Biology subjects

Kekenes-Huskey, P.

Publications and source records attributed to Kekenes-Huskey, P..

10 recordsLinked to original sources

bioRxiv2026

Allosteric Mechanisms Underlying Long QT Syndrome Type 2 (LQT2) Associated Mutations in hERG Channels

1Long QT syndrome Type 2 (LQT2) is a genetic disorder caused by missense mutations in the KCNH2 gene that encodes the potassium channel KV11.1. Previous studies have shown that most KV11.1 missense mutations with loss-of-function phenotypes result from impaired trafficking from the endoplasmic reticulum to the plasma membrane. To investigate the molecular basis of these defects, we used molecular dynamics simulations to analyze two sets of disease-associated missense mutations: those that suppress and those that maintain normal channel trafficking. We focused initially on the conformational and dynamics differences between wild-type and several mutants of KV11.1 via molecular dynamics simulations when two K+ were placed in the selectivity filter (SF). Our study reveals that missense mutations in the S4 helix allosterically disrupt the selectivity filter, a critical determinant for proper channel trafficking. Trafficking-competent variants largely retained a wild-type selectivity filter structure, whereas trafficking-deficient mutants exhibited pronounced structural perturbations in this region. These findings suggest that certain LQT2-associated missense mutations in KCNH2 impair channel trafficking by compromising the structural integrity of the selectivity filter. We additionally found that second-site variants Y652C in the drug binding vestibule can correct structural defects associated with some mistrafficking variants.

biophysics↗

bioRxiv2024

Molecular mechanisms of GSK3β-driven modulation of ABLIM1 and titin interactions in cardiac muscle

1The heart adapts to cardiac demand through various mechanisms, including chemical modifications of myofilament proteins responsible for cell contraction. Many of these modifications, such as phosphorylation, occur in unstructured, or intrinsically disordered, regions (IDRs) of proteins. Although often challenging to study, these IDRs are increasingly recognized as dynamic, tunable regulators of protein function. Given that cardiac dysfunction can involve changes in the post-translational modification (PTM) status of myofilament proteins, it is critical to assess how alterations within these disordered regions impact intact protein and myofilament behavior. We hypothesized that the function of ABLIM1, a myofilament protein containing an important IDR, is regulated by altering its IDR conformational ensemble through PTMs, primarily phosphorylation. We proposed that this conformational change would modulate its ability to bind to other myofilament proteins. To evaluate this hypothesis, we employed a multiscale modeling approach including molecular dynamics simulations. This was used to predict the conformational ensembles of ABLIM1 before and after phosphorylation, at sites known to be altered in a canine model of heart failure with reduced GSK3{beta} activity. We then used a state-based model of contraction to rationalize the physiological consequences of the molecular-scale predictions. Based on our data, we observed that local physicochemical alterations induced by phosphorylation in ABLIM1s intrinsically disordered regions significantly affect its overall conformational ensemble properties. This ensemble change subsequently influences the ability of its LIM domains to interact with titin. Furthermore, using the contraction model, we show that a reduced ability to recruit myosin heads for cross-bridge formation, resulting from the modified LIM domain/titin interactions, provides a mechanism that elucidates previous findings of diminished length-dependent activation. These findings offer crucial molecular insights, reframing IDRs not merely as structural noise but as key, tunable elements that control protein interactions and ultimately impact mechanical behavior in the sarcomere. This work bridges molecular disorder and biomechanical function, providing a new lens to understand dynamic control and dysfunction in cardiomyocyte contraction.

biophysics↗

bioRxiv2021

Prediction of hERG potassium channel PAS-domain variants trafficking via machine learning

1Congenital long QT syndrome (LQTS) is characterized by a prolonged QT-interval on an electrocardiogram (ECG). An abnormal prolongation in the QT-interval increases the risk for fatal arrhythmias despite otherwise normal metrics of cardiac function. Genetic variants in several different cardiac ion channel genes, including KCNH2, are known to cause LQTS. The population frequency of rare non-synonymous (missense) variants in LQTS-linked genes far outpaces the true incidence of the disease. Therefore, only a small percentage of missense variants identified in LQTS-linked genes are expected to associate with LQTS. Because of a lack of clear association between variants identified in LQTS-linked alleles and diseases, most variants are classified as variants of uncertain physiological significance (VUS). Here, we evaluated whether structure-based molecular dynamics (MD) simulations and machine learning (ML) can improve the identification of missense variants in LQTS-linked genes that associate with LQTS. To do this, we focused on investigating KCNH2 missense variants in the Kv11.1 channel protein shown to have wild type (WT) like or loss-of-function (LOF) phenotypes in vitro. We focused on KCNH2 missense variants that disrupt normal Kv11.1 channel protein trafficking, as it is the most common LOF phenotype for LQTS-associated variants. Specifically, we used these computational techniques to correlate structural and dynamic changes in the Kv11.1 channel protein PAS domain (PASD) with Kv11.1 channel protein trafficking phenotypes. These simulations unveiled several molecular features, including the numbers of hydrating waters and H-Bonds, as well as FoldX scores, that are predictive of trafficking. We then used statistical and ML (Decision tree (DT), Random forest (RF), and Support vector machine (SVM)) techniques to classify variants using these simulation-derived features. Together with bioinformatics data, such as sequence conservation and folding energies, we were able to predict with reasonable accuracy ({approx}75%) which KCNH2 variants do not traffic normally. We conclude, structure-based simulations of KCNH2 variants localized to the Kv11.1 channel PASD led to a significant improvement ({approx}10%) in classification accuracy and this approach should therefore be considered to complement the classification of VUS in the Kv11.1 channel PASD.

bioinformatics↗

bioRxiv2021

Live Monitoring of Inflammation Reveals Tissue and Sex-specific Responses to Western Diet and Butyrate treatment

Obesity is a current epidemic, affecting millions of individuals worldwide. Chronic obesity is characterized by a low-grade systemic inflammation besides not being a classic inflammatory disease. Many studies have tried to identify inflammatory insults dysregulated by a Westernized diet - consisted of high fat, high sucrose, and high cholesterol -mainly focusing on production and secretion of inflammatory cytokines. The gut microbiome and derived metabolites, including the short-chain fatty acid butyrate, have received increased attention as underlying some of the obesogenic features. In the present work, we utilized a novel biosensor mouse model capable of monitoring in vivo inflammation. We observed tissue- and sex- specific caspase-1 activation patterns in obese mice and treated with butyrate. Our work utilizing a caspase-1 biosensor mouse model, flow cytometry and computational analyses and offers new mechanistic insights underlying the effect of butyrate in obesity and its complications.

neuroscience↗

bioRxiv2021

Crowding within synaptic junctions influence the degradation of adenoside nucleotides by CD39 and CD73 ectonucleotidases

1Synapsed cells can communicate using exocytosed nucleotides like adenosine triphosphate (ATP). Ectonucleotidases localized to a synaptic junction degrade such nucleotides into metabolites like adenosine monophosphate (AMP) or adenosine, oftentimes in a sequential manner. CD39 and CD73 are a representative set of coupled ectonucleotidases, where CD39 first converts ATP and adenosine diphosphate (ADP) into AMP, after which the AMP product is dephosphorylated into adenosine by CD73. Hence, CD39/CD73 help shape cellular responses to extracellular ATP. In a previous study [1] we demonstrated that the rates of coupled CD39/CD73 activity within synapse-like junctions are strongly controlled by the enzymes co-localization, their surface charge densities, and the electrostatic potential of the surrounding cell membranes. In this study, we demonstrate that crowders within a synaptic junction, which can include globular proteins like cytokines and membrane-bound proteins, impact coupled CD39/CD73 electronucleotidase activity and in turn, the availability of intrasynapse ATP. Specifically, we simulated a spatially-explicit, reaction-diffusion model for the coupled conversion of ATP[->]AMP and AMP[->]adenosine in a model synaptic junction with crowders via the finite element method. Our modeling results suggest that the association rate for ATP to CD39 is strongly influenced by the density of intrasynaptic protein crowders, as increasing crowder density suppressed ATP association kinetics. Much of this suppression can be rationalized based on a loss of configurational entropy. The surface charges of crowders can further influence the association rate, with the surprising result that favorable crowder/nucleotide electrostatic interactions can yield CD39 association rates that are faster than crowder-free configurations. However, attractive crowder/nucleotide interactions decrease the rate and efficiency of adenosine production, which in turn increases the availability of ATP and AMP within the synapse relative to crowder-free configurations. These findings highlight how CD39/CD73 ectonucleotidase activity, electrostatics and crowding within synapses influence the availability of nucleotides for intercellular communication.

cell biology↗

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↗

bioRxiv2021

Molecular dynamics simulations of zfP2X4 receptors

The P2X4 receptor plays a prominent role in cellular responses to extracellular ATP. Through classical all-atom molecular dynamics (MD) simulations totaling 24 {micro}s we have investigated how metal-complexed ATP stabilizes the channels open state and prevents its closing. We have identified two metal-binding sites, magnesium (Mg2+) and potassium (K+), one at the intersection of the three subunits in the ectodomain (MBS1) and the second one near the ATP binding site (MBS2), similar to those characterized in Gulf coast P2X. Our data indicate that when Mg2+ and K+ ions are complexed with ATP, the channel is locked into an open state. Interestingly, irrespective of the number of bound ATP molecules, Mg2+ ions bound to the MBS2 resisted collapsing of the open state protein to a closed state by stabilizing the ATP-protein interactions. However, when Mg2+ in the MBS2 was replaced with K+ ions, as might be expected when in equilibrium with an extracellular solution, the interactions between the subunits were weakened and we found evidence of pore collapse. This collapse was apparent when fewer than two ATP were bound to MBS2 in the presence of K+. Therefore, the different capacities of common cations to stabilize the channel may underlie a mechanism governing P2X4 channel gating in physiological systems. This study provides structural insights into the differential modulation of ATP activation of P2X4 by Mg2+ and K+.

physiology↗

bioRxiv2020

Structural determinants of calcium binding beyond the EF-hand binding site: A study of alpha parvalbumins

1Parvalbumin (PV) is a calcium binding protein expressed in humans, fish and avian species. In these organisms, the calcium (Ca2+) affinities of specific PV isoforms can vary by orders of magnitude. Despite the availability of high resolution structural data for many PV isoforms, the structural bases for how such proteins confer widely-varying divalent Ca2+ affinities and selectivities against common ions like magnesium (Mg2+) has been difficult to rationalize. We therefore conducted molecular simulations of several -pavalbumin (-parvalbumin (PV)) constructs with Ca2+ affinities in the micromolar to nanomolar ranges to identify properties of conformations that contribute to their wide-ranging binding constants and selectivities against Mg2+. Specifically, we examined a D94S/G98E construct with a reported lower Ca2+ affinity ({approx} -18.2 kcal/mol) relative to the WT ({approx} -22 kcal/mol), an S55D/E59D variant with enhanced affinity ({approx} -24 kcal/mol), and a truncated variant of PV with weak affinity ({approx} -12.6 kcal/mol). We performed molecular dynamics simulations of these constructs and assessed their Ca2+ and Mg2+ binding properties using scores from molecular mechanics generalized Born approximation (MM/GBSA), ion/oxygen coordination patterns and thermodynamics via mean spherical approximation (MSA) theory, as well as via metrics of protein structure and hydration. Our key findings are that although MM/GBSA and MSA scores successfully rank-ordered the variants according to their previously-published affinities and Mg2+ selectivity, importantly, properties of Ca2+ loops in CBPs such as coordination, and charge are alone insufficient to rationalize their binding properties. Rather, Ca2+ affinity and selectivity against Mg2+ are emergent properties stemming from both local effects within the proteins ion binding sites as well as non-local contributions from protein folding and solubility. Our findings broaden our understanding of the molecular bases governing PV ion binding that are likely shared by many Ca2+ binding proteins.

biophysics↗

bioRxiv2019

Molecular basis of calmodulin-dependent calcineurin activation

Calcineurin (CaN) is a calcium-dependent phosphatase involved in numerous signaling pathways. Its activation by Ca2+ is in part driven by binding of calmodulin (CaM) to a Calmodulin (CaM)-recognition motif within the phosphatases regulatory domain (RD); how-ever, secondary interactions between CaM and the Calcineurin (CaN) regulatory domain may be necessary to fully activate CaN [1]. Specifically, it has been shown that the CaN regulatory domain folds upon CaM binding and that there is a region C-terminal to the canonical CaM-binding region, the distal helix, that assumes an helix fold and contributes to activation [1]. We hypothesized in Dunlap et al [1] that this putative helical distal helix is capable of binding CaM in a region distinct from the canonical CaM binding region (CaMBR) site, whereby CaN is activated. To test this hypothesis, we utilized molecular simulations including replica-exchange molecular dynamics, protein-protein docking and computational mutagene-sis to model distal helix conformations. From these simulations we have isolated a potential binding site on CaM (site D) that facilitates moderate affinity inter-protein interactions that may attenuate CaN auto-inhibition. Further, molecular simulations of the distal helix A454E mutation demonstrated weakened distal helix/CaM interactions that were previously shown to impair CaN activity. K30E and G40D mutations of CaM at site D presented similar de-creases in binding affinity predicted by simulations. The prediction was correlated with a phosphatase assay in which these two mutants show reduced CaN activity. This study there-fore provides a potential structural basis for the role of secondary CaM/CaN interactions in mediating CaN activation.

molecular biology↗

bioRxiv2018

Simulation of P2X-mediated calcium signaling in microglia

Microglia function is orchestrated through highly-coupled signaling pathways that depend on calcium (Ca2+). In response to extracellular adenosine triphosphate (ATP), transient increases in intracellular Ca2+ driven through the activation of purinergic receptors, P2X and P2Y, are sufficient to promote cytokine synthesis and potentially their release. While steps comprising the pathways bridging purinergic receptor activation with transcriptional responses have been probed in great detail, a quantitative model for how these steps collectively control cytokine production has not been established. Here we developed a minimal computational model that quantitatively links extracellular stimulation of two prominent ionotropic puriner-gic receptors, P2X4 and P2X7, with the graded production of a gene product, namely the tumor necrosis factor (TNF) cytokine. In addition to Ca2+ handling mechanisms common to eukaryotic cells, our model includes microglia-specific processes including ATP-dependent P2X4 and P2X7 activation, activation of NFAT transcription factors, and TNF production. Parameters for this model were optimized to reproduce published data for these processes, where available. With this model, we determined the propensity for TNF production in microglia, subject to a wide range of ATP exposure amplitudes, frequencies and durations that the cells could encounter in vivo. Furthermore, we have investigated the extent to which modulation of the signal transduction pathways influence TNF production. Our key findings are that TNF production via P2X4 is maximized at low ATP when subject to high frequency ATP stimulation, whereas P2X7 contributes most significantly at millimolar ATPranges. Given that Ca2+ homeostasis in microglia is profoundly important to its function, this computational model provides a quantitative framework to explore hypotheses pertaining to microglial physiology.

systems biology↗