Tejas Gupte - Academia.edu (original) (raw)
Papers by Tejas Gupte
Nature Communications, 2022
Established models of ternary complex formation between hormone, G protein coupled receptor (GPCR... more Established models of ternary complex formation between hormone, G protein coupled receptor (GPCR), and G protein assume that all interactions occur under equilibrium conditions. However, recent studies have established that the lifetimes of these interactions are comparable to the duration of hormone activated GPCR signaling. To simulate interactions during such non-equilibrium conditions, we propose a kinetic model wherein the receptor undergoes rate-limiting transitions between two hormone-bound active states. Simulations, using experimentally measured parameters, demonstrate transient states in ternary complex formation, and delineate the phenomenon of GPCR priming, wherein non-cognate G proteins substantially enhance cognate G protein signaling. Our model reveals that kinetic barriers of slow receptor interconversion can be overcome through allokairic modulation, a regulatory mechanism of ternary complex formation and downstream signaling.
During the cell cycle, mitochondria undergo regulated changes in morphology. Two particu-larly in... more During the cell cycle, mitochondria undergo regulated changes in morphology. Two particu-larly interesting events are first, mitochondrial hyperfusion during the G1-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented be-tween late G2- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the ‘mitotic mitochondrial fragmentation ’ checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophi-la S2R+ cell and cells in the pouch region of wing imaginal disc of Drosophila larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our expe...
Pharmacology Research & Perspectives
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Linkers in Biomacromolecules
Nature Communications
Despite the crowded nature of the cellular milieu, ligand-GPCR-G protein interactions are traditi... more Despite the crowded nature of the cellular milieu, ligand-GPCR-G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor-effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR-G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation. We find that interactions of the Gαs and Gαq C termini with the β 2-adrenergic receptor (β 2-AR), targeted at the G-protein-binding site, enhance Gs activation and cyclic AMP levels. β 2-AR-Gα C termini interactions alter receptor conformation, which persists for~90 s following Gα C terminus dissociation. Noncognate G-protein expression levels impact cognate signaling in cells. Our study demonstrates temporal allostery in GPCRs, with implications for the modulation of downstream responses through the canonical G-protein-binding interface.
Biophysical Journal, 2017
Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 2017
Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G prote... more Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimolecular event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming." Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, β2-adrenergic receptor (β2-AR) and D1 dopamine receptor (D1-R). Reciprocally, Gs enhances IP1 through vasopressin receptor (V1A-R) but not α1 adrenergic receptor (α1-AR), suggesting that GPCR priming is a receptor-specific phenomenon. The C terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C termini with the GPCR increases signaling potency, suggest...
Biophysical Journal, 2014
that the neonatal cardiac isoform of TnI, ssTnI, confers pH-insensitivity in this regard compared... more that the neonatal cardiac isoform of TnI, ssTnI, confers pH-insensitivity in this regard compared to the adult cTnI isoform. However ssTnI confers deleterious effects of impaired relaxation in the adult myocyte. Alignment and functional studies have demonstrated that this pH-insensitivity is derived from ssTnI residue H132. Introduction of a histidine at the cognate position in cTnI (A164H) mitigates the pH-sensitivity of the calcium-force relationship in cardiac myocytes while retaining relaxation enhancement via the Nterm domain relative to ssTnI. We are establishing a time-resolved fluorescence methodology for detecting alterations in the calcium sensitivity of the thin filament during ischemia. We have engineered a single cysteine mutation for labeling with environmentally sensitive fluorophores designed to detect Ca 2þ and pH-sensitive structural changes in cTnI and cTnC. We will discuss progress using this approach to interrogate troponin function in ischemia mimetic conditions.
PLOS ONE, 2015
During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly int... more During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G(1)-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G(2)- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the 'mitotic mitochondrial fragmentation' checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R(+) cell and cells in the pouch region of wing imaginal disc of Drosophila larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. We provide evidence for the importance of mitochondrial superoxide in this process. Our results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.
The Journal of biological chemistry, Jan 29, 2014
The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sa... more The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are thirty single residue mutations in TPM1, the gene encoding alpha-tropomyosin. We examined six mutant tropomyosins: E62Q, I172T, L185R, S215L, D230N and M281T, which were chosen based on their clinical severity and locations along the molecule. The goal of our study is to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn can provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca2+ sensitivity of myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca2+ activation and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca2+ concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that diff...
Biophysical Journal, 2014
Nature Communications, 2022
Established models of ternary complex formation between hormone, G protein coupled receptor (GPCR... more Established models of ternary complex formation between hormone, G protein coupled receptor (GPCR), and G protein assume that all interactions occur under equilibrium conditions. However, recent studies have established that the lifetimes of these interactions are comparable to the duration of hormone activated GPCR signaling. To simulate interactions during such non-equilibrium conditions, we propose a kinetic model wherein the receptor undergoes rate-limiting transitions between two hormone-bound active states. Simulations, using experimentally measured parameters, demonstrate transient states in ternary complex formation, and delineate the phenomenon of GPCR priming, wherein non-cognate G proteins substantially enhance cognate G protein signaling. Our model reveals that kinetic barriers of slow receptor interconversion can be overcome through allokairic modulation, a regulatory mechanism of ternary complex formation and downstream signaling.
During the cell cycle, mitochondria undergo regulated changes in morphology. Two particu-larly in... more During the cell cycle, mitochondria undergo regulated changes in morphology. Two particu-larly interesting events are first, mitochondrial hyperfusion during the G1-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented be-tween late G2- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the ‘mitotic mitochondrial fragmentation ’ checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophi-la S2R+ cell and cells in the pouch region of wing imaginal disc of Drosophila larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our expe...
Pharmacology Research & Perspectives
This is an open access article under the terms of the Creative Commons Attribution License, which... more This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Linkers in Biomacromolecules
Nature Communications
Despite the crowded nature of the cellular milieu, ligand-GPCR-G protein interactions are traditi... more Despite the crowded nature of the cellular milieu, ligand-GPCR-G protein interactions are traditionally viewed as spatially and temporally isolated events. In contrast, recent reports suggest the spatial and temporal coupling of receptor-effector interactions, with the potential to diversify downstream responses. In this study, we combine protein engineering of GPCR-G protein interactions with affinity sequestration and photo-manipulation of the crucial Gα C terminus, to demonstrate the temporal coupling of cognate and non-cognate G protein interactions through priming of the GPCR conformation. We find that interactions of the Gαs and Gαq C termini with the β 2-adrenergic receptor (β 2-AR), targeted at the G-protein-binding site, enhance Gs activation and cyclic AMP levels. β 2-AR-Gα C termini interactions alter receptor conformation, which persists for~90 s following Gα C terminus dissociation. Noncognate G-protein expression levels impact cognate signaling in cells. Our study demonstrates temporal allostery in GPCRs, with implications for the modulation of downstream responses through the canonical G-protein-binding interface.
Biophysical Journal, 2017
Proceedings of the National Academy of Sciences of the United States of America, Jan 4, 2017
Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G prote... more Although individual G-protein-coupled receptors (GPCRs) are known to activate one or more G proteins, the GPCR-G-protein interaction is viewed as a bimolecular event involving the formation of a ternary ligand-GPCR-G-protein complex. Here, we present evidence that individual GPCR-G-protein interactions can reinforce each other to enhance signaling through canonical downstream second messengers, a phenomenon we term "GPCR priming." Specifically, we find that the presence of noncognate Gq protein enhances cAMP stimulated by two Gs-coupled receptors, β2-adrenergic receptor (β2-AR) and D1 dopamine receptor (D1-R). Reciprocally, Gs enhances IP1 through vasopressin receptor (V1A-R) but not α1 adrenergic receptor (α1-AR), suggesting that GPCR priming is a receptor-specific phenomenon. The C terminus of either the Gαs or Gαq subunit is sufficient to enhance Gα subunit activation and cAMP levels. Interaction of Gαs or Gαq C termini with the GPCR increases signaling potency, suggest...
Biophysical Journal, 2014
that the neonatal cardiac isoform of TnI, ssTnI, confers pH-insensitivity in this regard compared... more that the neonatal cardiac isoform of TnI, ssTnI, confers pH-insensitivity in this regard compared to the adult cTnI isoform. However ssTnI confers deleterious effects of impaired relaxation in the adult myocyte. Alignment and functional studies have demonstrated that this pH-insensitivity is derived from ssTnI residue H132. Introduction of a histidine at the cognate position in cTnI (A164H) mitigates the pH-sensitivity of the calcium-force relationship in cardiac myocytes while retaining relaxation enhancement via the Nterm domain relative to ssTnI. We are establishing a time-resolved fluorescence methodology for detecting alterations in the calcium sensitivity of the thin filament during ischemia. We have engineered a single cysteine mutation for labeling with environmentally sensitive fluorophores designed to detect Ca 2þ and pH-sensitive structural changes in cTnI and cTnC. We will discuss progress using this approach to interrogate troponin function in ischemia mimetic conditions.
PLOS ONE, 2015
During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly int... more During the cell cycle, mitochondria undergo regulated changes in morphology. Two particularly interesting events are first, mitochondrial hyperfusion during the G(1)-S transition and second, fragmentation during entry into mitosis. The mitochondria remain fragmented between late G(2)- and mitotic exit. This mitotic mitochondrial fragmentation constitutes a checkpoint in some cell types, of which little is known. We bypass the 'mitotic mitochondrial fragmentation' checkpoint by inducing fragmented mitochondrial morphology and then measure the effect on cell cycle progression. Using Drosophila larval hemocytes, Drosophila S2R(+) cell and cells in the pouch region of wing imaginal disc of Drosophila larvae we show that inhibiting mitochondrial fusion, thereby increasing fragmentation, causes cellular hyperproliferation and an increase in mitotic index. However, mitochondrial fragmentation due to over-expression of the mitochondrial fission machinery does not cause these changes. Our experiments suggest that the inhibition of mitochondrial fusion increases superoxide radical content and leads to the upregulation of cyclin B that culminates in the observed changes in the cell cycle. We provide evidence for the importance of mitochondrial superoxide in this process. Our results provide an insight into the need for mitofusin-degradation during mitosis and also help in understanding the mechanism by which mitofusins may function as tumor suppressors.
The Journal of biological chemistry, Jan 29, 2014
The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sa... more The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are thirty single residue mutations in TPM1, the gene encoding alpha-tropomyosin. We examined six mutant tropomyosins: E62Q, I172T, L185R, S215L, D230N and M281T, which were chosen based on their clinical severity and locations along the molecule. The goal of our study is to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn can provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca2+ sensitivity of myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca2+ activation and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca2+ concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that diff...
Biophysical Journal, 2014