart failure.

physiology↗

bioRxiv2023

Activation mechanism of the cardiac calcium pump by a small-molecule allosteric modulator

The discovery of small-molecule 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 (TCSPC) imaging approach to investigate the activation mechanism of a druggable protein by a small-molecule allosteric modulator. We tested this approach using the cardiac sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), an important pharmacological target that transports Ca2+ at the expense of ATP hydrolysis in the heart. We found that CDN1163, a validated SERCA2a activator, does not dissociate the endogenous complex between SERCA2a and its regulator phospholamban (PLN) in the presence of either Ca2+ or AMP-PCP, a non-hydrolyzable ATP analog. CDN1163 does not influence SERCA2as affinity for Ca2+ ions at functionally relevant conditions. Global analysis of the fluorescence lifetimes showed that ATP is both a substrate and a modulator that populates competent SERCA2a conformations. Interestingly, CDN1163 alone does not significantly induce changes in the structural populations of SERCA2a. Instead, CDN1163 potentiates the effects of ATP to further shift the equilibrium toward a competent SERCA2a conformation. Importantly, this population shift occurs at sub-physiological conditions, and within physiological Ca2+ concentrations at which SERCA2a operates. We propose an activation mechanism whereby a small-molecule modulator synergizes with ATP to stabilize a conformation of SERCA2a primed for activation. This study demonstrates the power of TCSPC to reveal novel insights into how structural and biochemical states are coupled to allosterically activate a pharmacological target in the heart.

biophysics↗

bioRxiv2023

Phospholamban Inhibits the Cardiac Calcium Pump Through Reversing the Allosteric Enhancement of Calcium Affinity by ATP

Phospholamban (PLB) is a transmembrane micropeptide that regulates the Ca2+ pump 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 the Ca2+-binding step in the SERCA enzymatic cycle, altering the Ca2+ dependence of cycling without affecting the true affinity of the Ca2+ binding 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 interrupted by PLB binding. Thus, PLB reduces the true Ca2+ affinity of SERCA by reversing allosteric activation of the pump by ATP. O_TEXTBOXSignificance StatementThe micropeptide phospholamban (PLB) serves a vital role as the "adrenaline trigger" for the heart. PLB inhibits the calcium pump SERCA at rest, and relief of this inhibition increases cardiac performance during exercise. Still, the physical mechanism for PLB inhibition of SERCA remains poorly understood. Here, our results reveal that PLB regulates SERCA by reversing an allosteric effect of ATP that enhances SERCAs calcium affinity. We mapped a novel allosteric pathway that structurally couples SERCAs ATP- and calcium-binding sites. PLB binds residues along this pathway to disrupt communication between the ligand-binding sites. The data reveal an allosteric mechanism that may be conserved in other ATP-dependent ion pumps. This allosteric pathway may provide new opportunities for pharmacological targeting of SERCA. C_TEXTBOX

biochemistry↗

bioRxiv2023

RyR2 Binding of an Antiarrhythmic Cyclic Depsipeptide Mapped Using Confocal Fluorescence Lifetime Detection of FRET

Hyperactivity of cardiac sarcoplasmic reticulum (SR) ryanodine receptor (RyR2) Ca2+-release channels contributes to heart failure and arrhythmias. Reducing 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 Forster 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). Multi-exponential 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.

biophysics↗

bioRxiv2021

STOICHIOMETRY OF THE SODIUM PUMP-PHOSPHOLEMMAN REGULATORY COMPLEX

The sodium-potassium ATPase (NKA) establishes ion gradients that facilitate many physiological processes. In the heart, NKA activity is regulated by its interaction with phospholemman (PLM, FXYD1). Here we used a novel fluorescence lifetime-based assay to investigate the structure, stoichiometry, and affinity of the NKA-PLM regulatory complex. We observed concentration dependent association of the subunits of NKA-PLM regulatory complex, with avid association of the alpha subunit with the essential beta subunit followed by lower affinity alpha-alpha and alpha-PLM interactions. The data provide the first evidence that the regulatory complex is composed of two alpha subunits associated with two beta subunits, decorated with two PLM regulatory subunits in intact cells. Docking and molecular dynamics 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 stoichiometry (alpha-beta-PLM)2.

biophysics↗

bioRxiv2021

Inhibitory and Stimulatory Micropeptides Preferentially Bind to Different Conformations of the Cardiac Calcium Pump

The ATP-dependent ion pump SERCA sequesters Ca2+ in the endoplasmic reticulum to establish a reservoir for cell signaling. Because of its central importance in physiology, this transporter is tightly controlled by physical interactions with tissue-specific regulatory micropeptides that tune SERCA function to match changing physiological conditions. In the heart, 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. The distinct functions of these peptides may relate to their reciprocal preferences for SERCA binding. While SERCA binds PLB more avidly at low cytoplasmic Ca2+, it binds DWORF better at high Ca2+. In the present study, we determined that this opposing Ca2+ sensitivity is due to preferential binding of DWORF and PLB to different intermediate conformations that the pump samples during the Ca2+ transport cycle. The results suggest a mechanistic basis for inhibitory and stimulatory micropeptide function. In addition, fluorescence resonance energy transfer (FRET) measurements revealed dynamic shifts in SERCA-micropeptide binding equilibria during cellular Ca2+ elevations. The data suggest Ca2+-dependent dynamic exchange of inhibitory and stimulatory micropeptides from SERCA during the cardiac cycle. Together, these mechanisms provide beat-to-beat modulation of cardiac Ca2+ handling and contribute to the hearts adaptation to the increased physiological demands of exercise. O_TEXTBOXSignificance Interactions between SERCA and its regulatory micropeptides modulate cardiac performance. A previous study demonstrated that while the SERCA inhibitor, phospholamban, loses affinity during intracellular Ca2+ elevations, its competitor DWORF stimulates SERCA and binds better at elevated Ca2+. Here, we found this Ca2+-dependent difference in affinity is driven by preferential micropeptide binding to different SERCA conformations sampled during its enzymatic cycle. Tight binding to different conformations of the pump may underly the distinct mechanistic functions of these regulators. Lower affinity during alternating phases of the cardiac cycle may drive dynamic exchange of inhibitory and stimulatory micropeptides during contraction and relaxation. Our results reveal mechanisms that modulate cardiac Ca2+ handling on a beat-to-beat basis and help the heart adapt to exercise. C_TEXTBOX

biophysics↗