Many receptors are able to undergo heteromerisation, leading to the formation of receptor complex... more Many receptors are able to undergo heteromerisation, leading to the formation of receptor complexes that may have pharmacological profiles distinct from those of the individual receptors. As a consequence of this, receptor heteromers can be classed as new drug targets, with the potential for achieving greater specificity and selectivity over targeting their constituent receptors. We have developed the Receptor-Heteromer Investigation Technology (Receptor-HIT), which enables the detection of receptor heteromers using a proximity-based reporter system such as bioluminescence resonance energy transfer (BRET). Receptor-HIT detects heteromers in live cells and in real time, by utilising ligand-induced signals that arise from altered interactions with specific biomolecules, such as ligands or proteins. Furthermore, monitoring the interaction between the receptors and the specific biomolecules generates functional information about the heteromer that can be pharmacologically quantified. Th...
Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components... more Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components of honeybee (Apis mellifera) venom as anticancer agents remain largely unknown. Here, we demonstrate that honeybee venom and its major component melittin potently induce cell death, particularly in the aggressive triple-negative and HER2-enriched breast cancer subtypes. Honeybee venom and melittin suppress the activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells. Mutational studies reveal that a positively charged C-terminal melittin sequence mediates plasma membrane interaction and anticancer activity. Engineering of an RGD motif further enhances targeting of melittin to malignant cells with minimal toxicity to normal cells. Lastly, administration of melittin enhances the effect of docetaxel in suppressing breast tumor growth in an allograft model. Our work unveils a molecular mechanism underpinning the ...
Förster resonance energy transfer (FRET) technology has been widely used in biological and biomed... more Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research, and it is a very powerful tool for elucidating protein interactions in either dynamic or steady state. SUMOylation (the process of SUMO [small ubiquitin-like modifier] conjugation to substrates) is an important posttranslational protein modification with critical roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENPs) act as an endopeptidase to process the pre-SUMO or as an isopeptidase to deconjugate SUMO from its substrate. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. Here, we report a novel development of a quantitative FRET-based protease assay for SENP1 kinetic parameter determination. The assay is based on the quantitative analysis of the FRET signal from the total fluorescent signal at acceptor emission wavelength, which consists of three components: donor (CyPet-SUMO1) emission, acceptor (YPet) emission, and FRET signal during the digestion process. Subsequently, we developed novel theoretical and experimental procedures to determine the kinetic parameters, k(cat), K(M), and catalytic efficiency (k(cat)/K(M)) of catalytic domain SENP1 toward pre-SUMO1. Importantly, the general principles of this quantitative FRET-based protease kinetic determination can be applied to other proteases.
Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the p... more Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII’s kD by 2.6 fold with no effect on its Bmax. Molecular docking and MD simulations indicated that the binding of LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets residues on intracellular loops 2 and 3 of AT1R, w...
The Journal of clinical investigation, Jan 10, 2018
Activation of the type 1 angiotensin II receptor (AT1) triggers proinflammatory signaling through... more Activation of the type 1 angiotensin II receptor (AT1) triggers proinflammatory signaling through pathways independent of classical Gq signaling that regulate vascular homeostasis. Here, we report that the AT1 receptor preformed a heteromeric complex with the receptor for advanced glycation endproducts (RAGE). Activation of the AT1 receptor by angiotensin II (Ang II) triggered transactivation of the cytosolic tail of RAGE and NF-κB-driven proinflammatory gene expression independently of the liberation of RAGE ligands or the ligand-binding ectodomain of RAGE. The importance of this transactivation pathway was demonstrated by our finding that adverse proinflammatory signaling events induced by AT1 receptor activation were attenuated when RAGE was deleted or transactivation of its cytosolic tail was inhibited. At the same time, classical homeostatic Gq signaling pathways were unaffected by RAGE deletion or inhibition. These data position RAGE transactivation by the AT1 receptor as a ta...
Chemokines and their receptors collectively orchestrate the trafficking of leukocytes in normal i... more Chemokines and their receptors collectively orchestrate the trafficking of leukocytes in normal immune function and inflammatory diseases. Different chemokines can induce distinct responses at the same receptor. In comparison to monocyte chemoattractant protein-1 (MCP-1; also known as CCL2), the chemokines MCP-2 (CCL8) and MCP-3 (CCL7) are partial agonists of their shared receptor CCR2, a key regulator of the trafficking of monocytes and macrophages that contribute to the pathology of atherosclerosis, obesity, and type 2 diabetes. Through experiments with chimeras of MCP-1 and MCP-3, we identified the chemokine amino-terminal region as being the primary determinant of both the binding and signaling selectivity of these two chemokines at CCR2. Analysis of CCR2 mutants showed that the chemokine amino terminus interacts with the major subpocket in the transmembrane helical bundle of CCR2, which is distinct from the interactions of some other chemokines with the minor subpockets of thei...
Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a genetic disease first described i... more Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a genetic disease first described in 2 unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. Here, we describe and functionally characterize a novel vasopressin type 2 receptor (V2R) gain-of-function mutation. An L312S substitution in the seventh transmembrane domain was identified in a boy presenting with water-induced hyponatremic seizures at the age of 5.8 years. We show that, compared with wild-type V2R, the L312S mutation results in the constitutive production of cAMP, indicative of the gain-of-function NSIAD profile. Interestingly, like the previously described F229V and I130N NSIAD-causing mutants, this appears to both occur in the absence of notable constitutive β-arrestin2 recruitment and can be reduced by the inverse agonist Tolvaptan. In addition, ...
beta-Arrestins bind to phosphorylated, seven-transmembrane-spanning, G protein-coupled receptors ... more beta-Arrestins bind to phosphorylated, seven-transmembrane-spanning, G protein-coupled receptors (GPCRs), including the type 1 angiotensin II receptor (AT(1)R), to promote receptor desensitization and internalization. The AT(1) R is a class B GPCR that recruits both beta-arrestin1 and beta-arrestin2, forming stable complexes that cotraffic to deep-core endocytic vesicles. beta-Arrestins contain one amphipathic and potentially amphitropic (membrane-targeting) alpha-helix (helix I) that may promote translocation to the membrane or influence receptor internalization or trafficking. Here, we investigated the trafficking and function of beta-arrestin1 and beta-arrestin2 mutants bearing substitutions in both the hydrophobic and positively charged faces of helix I. The level of expression of these mutants and their cytoplasmic localization (in the absence of receptor activation) was similar to wild-type beta-arrestins. After angiotensin II stimulation, both wild-type and beta-arrestin mutants translocated to the cell membrane, although recruitment was weaker for mutants of the hydrophobic face of helix I. For all beta-arrestin mutants, the formation of deep-core vesicles was less observed compared with wild-type beta-arrestins. Furthermore, helix I conjugated to green fluorescent protein is not membrane-localized, suggesting that helix I, in isolation, is not amphitropic. Bioluminescence resonance energy transfer analysis revealed that both wild-type and beta-arrestin mutants retained a capacity to interact with the AT(1)R, although the interaction with the mutants was less stable. Finally, wild-type and mutant beta-arrestins fully supported receptor internalization in human embryonic kidney cells and mouse embryonic fibroblasts deficient in beta-arrestin1 and -2. Thus, helix I is implicated in postmembrane trafficking but is not strongly amphitropic.
Many receptors are able to undergo heteromerisation, leading to the formation of receptor complex... more Many receptors are able to undergo heteromerisation, leading to the formation of receptor complexes that may have pharmacological profiles distinct from those of the individual receptors. As a consequence of this, receptor heteromers can be classed as new drug targets, with the potential for achieving greater specificity and selectivity over targeting their constituent receptors. We have developed the Receptor-Heteromer Investigation Technology (Receptor-HIT), which enables the detection of receptor heteromers using a proximity-based reporter system such as bioluminescence resonance energy transfer (BRET). Receptor-HIT detects heteromers in live cells and in real time, by utilising ligand-induced signals that arise from altered interactions with specific biomolecules, such as ligands or proteins. Furthermore, monitoring the interaction between the receptors and the specific biomolecules generates functional information about the heteromer that can be pharmacologically quantified. Th...
Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components... more Despite decades of study, the molecular mechanisms and selectivity of the biomolecular components of honeybee (Apis mellifera) venom as anticancer agents remain largely unknown. Here, we demonstrate that honeybee venom and its major component melittin potently induce cell death, particularly in the aggressive triple-negative and HER2-enriched breast cancer subtypes. Honeybee venom and melittin suppress the activation of EGFR and HER2 by interfering with the phosphorylation of these receptors in the plasma membrane of breast carcinoma cells. Mutational studies reveal that a positively charged C-terminal melittin sequence mediates plasma membrane interaction and anticancer activity. Engineering of an RGD motif further enhances targeting of melittin to malignant cells with minimal toxicity to normal cells. Lastly, administration of melittin enhances the effect of docetaxel in suppressing breast tumor growth in an allograft model. Our work unveils a molecular mechanism underpinning the ...
Förster resonance energy transfer (FRET) technology has been widely used in biological and biomed... more Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research, and it is a very powerful tool for elucidating protein interactions in either dynamic or steady state. SUMOylation (the process of SUMO [small ubiquitin-like modifier] conjugation to substrates) is an important posttranslational protein modification with critical roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENPs) act as an endopeptidase to process the pre-SUMO or as an isopeptidase to deconjugate SUMO from its substrate. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. Here, we report a novel development of a quantitative FRET-based protease assay for SENP1 kinetic parameter determination. The assay is based on the quantitative analysis of the FRET signal from the total fluorescent signal at acceptor emission wavelength, which consists of three components: donor (CyPet-SUMO1) emission, acceptor (YPet) emission, and FRET signal during the digestion process. Subsequently, we developed novel theoretical and experimental procedures to determine the kinetic parameters, k(cat), K(M), and catalytic efficiency (k(cat)/K(M)) of catalytic domain SENP1 toward pre-SUMO1. Importantly, the general principles of this quantitative FRET-based protease kinetic determination can be applied to other proteases.
Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the p... more Hemorphins are known for their role in the control of blood pressure. Recently, we revealed the positive modulation of the angiotensin II (AngII) type 1 receptor (AT1R) by LVV-hemorphin-7 (LVV-H7) in human embryonic kidney (HEK293) cells. Here, we examined the molecular binding behavior of LVV-H7 on AT1R and its effect on AngII binding using a nanoluciferase-based bioluminescence resonance energy transfer (NanoBRET) assay in HEK293FT cells, as well as molecular docking and molecular dynamics (MD) studies. Saturation and real-time kinetics supported the positive effect of LVV-H7 on the binding of AngII. While the competitive antagonist olmesartan competed with AngII binding, LVV-H7 slightly, but significantly, decreased AngII’s kD by 2.6 fold with no effect on its Bmax. Molecular docking and MD simulations indicated that the binding of LVV-H7 in the intracellular region of AT1R allosterically potentiates AngII binding. LVV-H7 targets residues on intracellular loops 2 and 3 of AT1R, w...
The Journal of clinical investigation, Jan 10, 2018
Activation of the type 1 angiotensin II receptor (AT1) triggers proinflammatory signaling through... more Activation of the type 1 angiotensin II receptor (AT1) triggers proinflammatory signaling through pathways independent of classical Gq signaling that regulate vascular homeostasis. Here, we report that the AT1 receptor preformed a heteromeric complex with the receptor for advanced glycation endproducts (RAGE). Activation of the AT1 receptor by angiotensin II (Ang II) triggered transactivation of the cytosolic tail of RAGE and NF-κB-driven proinflammatory gene expression independently of the liberation of RAGE ligands or the ligand-binding ectodomain of RAGE. The importance of this transactivation pathway was demonstrated by our finding that adverse proinflammatory signaling events induced by AT1 receptor activation were attenuated when RAGE was deleted or transactivation of its cytosolic tail was inhibited. At the same time, classical homeostatic Gq signaling pathways were unaffected by RAGE deletion or inhibition. These data position RAGE transactivation by the AT1 receptor as a ta...
Chemokines and their receptors collectively orchestrate the trafficking of leukocytes in normal i... more Chemokines and their receptors collectively orchestrate the trafficking of leukocytes in normal immune function and inflammatory diseases. Different chemokines can induce distinct responses at the same receptor. In comparison to monocyte chemoattractant protein-1 (MCP-1; also known as CCL2), the chemokines MCP-2 (CCL8) and MCP-3 (CCL7) are partial agonists of their shared receptor CCR2, a key regulator of the trafficking of monocytes and macrophages that contribute to the pathology of atherosclerosis, obesity, and type 2 diabetes. Through experiments with chimeras of MCP-1 and MCP-3, we identified the chemokine amino-terminal region as being the primary determinant of both the binding and signaling selectivity of these two chemokines at CCR2. Analysis of CCR2 mutants showed that the chemokine amino terminus interacts with the major subpocket in the transmembrane helical bundle of CCR2, which is distinct from the interactions of some other chemokines with the minor subpockets of thei...
Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a genetic disease first described i... more Nephrogenic syndrome of inappropriate antidiuresis (NSIAD) is a genetic disease first described in 2 unrelated male infants with severe symptomatic hyponatremia. Despite undetectable arginine vasopressin levels, patients have inappropriately concentrated urine resulting in hyponatremia, hypoosmolality, and natriuresis. Here, we describe and functionally characterize a novel vasopressin type 2 receptor (V2R) gain-of-function mutation. An L312S substitution in the seventh transmembrane domain was identified in a boy presenting with water-induced hyponatremic seizures at the age of 5.8 years. We show that, compared with wild-type V2R, the L312S mutation results in the constitutive production of cAMP, indicative of the gain-of-function NSIAD profile. Interestingly, like the previously described F229V and I130N NSIAD-causing mutants, this appears to both occur in the absence of notable constitutive β-arrestin2 recruitment and can be reduced by the inverse agonist Tolvaptan. In addition, ...
beta-Arrestins bind to phosphorylated, seven-transmembrane-spanning, G protein-coupled receptors ... more beta-Arrestins bind to phosphorylated, seven-transmembrane-spanning, G protein-coupled receptors (GPCRs), including the type 1 angiotensin II receptor (AT(1)R), to promote receptor desensitization and internalization. The AT(1) R is a class B GPCR that recruits both beta-arrestin1 and beta-arrestin2, forming stable complexes that cotraffic to deep-core endocytic vesicles. beta-Arrestins contain one amphipathic and potentially amphitropic (membrane-targeting) alpha-helix (helix I) that may promote translocation to the membrane or influence receptor internalization or trafficking. Here, we investigated the trafficking and function of beta-arrestin1 and beta-arrestin2 mutants bearing substitutions in both the hydrophobic and positively charged faces of helix I. The level of expression of these mutants and their cytoplasmic localization (in the absence of receptor activation) was similar to wild-type beta-arrestins. After angiotensin II stimulation, both wild-type and beta-arrestin mutants translocated to the cell membrane, although recruitment was weaker for mutants of the hydrophobic face of helix I. For all beta-arrestin mutants, the formation of deep-core vesicles was less observed compared with wild-type beta-arrestins. Furthermore, helix I conjugated to green fluorescent protein is not membrane-localized, suggesting that helix I, in isolation, is not amphitropic. Bioluminescence resonance energy transfer analysis revealed that both wild-type and beta-arrestin mutants retained a capacity to interact with the AT(1)R, although the interaction with the mutants was less stable. Finally, wild-type and mutant beta-arrestins fully supported receptor internalization in human embryonic kidney cells and mouse embryonic fibroblasts deficient in beta-arrestin1 and -2. Thus, helix I is implicated in postmembrane trafficking but is not strongly amphitropic.
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Papers by K. Pfleger