The Experts below are selected from a list of 102 Experts worldwide ranked by ideXlab platform
David D. Thomas - One of the best experts on this subject based on the ideXlab platform.
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Probing the Molecular Mechanism of SERCA-PLB Regulation by Time-Resolved FRET
Biophysical Journal, 2011Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. ThomasAbstract:We are using solid-phase peptide synthesis, Membrane Reconstitution, an enzyme-coupled Ca-ATPase activity assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After Reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA.
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Probing the Mechanism of SERCA-PLB Regulation by Time-Resolved Fret
Biophysical Journal, 2010Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. ThomasAbstract:We are using solid-phase peptide synthesis, Membrane Reconstitution, an enzyme-coupled Ca-ATPase activity assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After Reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight but opposite effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA, with distinct mechanisms for relief by Ca2+ and PLB phosphorylation.
Ronald D Vale - One of the best experts on this subject based on the ideXlab platform.
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in vitro Membrane Reconstitution of the t cell receptor proximal signaling network
Nature Structural & Molecular Biology, 2014Co-Authors: Ronald D ValeAbstract:Different kinases and phosphatases control the triggering of T-cell receptors. This signaling network is now reconstituted in vitro with CD3ζ, Lck, CD45, Csk and liposomes. Rigorous quantitative analyses reveal how the system is maintained in a quiescent state and how the kinase-phosphatase balance can be modulated by different events to allow activation in an ultrasensitive manner.
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In vitro Membrane Reconstitution of the T-cell receptor proximal signaling network
Nature Structural & Molecular Biology, 2014Co-Authors: Enfu Hui, Ronald D ValeAbstract:T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45 and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the Membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over an ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that co-clustering TCR and Lck or detaching Csk from the Membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs. Different kinases and phosphatases control the triggering of T-cell receptors. This signaling network is now reconstituted in vitro with CD3ζ, Lck, CD45, Csk and liposomes. Rigorous quantitative analyses reveal how the system is maintained in a quiescent state and how the kinase-phosphatase balance can be modulated by different events to allow activation in an ultrasensitive manner.
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in vitro Membrane Reconstitution of the t cell receptor proximal signaling network
Nature Structural & Molecular Biology, 2014Co-Authors: Enfu Hui, Ronald D ValeAbstract:T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45 and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the Membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over an ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that co-clustering TCR and Lck or detaching Csk from the Membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs.
Enfu Hui - One of the best experts on this subject based on the ideXlab platform.
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Understanding T cell signaling using Membrane Reconstitution
Immunological Reviews, 2019Co-Authors: Enfu HuiAbstract:T cells are central players of our immune system, as their functions range from killing tumorous and virus-infected cells to orchestrating the entire immune response. In order for T cells to divide and execute their functions, they must be activated by antigen-presenting cells (APCs) through a cell-cell junction. Extracellular interactions between receptors on T cells and their ligands on APCs trigger signaling cascades comprised of protein-protein interactions, enzymatic reactions, and spatial reorganization events, to either stimulate or repress T cell activation. Plasma Membrane is the major platform for T cell signaling. Recruitment of cytosolic proteins to Membrane-bound receptors is a common critical step in many signaling pathways. Membranes decrease the dimensionality of protein-protein interactions to enable weak yet biologically important interactions. Membrane resident proteins can phase separate into micro-islands that promote signaling by enriching or excluding signal regulators. Moreover, some Membrane lipids can either mediate or regulate cell signaling by interacting with signaling proteins. While it is critical to investigate T cell signaling in a cellular environment, the large number of signaling pathways involved and potential crosstalk have made it difficult to obtain precise, quantitative information on T cell signaling. Reconstitution of purified proteins to model Membranes provides a complementary avenue for T cell signaling research. Here, I review recent progress in studying T cell signaling using Membrane Reconstitution approaches.
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abstract a226 cell free Membrane Reconstitution system for cis and trans interaction of t cell co receptors and ligands
Cancer immunology research, 2019Co-Authors: Yunlong Zhao, Enfu HuiAbstract:Immunotherapy, which harnesses the immune system to combat cancers, is a paradigm-shifting approach in oncology. One important branch of immunotherapy is to use drugs to perturb molecular interactions at the T-cell–antigen presenting cell (APC) interface. In particular, antibodies that block the T-cell coinhibitory receptor PD-1 or its ligand PD-L1 have produced unprecedented clinical activities against a subset of human cancers in a fraction of patients. Extending immunotherapy to a larger patient population requires a better understanding of the interaction network at the T-cell-APC interface. Typically, it is believed that ligands from APCs bind to receptors on T-cells (in trans) to trigger intracellular signaling. A largely overlooked fact is that many receptors often co-exist with ligands on T-cells and APCs, including PD-1/PD-L1 and CD28/B7.1. Using novel Reconstitution approaches, we recently discovered that co-expressed PD- 1 and PD-L1 bind to each other in cis and this cis interaction competes with their trans interaction to inhibit PD-1 signaling. We hypothesize that cis interaction occurs with many other ligand-receptor pairs and may regulate other aspects of tumor immunity by perturbing their trans interactions. We are currently using our robust Reconstitution systems to ask whether other immunoreceptors and ligands interact in cis and how the putative cis interaction affect their trans interactions. Specifically, to detect cis interaction, we co-attach the ligand and receptor to liposomes, and probe their cis interaction using a fluorescence resonance energy transfer (FRET) readout. To detect trans interaction, we have developed a liposome-lipid bilayer conjugation assay, in which binding between ligand- labeled liposomes and receptor-labeled supported lipid bilayer (SLB) is used as an index for trans interaction. To determine whether cis interaction inhibits trans interaction, we ask in the liposome-bilayer system if addition of receptor on liposomes decreases liposome-SLB conjugation. Our preliminary data revealed that CD28 only binds B7.1 in trans, but not in cis. In contrast, PD-1 binds with PD-L1 both in cis and trans, and the two modes of interaction compete with each other. Further experiments in cells confirmed our finding in vitro, demonstrating the utility of our cell-free Reconstitution systems in detecting cis and trans interaction of T-cell co-receptors. Insights from this study will help us clarify the ligand-receptor interaction network at the immunological synapse. Citation Format: Yunlong Zhao, Enfu Hui. Cell-free Membrane Reconstitution system for cis and trans interaction of T-cell co-receptors and ligands [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A226.
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In vitro Membrane Reconstitution of the T-cell receptor proximal signaling network
Nature Structural & Molecular Biology, 2014Co-Authors: Enfu Hui, Ronald D ValeAbstract:T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45 and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the Membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over an ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that co-clustering TCR and Lck or detaching Csk from the Membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs. Different kinases and phosphatases control the triggering of T-cell receptors. This signaling network is now reconstituted in vitro with CD3ζ, Lck, CD45, Csk and liposomes. Rigorous quantitative analyses reveal how the system is maintained in a quiescent state and how the kinase-phosphatase balance can be modulated by different events to allow activation in an ultrasensitive manner.
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in vitro Membrane Reconstitution of the t cell receptor proximal signaling network
Nature Structural & Molecular Biology, 2014Co-Authors: Enfu Hui, Ronald D ValeAbstract:T-cell receptor (TCR) phosphorylation is controlled by a complex network that includes Lck, a Src family kinase (SFK), the tyrosine phosphatase CD45 and the Lck-inhibitory kinase Csk. How these competing phosphorylation and dephosphorylation reactions are modulated to produce T-cell triggering is not fully understood. Here we reconstituted this signaling network using purified enzymes on liposomes, recapitulating the Membrane environment in which they normally interact. We demonstrate that Lck's enzymatic activity can be regulated over an ~10-fold range by controlling its phosphorylation state. By varying kinase and phosphatase concentrations, we constructed phase diagrams that reveal ultrasensitivity in the transition from the quiescent to the phosphorylated state and demonstrate that co-clustering TCR and Lck or detaching Csk from the Membrane can trigger TCR phosphorylation. Our results provide insight into the mechanism of TCR signaling as well as other signaling pathways involving SFKs.
Elizabeth L. Lockamy - One of the best experts on this subject based on the ideXlab platform.
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Probing the Molecular Mechanism of SERCA-PLB Regulation by Time-Resolved FRET
Biophysical Journal, 2011Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. ThomasAbstract:We are using solid-phase peptide synthesis, Membrane Reconstitution, an enzyme-coupled Ca-ATPase activity assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After Reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA.
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Probing the Mechanism of SERCA-PLB Regulation by Time-Resolved Fret
Biophysical Journal, 2010Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. ThomasAbstract:We are using solid-phase peptide synthesis, Membrane Reconstitution, an enzyme-coupled Ca-ATPase activity assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After Reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight but opposite effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA, with distinct mechanisms for relief by Ca2+ and PLB phosphorylation.
Christine B. Karim - One of the best experts on this subject based on the ideXlab platform.
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Probing the Molecular Mechanism of SERCA-PLB Regulation by Time-Resolved FRET
Biophysical Journal, 2011Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. ThomasAbstract:We are using solid-phase peptide synthesis, Membrane Reconstitution, an enzyme-coupled Ca-ATPase activity assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After Reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA.
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Probing the Mechanism of SERCA-PLB Regulation by Time-Resolved Fret
Biophysical Journal, 2010Co-Authors: Elizabeth L. Lockamy, Razvan L. Cornea, Christine B. Karim, David D. ThomasAbstract:We are using solid-phase peptide synthesis, Membrane Reconstitution, an enzyme-coupled Ca-ATPase activity assay, and time-resolved fluorescence resonance energy transfer (TR-FRET) to investigate the molecular mechanism by which the cardiac Ca-ATPase (SERCA) is regulated by phospholamban (PLB). In human heart failure, SERCA activity is inadequate, and current therapeutic research focuses on the goal of increasing SERCA activity by reducing PLB inhibition of SERCA. PLB inhibition is relieved by [Ca2+] > μM or by phosphorylation of S16 by PKA. It has been proposed that relief of this inhibition requires dissociation of the SERCA-PLB complex. To test this hypothesis, we have designed and synthesized monomeric PLB variants with a FRET acceptor (DABCYL), with and without phosphorylation at S16, and then reconstituted them with SERCA labeled with a FRET donor (IAEDANS). After Reconstitution, the interactions of these PLB variants with SERCA were characterized both functionally (Ca-ATPase activity) and physically (TR-FRET), as affected by Ca2+ and PLB phosphorylation. We found that Ca2+ completely relieves SERCA inhibition, while phosphorylation partially relieves SERCA inhibition. We also found that Ca2+ and phosphorylation have slight but opposite effects on FRET. Time resolution provided independent measurements of protein association and structure. We conclude that inhibition of SERCA is relieved by structural rearrangement within the SERCA-PLB complex, without dissociation of PLB from SERCA, with distinct mechanisms for relief by Ca2+ and PLB phosphorylation.