Biology subjects

Desetty, R.

Publications and source records attributed to Desetty, R..

4 recordsLinked to original sources

bioRxiv2026

AZD5069 Inhibits Angiogenesis Without Cytotoxicity In Human Endothelial Cell Culture

BackgroundThe CXCR2 receptor pathway plays a major role in inflammatory and invasive angiogenesis in human disease. ObjectiveWe evaluated AZD5069, a selective CXCR2 antagonist, as an angiogenesis inhibitor in human cell culture. MethodsHuman Umbilical Venous Endothelial Cells (HUVECs), Human Aortic Endothelial Cells (HAECs), and Human Pulmonary Artery Endothelial Cells (HPAECs) were cultured with standard in vitro techniques. AZD5069 (0, 8, 16, 32, 64, 128, 256 M) was evaluated as an angiogenesis inhibitor with fluorescent-labeled 5-Ethynyl-2-deoxyuridine (EdU) uptake to quantify endothelial cell proliferation, scratch assay to quantify endothelial cell migration, and Geltrex assay to quantify endothelial cell tubule and hub formation. AZD5069 cytotoxicity was evaluated with in situ terminal deoxynucleotidyl transferase 2-Deoxyuridine triphosphate- 5 (dUTP) nick-end labeling (TUNEL) to quantify apoptosis and membrane-impermeable cyanine dye uptake to quantify necrotic cell death. ResultsAZD5069 significantly reduced HUVEC, HAEC, and HPAEC proliferation, migration, tubule count, total tubule length, and node count with a dose-response. AZD5069 did not cause apoptosis nor necrotic cell death. ConclusionsAZD5069 inhibited angiogenesis without cytotoxicity in human endothelial cell culture. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility in cardiovascular, oncologic, and inflammatory disease. Condensed AbstractThe CXCR2 receptor pathway plays a major role regulating angiogenesis in inflammation and cancer. The CXCR2 receptor pathway has been evaluated in humans as a target for therapy in inflammatory disease and cancer but not as a therapeutic approach to block pathologic angiogenesis. AZD5069 is a clinical stage, direct CXCR2 antagonist. In human endothelial cell culture, AZD5069 inhibited angiogenesis without causing apoptosis or necrotic cell death. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility as a novel angiogenesis blocker in human disease. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=137 SRC="FIGDIR/small/731993v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@1b0bd3forg.highwire.dtl.DTLVardef@58ad69org.highwire.dtl.DTLVardef@1e0cdcdorg.highwire.dtl.DTLVardef@1e7708b_HPS_FORMAT_FIGEXP M_FIG Visual Abstract: AZD5069, a selective CXCR2 antagonist, significantly reduced endothelial cell proliferation, migration, and vascular tubule formation without causing necrotic or apoptotic cell death. The endothelial cell CXCR2 receptor pathway may be a novel target for anti-angiogenesis therapy. AZD5069 may have clinical utility in human cardiovascular, oncologic, and inflammatory disease with pathologic, dysregulated, or excessive angiogenesis. C_FIG

pharmacology and toxicology↗

bioRxiv2023

Dilated cardiomyopathy mutation in beta-cardiac myosin enhances actin activation of the power stroke and phosphate release

Inherited mutations in human beta-cardiac myosin (M2{beta}) can lead to severe forms of heart failure. The E525K mutation in M2{beta} is associated with dilated cardiomyopathy (DCM) and was found to stabilize the interacting heads motif (IHM) and autoinhibited super-relaxed (SRX) state in dimeric heavy meromyosin. However, in monomeric M2{beta} subfragment 1 (S1) we found that E525K enhances (3-fold) the maximum steady-state actin-activated ATPase activity (kcat) and decreases (6-fold) the actin concentration at which ATPase is one-half maximal (KATPase). We also found a 3 to 4-fold increase in the actin-activated power stroke and phosphate release rate constants at 30 M actin, which overall enhanced the duty ratio 3-fold. Loaded motility assays revealed that the enhanced intrinsic motor activity translates to increased ensemble force in M2{beta} S1. Glutamate 525, located near the actin binding region in the so-called activation loop, is highly conserved and predicted to form a salt-bridge with another conserved residue (lysine 484) in the relay helix. Enhanced sampling molecular dynamics simulations predict that the charge reversal mutation disrupts the E525-K484 salt-bridge, inducing conformations with a more flexible relay helix and a wide phosphate release tunnel. Our results highlight a highly conserved allosteric pathway associated with actin activation of the power stroke and phosphate release and suggest an important feature of the autoinhibited IHM is to prevent this region of myosin from interacting with actin. The ability of the E525K mutation to stabilize the IHM likely overrides the enhanced intrinsic motor properties, which may be key to triggering DCM pathogenesis. Significance StatementHeart disease can be caused by inherited mutations in beta-cardiac myosin, the molecular motor that powers systolic contraction in the ventricles of the heart. However, it remains unclear how these mutations lead to contractile dysfunction and pathogenic remodeling of the heart. We investigated a unique dilated cardiomyopathy mutation (E525K) that dramatically stabilizes the autoinhibited state while enhancing intrinsic motor function. Thus, we examined how this mutation impacts transient kinetic steps of the ATPase cycle, motile properties, and structural changes associated with the power stroke and phosphate release. Our results provide a kinetic and structural basis for how beta-cardiac myosin mutations may disrupt molecular-level contractile function in complex ways, which may inform the development of targeted therapeutics.

biophysics↗

bioRxiv2022

Dilated cardiomyopathy mutation E525K in human beta-cardiac myosin stabilizes the interacting heads motif and super-relaxed state of myosin

The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain EM and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin. Significance StatementDilated cardiomyopathy can be caused by single point mutations in cardiac muscle myosin, the motor protein that powers contraction of the myocardium. We found that the E525K DCM mutation in the cardiac myosin heavy chain stabilizes the auto-inhibited, super-relaxed state, suggesting a mechanism by which this mutation reduces muscle force and power. The E525K mutation also highlights critical electrostatic interactions important for forming the conserved, auto-inhibited conformational state of striated muscle myosins.

biophysics↗

bioRxiv2020

Cardiomyopathy Mutations Impact the Power Stroke of Human Cardiac Myosin

Cardiac muscle contraction is driven by the molecular motor myosin that uses the energy from ATP hydrolysis to generate a power stroke when interacting with actin filaments, while it is unclear how this mechanism is impaired by mutations in myosin that can lead to heart failure. We have applied a Forster resonance energy transfer (FRET) strategy to investigate structural changes in the lever arm domain of human {beta}-cardiac myosin subfragment 1 (M2{beta}-S1). We exchanged the human ventricular regulatory light chain labeled at a single cysteine (V105C) with Alexa 488 onto M2{beta}-S1, which served as a donor for Cy3ATP bound to the active site. We monitored the FRET signal during the actin-activated product release steps using transient kinetic stopped-flow measurements. We proposed that the fast phase measured with our FRET probes represents the structural change associated with rotation of the lever arm during the power stroke in M2{beta}-S1. Our results demonstrated human cardiac muscle myosin has a slower power stroke compared with fast skeletal muscle myosin and myosin V. Measurements at different temperatures comparing the rate constants of the power stroke and phosphate release revealed that the power stroke occurs before phosphate release, and the two steps are tightly coupled. We speculate that the slower power stroke rate constant in cardiac myosin may correlate with the slower force development and/or unique thin filament activation properties in cardiac muscle. Additionally, we demonstrated that HCM (R723G) and DCM (F764L) associated mutations both reduced actin-activation of the power stroke in M2{beta}-S1. We also demonstrate that both mutations decrease ensemble force development in the loaded in vitro motility assay. Thus, examining the structural kinetics of the power stroke in M2{beta}-S1 has revealed critical mutation-associated defects in the myosin ATPase pathway, suggesting these measurements will be extremely important for establishing structure-based mechanisms of contractile dysfunction. SignificanceMutations in human beta-cardiac myosin are known to cause various forms of heart disease, while it is unclear how the mutations lead to contractile dysfunction and pathogenic remodeling of the heart. In this study, we investigated two mutations with opposing phenotypes and examined their impact on ATPase cycle kinetics, structural changes associated with the myosin power stroke, and ability to slide actin filaments in the presence of load. We found that both mutations impair the myosin power stroke and other key kinetic steps as well as the ability to produce ensemble force. Thus, our results provide a structural basis for how mutations disrupt molecular level contractile dysfunction.

biophysics↗