Biology subjects

Dubey, V.

Publications and source records attributed to Dubey, V..

6 recordsLinked to original sources

bioRxiv2025

Understanding the Mechanisms Behind the Annuloplasty Effect in Tricuspid Valve TEER: A Computational Study

BackgroundAn annuloplasty effect has been observed following tricuspid transcatheter edge-to-edge repair (TEER) and has been shown to have a therapeutic benefit. However, the mechanisms underlying the annuloplasty effect remain unknown. ObjectivesWe investigate the impact of TEER-induced annular forces on the annuloplasty effect. Additionally, we explored the impact of clip size, clip orientation, leaflet pair, and leaflet site on TEER-induced annular forces. MethodsWe simulated 36 TEER repairs in finite element models of three human tricuspid valves. We used either a NTW or XTW TriClip. The clip was placed between either the anterior-septal or anterior-posterior leaflet pairs, in either the central or near-annulus site. For each scenario, we quantified the reduction in annular area, septal-lateral (SL) diameter, and anterior-posterior (AP) diameter. We also report the total annular force, orientation of maximum annular force, total papillary muscle force, leaflet stress, and coaptation area ratio following TEER. ResultsTEER induced annular forces, which strongly predicted the annuloplasty effect as measured by reduction in annular area and SL diameter. The maximum annular force aligned with the orientation of the clip. Furthermore, XTW clips induced more annular force and leaflet stress than NTW clips. A central anterior-posterior site induced more force than a near-annulus anterior-posterior site, but showed no difference from an anterior-septal pair. ConclusionsTEER procedural parameters, such as clip size and site, strongly influence the magnitude of induced annular forces, which, in turn, correlate with the degree of annuloplasty effect.

bioengineering↗

bioRxiv2025

In vivo anti-tumor activity of high-dose parenteral ascorbic acid is mediated primarily via cofactor activity, not via oxidative stress.

The anti-tumor effect of high-dose ascorbic acid (AA) has been demonstrated in multiple in vitro and in vivo cancer models with the postulation of two primary categories of mechanisms: antioxidant/cofactor activity and H2O2-mediated oxidative damage. Both mechanisms have been conclusively demonstrated in vitro. However, while parenteral high-dose AA-induced cofactor activity (TET-mediated DNA demethylation and prolyl/asparaginyl hydroxylase-mediated HIF activity inhibition via reduction of enzymatic Fe3+ to Fe2+) has been demonstrated intratumorally in vivo in multiple models, the cumulative data on parenteral high-dose AA-induced intratumoral oxidative damage in vivo has been inconclusive. Furthermore, the relative contribution of the seemingly opposing mechanisms towards in vivo anti-cancer activity has not been studied concurrently. We therefore sought to definitively delineate the roles of both antioxidant/cofactor activity and prooxidant functions of high-dose AA in the in vivo anti-tumor response. Using two syngeneic mouse tumor models, the AA-sensitive A20 model and the AA-resistant Renca model, we assessed markers of DNA and lipid oxidative damage as well as the specific roles of TET2 and AA transporter SLC23A2 in the anti-tumor response to parenteral high-dose AA. In the sensitive A20 model, loss of either Tet2 or Slc23a2 fully reversed anti-tumor activity. Similarly, overexpression of Tet2 in the resistant Renca model (which expresses high baseline levels of AA transporters SLC23A1 and SLC23A2, but does not express TET2), resulted in increased CD8+T cell infiltration and dramatic reduction in tumor growth overall. In both A20 and Renca models, high-dose parenteral AA increased total intratumoral antioxidant capacity, and this was attenuated by Slc23a2 knockdown in A20. High-dose AA treatment also resulted in a Tet2- and Slc23a2-dependent increase in intratumoral 5-hydroxymethylcytosine. Intracellular oxidative damage markers, 8-OHdG and 4-HNE, were not induced in tumors by high-dose AA in either model. In contrast, these markers were robustly induced in vitro by high-dose AA in A20 and Renca cells. Using dynamic real-time extracellular H2O2 measurements with high-dose AA, difference in molecular oxygen concentration between standard in vitro and hypoxic in vivo conditions was identified as an important factor underlying the marked discrepancy between the abundant in vitro and absent in vivo intratumoral oxidative stress with high-dose AA. Furthermore, using additional syngeneic models resistant (MB49) and sensitive (MC38) to AA-induced potentiation of anti-PD1 checkpoint inhibition, we demonstrate that very low catalase expression does not confer sensitivity to high-dose AA in vivo (further arguing against the H2O2 mechanism in vivo), that TET2 expression alone is not sufficient to drive an AA-induced anti-tumor response (either as a single agent or in combination with immunotherapy), and that high-dose AA can significantly enhance the efficacy of anti-PD1 immunotherapy even in the absence of single-agent activity. Our data strongly indicate that the in vivo anti-tumor effect of high-dose parenteral AA-including potentiation of immunotherapy-is mediated primarily by its specific antioxidant/cofactor activity (with TET2 expression likely being necessary but certainly not sufficient), and not via oxidative stress. Collectively, the study represents a paradigm shift in our understanding of the cumulative mechanisms of in vivo anti-cancer activity of high-dose AA, with critical implications not just for the clinical translation of AA as an anti-cancer agent (including in enhancing immunotherapy efficacy) but also the field of free radical biology. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/655101v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@efc305org.highwire.dtl.DTLVardef@ecb41corg.highwire.dtl.DTLVardef@24ac2dorg.highwire.dtl.DTLVardef@1a3f0b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2021

An intracellular pathway controlled by the N-terminus of the pump subunit inhibits the bacterial KdpFABC ion pump in high K+ conditions

The heterotetrameric bacterial KdpFABC transmembrane protein complex is an ion channel-pump hybrid that consumes ATP to import K+ against its transmembrane chemical potential gradient in low external K+ environments. The KdpB ion-pump subunit of KdpFABC is a P-type ATPase, and catalyses ATP hydrolysis. Under high external K+ conditions, K+ can diffuse into the cells through passive ion channels. KdpFABC must therefore be inhibited in high K+ conditions to conserve cellular ATP. Inhibition is thought to occur via unusual phosphorylation of residue Ser162 of the TGES motif of the cytoplasmic A domain. It is proposed that phosphorylation most likely traps KdpB in an inactive E1-P like conformation, but the molecular mechanism of phosphorylation-mediated inhibition and the allosteric links between phosphorylation on the A domain and the inactivation of the pump remain unknown. Here, we employ molecular dynamics (MD) simulations of the dephosphorylated and phosphorylated versions of KdpFABC to demonstrate that phosphorylated KdpB is trapped in a conformation where the ion-binding site is hydrated by an intracellular pathway between transmembrane helices M1 and M2 which opens in response to the rearrangement of cytoplasmic domains resulting from phosphorylation. Cytoplasmic access of water to the ion-binding site is accompanied by a remarkable loss of secondary structure of the KdpB N-terminus and disruption of a key salt bridge between Glu87 in the A domain and Arg212 in the P domain. Our results provide the molecular basis of a unique mechanism of regulation amongst P-type ATPases, and suggest that the N-terminus has a significant role to play in the conformational cycle and regulation of KdpFABC

biophysics↗

bioRxiv2019

Cholesterol binding to the sterol-sensing region of Niemann Pick C1 protein confines dynamics of its N-terminal domain

Lysosomal accumulation of cholesterol is a hallmark of Niemann Pick type C (NPC) disease caused by mutations primarily in the lysosomal membrane protein NPC1. NPC1 contains a transmembrane sterol sensing domain (SSD), which is supposed to regulate protein activity upon cholesterol binding, but the mechanisms underlying this process are poorly understood. Using atomistic simulations, we show that the binding of cholesterol to the SSD of NPC1 suppresses conformational dynamics of the luminal domains which otherwise bring the luminal N-terminal domain (NTD) closer to the lipid bilayer. The presence of an additional 20% membrane cholesterol has negligible impact on this process. We propose that cholesterol acts as an allosteric effector, and the modulation of NTD dynamics by the SSD-bound cholesterol constitutes an allosteric feedback mechanism in NPC1 which controls cholesterol abundance in the lysosomal membrane.

biophysics↗

bioRxiv2019

A single K+-binding site in the crystal structure of the gastric proton pump

The gastric proton pump (H+,K+-ATPase), a P-type ATPase responsible for gastric acidification, mediates electro-neutral exchange of H+ and K+ coupled with ATP hydrolysis, but with an as yet undetermined transport stoichiometry. Here we show crystal structures at a resolution of 2.5 [A] of the pump in the E2-P transition state, in which the counter-transporting cation is occluded. We found a single K+ bound to the cation-binding site of H+,K+-ATPase, indicating an exchange of 1H+/1K+ per hydrolysis of one ATP molecule. This fulfils the energy requirement for the generation of a six pH unit gradient across the membrane. The structural basis of K+recognition is resolved, supported by molecular dynamics simulations, and this establishes how H+,K+-ATPase overcomes the energetic challenge to generate an H+ gradient of more than a million-fold - the highest cation gradient known in any mammalian tissue - across the membrane.

biochemistry↗

bioRxiv2019

Flexible pivoting of the pleckstrin domain domain in dynamin catalyzes fission by affecting membrane properties: Insights into the molecular degrees of freedom

The neuronal dynamin1 functions in the release of synaptic vesicles by orchestrating the process of GTPase-dependent membrane fission. Dynamin1 associates with the plasma membrane-localized phosphatidylinositol-4,5-bisphosphate (PIP2) with its centrally-located pleckstrin homology domain (PHD). The PHD is dispensable as fission can be managed, albeit at much slower rates, even when the PHD-PIP2 interaction is replaced by a generic polyhistidine- or polylysine-lipid interaction. However, even when the PHD is present, the length of the dynamin scaffold and in turn the membrane remodeling and fission rates are severely restricted with mutations such as I533A on membrane-interacting variable loop 1 (VL1) of PHD. These observations suggest that PIP2-containing membrane interactions of PHD could have evolved to expedite fission to fulfill the requirement of rapid kinetics of synaptic vesicle recycling. Here, we use a suite of multiscale modeling approaches that combine atomistic molecular dynamics simulations, mixed resolution membrane mimetic models, coarse-grained molecular simulations and advanced free-energy sampling methods (metadynamics and umbrella sampling) to explore PHD-membrane interactions. Our results reveal that: (a) the binding of PHD to PIP2-containing membranes modulates the lipids towards fission-favoring conformations and softens the membrane, (b) that PHD engages another loop (VL4) for membrane association, which acts as an auxiliary pivot and modulates the orientation flexibility of PHD on the membrane - a mechanism we believe may be important for high fidelity dynamin collar assembly on the membrane. (c) Through analyses of our trajectories data and free-energy calculations on membrane-bound WT and mutant systems, we also identify key residues on multiple VLs that stabilizes PHD membrane association. And we suggest experiments to explore the ability of PHD to associate with membrane in orientations that favors faster fission. Together, these insights provide a molecular-level understanding of the "catalytic" role of the PHD in dynamin-mediated membrane fission. SIGNIFICANCEDynamin, a large multi-domain GTPase, remodels the membrane by self-assembling onto the neck of a budding vesicle and induces fission by its energy driven conformational changes. In this work, we use multi-scale molecular simulations to probe the role of dynamins pleckstrin-homology domain (PHD), which facilitates membrane interactions. Notably, PHD is dispensable for fission as is the case with extant bacterial and mitochondrial dynamins. However, reconstitution experiments suggest that the functional role of PHD in neuronal-membrane goes beyond that of an adaptor domain as it possibly expedites the fission reaction during synaptic vesicle recycling. We provide a molecular-dynamics picture of how PHDs make membranes more pliable for fission and suggest new insights into the molecular-level processes driving the expedited fission behavior.

biophysics↗