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Jose Requejoisidro - One of the best experts on this subject based on the ideXlab platform.
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a complex interplay of anionic phospholipid binding regulates 3 phosphoinositide dependent kinase 1 Homodimer activation
Scientific Reports, 2019Co-Authors: Gloria De Las Herasmartinez, Veronique Calleja, Banafshe Larijani, Jose Requejoisidro, Remy Bailly, Jean DessolinAbstract:3′-Phosphoinositide-dependent-Kinase-1 (PDK1) is a master regulator whereby its PI3-kinase-dependent dysregulation in human pathologies is well documented. Understanding the direct role for PtdIns(3,4,5)P3 and other anionic phospholipids in the regulation of PDK1 conformational dynamics and its downstream activation remains incomplete. Using advanced quantitative-time-resolved imaging (Fluorescence Lifetime Imaging and Fluorescence Correlation Spectroscopy) and molecular modelling, we show an interplay of antagonistic binding effects of PtdIns(3,4,5)P3 and other anionic phospholipids, regulating activated PDK1 Homodimers. We demonstrate that phosphatidylserine maintains PDK1 in an inactive conformation. The dysregulation of the PI3K pathway affects the spatio-temporal and conformational dynamics of PDK1 and the activation of its downstream substrates. We have established a new anionic-phospholipid-dependent model for PDK1 regulation, depicting the conformational dynamics of multiple Homodimer states. We show that the dysregulation of the PI3K pathway perturbs equilibrium between the PDK1 Homodimer conformations. Our findings provide a role for the PtdSer binding site and its previously unrewarding role in PDK1 downregulation, suggesting a possible therapeutic strategy where the constitutively active dimer conformer of PDK1 may be rendered inactive by small molecules that drive it to its PtdSer-bound conformer.
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a complex interplay of anionic phospholipid binding regulates 3 phosphoinositide dependent kinase 1 Homodimer activation
bioRxiv, 2019Co-Authors: Güemes Heras, Veronique Calleja, Jose Requejoisidro, Remy Bailly, Jean Dessolin, Banafshe LarijaniAbstract:3-Phosphoinositide-dependent-Kinase-1 is a master regulator whereby its PI3- kinase-dependent dysregulation in human pathologies is well documented. Understanding the direct role for PtdIns(3,4,5)P3 and other anionic phospholipids in the regulation of PDK1 conformational dynamics and its downstream activation remains incomplete. Using advanced quantitative-time-resolved imaging, FCS and molecular modelling, we show an interplay of antagonistic binding effects of PtdIns(3,4,5)P3 and other anionic phospholipids, regulating activated PDK1 Homodimers. We demonstrate that phosphatidylserine maintains PDK1 in an inactive conformation. The dysregulation of the PI3K pathway affects the spatio-temporal and conformational dynamics of PDK1 and the activation of its downstream substrates. We establish an anionic-phospholipid-dependent model for PDK1 regulation, depicting the conformational dynamics of multiple Homodimer states. The dysregulation of the PI3K pathway perturbs equilibrium between the PDK1 Homodimer conformations. Our findings indicate that the alteration of specific basic residues of PDK1-PH domain leads to its constitutive activation, potential significance in different types of carcinomas.
Laura Domínguez - One of the best experts on this subject based on the ideXlab platform.
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impact of membrane lipid composition on the structure and stability of the transmembrane domain of amyloid precursor protein
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Leigh Foster, Laura Domínguez, John E Straub, Dave ThirumalaiAbstract:Cleavage of the amyloid precursor protein (APP) by γ-secretase is a crucial first step in the evolution of Alzheimer’s disease. To discover the cleavage mechanism, it is urgent to predict the structures of APP monomers and dimers in varying membrane environments. We determined the structures of the C9923−55 monomer and Homodimer as a function of membrane lipid composition using a multiscale simulation approach that blends atomistic and coarse-grained models. We demonstrate that the C9923−55 Homodimer structures form a heterogeneous ensemble with multiple conformational states, each stabilized by characteristic interpeptide interactions. The relative probabilities of each conformational state are sensitive to the membrane environment, leading to substantial variation in Homodimer peptide structure as a function of membrane lipid composition or the presence of an anionic lipid environment. In contrast, the helicity of the transmembrane domain of monomeric C991−55 is relatively insensitive to the membrane lipid composition, in agreement with experimental observations. The dimer structures of human EphA2 receptor depend on the lipid environment, which we show is linked to the location of the structural motifs in the dimer interface, thereby establishing that both sequence and membrane composition modulate the complete energy landscape of membrane-bound proteins. As a by-product of our work, we explain the discrepancy in structures predicted for C99 congener Homodimers in membrane and micelle environments. Our study provides insight into the observed dependence of C99 protein cleavage by γ-secretase, critical to the formation of amyloid-β protein, on membrane thickness and lipid composition.
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structural heterogeneity in transmembrane amyloid precursor protein Homodimer is a consequence of environmental selection
Journal of the American Chemical Society, 2014Co-Authors: Laura Domínguez, Leigh Foster, John E Straub, Stephen C Meredith, D ThirumalaiAbstract:The 99 amino acid C-terminal fragment of amyloid precursor protein (C99), consisting of a single transmembrane (TM) helix, is known to form Homodimers. Homodimers can be processed by γ-secretase to produce amyloid-β (Aβ) protein, which is implicated in Alzheimer’s disease (AD). While knowledge of the structure of C99 Homodimers is of great importance, experimental NMR studies and simulations have produced varying structural models, including right-handed and left-handed coiled-coils. In order to investigate the structure of this critical protein complex, simulations of the C9915–55 Homodimer in POPC membrane bilayer and DPC surfactant micelle environments were performed using a multiscale approach that blends atomistic and coarse-grained models. The C9915–55 Homodimer adopts a dominant right-handed coiled-coil topology consisting of three characteristic structural states in a bilayer, only one of which is dominant in the micelle. Our structural study, which provides a self-consistent framework for underst...
Milt Teitler - One of the best experts on this subject based on the ideXlab platform.
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determining the oligomer number of native gpcr using florescence correlation spectroscopy and drug induced inactivation reactivation
Current Pharmaceutical Biotechnology, 2014Co-Authors: Milt Teitler, Katharine HerrickdavisAbstract:GPCRs are a major family of homologous proteins and are key mediators of the effects of numerous endogenous neurotransmitters, hormones, cytokines, therapeutic drugs, and drugs-of-abuse. Despite the enormous amount of research on the pharmacological and biochemical properties of GPCRs, there is surprisingly little information on GPCR dimer structure and function in primary cell culture or in vivo. We have used two novel approaches to develop methods to detect and study GPCR dimer function: FCS/PCH and "inactivation-reactivation". This review will focus on the data we have developed and our interpretations of those data. Using FCS/PCH 5-HT2C receptors have been detected directly and appear to exist as dimers, consistent with the inactivation-reactivation data on 5-HT7 and 5-HT2A receptors. Studies of the 5-HT7 and 5-HT2A serotonin receptors have revealed that binding of a pseudo-irreversible antagonist ("inactivator") to one of the orthosteric sites of a Homodimer abolishes all receptor activity, and subsequent binding of a competitive antagonist to the orthosteric site of the second protomer releases the inactivator, allowing the receptor to return to an active state. This approach demonstrates allosteric crosstalk between protomers of native GPCR Homodimers, indicating that GPCRs do exist and function as Homodimers in both recombinant cells and rat primary astrocytes. This technique can be applied universally using intact recombinant or primary cells in culture, membrane homogenate preparations and, potentially, in vivo. This approach can be applied to heterodimers as well as Homodimers and may aid in the development of novel drugs with heterodimer selectivity.
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A new approach for studying GPCR dimers: drug-induced inactivation and reactivation to reveal GPCR dimer function in vitro, in primary culture, and in vivo
Pharmacology & Therapeutics, 2011Co-Authors: Milt Teitler, Michael T. KleinAbstract:GPCRs are a major family of homologous proteins and are key mediators of the effects of numerous endogenous neurotransmitters, hormones, cytokines, therapeutic drugs, and drugs-of-abuse. Despite the enormous amount of research on the pharmacological and biochemical properties of GPCRs, the question as to whether they exist as monomers, dimers, or higher order structures in the body is unanswered. The GPCR dimer field has been dominated by techniques involving recombinant cell lines expressing mutant receptors, often involving the solubilization of the receptors. These techniques cannot be applied in vivo or even to primary cell cultures. This review will focus on a novel approach to exploring the functional properties of Homodimers. Studies of the 5-HT7 and 5-HT2A serotonin receptors have revealed that binding of a pseudo-irreversible antagonist (“inactivator”) to one of the orthosteric sites of a Homodimer abolishes all receptor activity, and subsequent binding of a competitive antagonist to the orthosteric site of the second protomer releases the inactivator, allowing the receptor to return to an active state. This approach demonstrates allosteric crosstalk between protomers of native GPCR Homodimers, indicating that GPCRs do exist and function as Homodimers in both recombinant cells and rat primary astrocytes. This technique can be applied universally using intact recombinant or primary cells in culture, membrane homogenate preparations and, potentially, in vivo. The data obtained using the 5-HT7 and 5-HT2A receptors are strongly supportive of a GPCR Homodimer structure, with little evidence of monomer involvement in the function of these receptors.
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risperidone induced inactivation and clozapine induced reactivation of rat cortical astrocyte 5 hydroxytryptamine7 receptors evidence for in situ g protein coupled receptor Homodimer protomer cross talk
Molecular Pharmacology, 2011Co-Authors: Carol Smith, Michael T. Klein, Nicole Toohey, Jessica A Knight, Milt TeitlerAbstract:We have reported previously novel drug-induced inactivation and reactivation of human 5-hydroxytryptamine7 (5-HT7) receptors in a recombinant cell line. To explain these novel observations, a Homodimer structure displaying protomer-protomer cross-talk was proposed. To determine whether these novel observations and interpretations are due to an artifactual G protein-coupled receptor (GPCR) mechanism unique to the recombinant cell line, we explored the properties of r5-HT7 receptors expressed by cortical astrocytes in primary culture. As in the recombinant cell line, risperidone, 9-OH-risperidone, methiothepin, and bromocriptine were found to potently inactivate r5-HT7 receptors. As in the recombinant cell line, exposure of risperidone-inactivated astrocyte r5-HT7 receptors to competitive antagonists resulted in the reactivation of r5-HT7 receptors. The potencies of the reactivating drugs closely correlated with their affinities for h5-HT7 receptors. These results indicate the novel inactivating and reactivating property of drugs is not due to an artifact of the recombinant cell line expressing h5-HT7 receptors but is an intrinsic property of 5-HT7 receptors in vitro and ex vivo. This evidence suggests that a native (nonmutated) GPCR, in its native membrane environment (cortical astrocyte primary culture), can function as a Homodimer with protomer-protomer cross-talk. Homodimers may be a common GPCR structure. The experimental design used in our studies can be used to explore the properties of other GPCRs in their native forms in recombinant cells, primary cultures expressing the endogenous GPCRs, and possibly in vivo. The Homodimer structure and protomer-protomer cross-talk offer new avenues of research into receptor dysfunction in disease states and the development of novel drugs.
Weihong Qiu - One of the best experts on this subject based on the ideXlab platform.
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the preprophase band associated kinesin 14 oskch2 is a processive minus end directed microtubule motor
Nature Communications, 2018Co-Authors: Kuofu Tseng, Bo Liu, Pan Wang, Yuhru Julie Lee, Joel Bowen, Allison M Gicking, Lijun Guo, Weihong QiuAbstract:In animals and fungi, cytoplasmic dynein is a processive minus-end-directed motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a Homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual Homodimers. We find that CC1, but not CC2, forms the coiled-coil to enable OsKCH2 dimerization. Instead, our results reveal that removing CC2 renders OsKCH2 a nonprocessive motor. Collectively, these results show that land plants have evolved unconventional kinesin-14 Homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.
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the preprophase band associated kinesin 14 oskch2 is a processive minus end directed microtubule motor
bioRxiv, 2017Co-Authors: Kuofu Tseng, Bo Liu, Pan Wang, Yuhru Julie Lee, Joel Bowen, Allison M Gicking, Lijun Guo, Weihong QiuAbstract:In animals and fungi, cytoplasmic dynein is a processive motor that plays dominant roles in various intracellular processes. In contrast, land plants lack cytoplasmic dynein but contain many minus-end-directed kinesin-14s. No plant kinesin-14 is known to produce processive motility as a Homodimer. OsKCH2 is a plant-specific kinesin-14 with an N-terminal actin-binding domain and a central motor domain flanked by two predicted coiled-coils (CC1 and CC2). Here, we show that OsKCH2 specifically decorates preprophase band microtubules in vivo and transports actin filaments along microtubules in vitro. Importantly, OsKCH2 exhibits processive minus-end-directed motility on single microtubules as individual Homodimers. We find that CC1 but not CC2 forms the coiled-coil for OsKCH2 dimerization. Instead, CC2 functions to enable OsKCH2 processivity by enhancing its binding to microtubules. Collectively, these results show that land plants have evolved unconventional kinesin-14 Homodimers with inherent minus-end-directed processivity that may function to compensate for the loss of cytoplasmic dynein.
Paul J Carter - One of the best experts on this subject based on the ideXlab platform.
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stable heterodimers from remodeling the domain interface of a Homodimer using a phage display library
Journal of Molecular Biology, 1997Co-Authors: Shane Atwell, John B Ridgway, James A Wells, Paul J CarterAbstract:Structure-guided phage display was used to select for combinations of interface residues for antibody CH3 domains that promote the formation of stable heterodimers. A CH3 “knob” mutant was made by replacement of a small residue, threonine, with a larger one, tryptophan: T366W. A library of CH3 “hole” mutants was then created by randomizing residues 366, 368 and 407, which are in proximity to the knob on the partner CH3 domain. The CH3 knob mutant was fused to a peptide flag and the CH3 hole library was fused to M13 gene III. Phage displaying stable CH3 heterodimers were recovered by panning using an anti-flag antibody. Phage-selected CH3 heterodimers differed in sequence from the previously designed heterodimer T366W-Y407′ A, and most clones tested were more stable to guanidine hydrochloride denaturation. The thermal stability of individual CH3 domains secreted from Escherichia coli was analyzed by differential scanning calorimetry. One heterodimer, T366W-T366′S:L368′A:Y407′V, had a tm of 69.4°C, which is 4.0deg.C higher than that for the designed heterodimer and 11.0deg.C lower than that for the wild-type Homodimer. The phage-selected CH3 mutant maintained the preference for forming heterodimers over Homodimers as judged by near-quantitative formation of an antibody/immunoadhesin hybrid in a cotransfection assay. Phage optimization provides a complementary and more comprehensive strategy to rational design for engineering Homodimers for heterodimerization.