The Experts below are selected from a list of 4251 Experts worldwide ranked by ideXlab platform
Isabelle Vernos - One of the best experts on this subject based on the ideXlab platform.
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allosteric inhibition of aurora a kinase by a synthetic vnar domain
Open Biology, 2016Co-Authors: Isabelle Vernos, Selena G Burgess, Arkadiusz Oleksy, Tommaso Cavazza, Mark W Richards, David Matthews, Richard BaylissAbstract:The vast majority of clinically approved protein kinase inhibitors target the ATP-binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, vNAR single domain scaffold, vNAR-D01. Biochemical studies and a crystal structure of the Aurora-A/vNAR-D01 complex show that the vNAR domain overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the vNAR domain stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how single domain antibodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors.
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allosteric inhibition of aurora a kinase by a synthetic vnar nanobody
bioRxiv, 2016Co-Authors: Selena G Burgess, Isabelle Vernos, Arkadiusz Oleksy, Tommaso Cavazza, Mark W Richards, David J Matthews, Richard BaylissAbstract:The vast majority of clinically-approved protein kinase inhibitors target the ATP binding pocket directly. Consequently, many inhibitors have broad selectivity profiles and most have significant off-target effects. Allosteric inhibitors are generally more selective, but are difficult to identify because allosteric binding sites are often unknown or poorly characterized, and there is no clearly preferred approach to generating hit matter. Aurora-A is activated through binding of TPX2 to an allosteric site on the kinase catalytic domain, and this knowledge could be exploited to generate an inhibitor. However, it is currently unclear how to design such a compound because a small molecule or peptide mimetic of TPX2 would be expected to activate, not inhibit the kinase. Here, we generated an allosteric inhibitor of Aurora-A kinase based on a synthetic, VNAR single domain nanobody scaffold, IgNARV-D01. Biochemical studies and a crystal structure of the Aurora-A/IgNARV-D01 complex show that the nanobody overlaps with the TPX2 binding site. In contrast with the binding of TPX2, which stabilizes an active conformation of the kinase, binding of the nanobody stabilizes an inactive conformation, in which the αC-helix is distorted, the canonical Lys-Glu salt bridge is broken, and the regulatory (R-) spine is disrupted by an additional hydrophobic side chain from the activation loop. These studies illustrate how nanobodies can be used to characterize the regulatory mechanisms of kinases and provide a rational basis for structure-guided design of allosteric Aurora-A kinase inhibitors.
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microtubule nucleation in mitosis by a rangtp dependent protein complex
Current Biology, 2015Co-Authors: Jacopo Scrofani, Isabelle Vernos, Teresa Sardon, Sylvain MeunierAbstract:Summary Background The γ-tubulin ring complex (γTuRC) is a multisubunit complex responsible for microtubule (MT) nucleation in eukaryotic cells. During mitosis, its spatial and temporal regulation promotes MT nucleation through different pathways. One of them is triggered around the chromosomes by RanGTP. Chromosomal MTs are essential for functional spindle assembly, but the mechanism by which RanGTP activates MT nucleation has not yet been resolved. Results We used a combination of Xenopus egg extracts and in vitro experiments to dissect the mechanism by which RanGTP triggers MT nucleation. In egg extracts, NEDD1-coated beads promote MT nucleation only in the presence of RanGTP. We show that RanGTP promotes a direct interaction between one of its targets, TPX2, and XRHAMM that defines a specific γTuRC subcomplex. Through depletion/add-back experiments using mutant forms of TPX2 and NEDD1, we show that the activation of MT nucleation by RanGTP requires both NEDD1 phosphorylation on S405 by the TPX2-activated Aurora A and the recruitment of the complex through a TPX2-dependent mechanism. Conclusions The XRHAMM-γTuRC complex is the target for activation by RanGTP that promotes an interaction between TPX2 and XRHAMM. The resulting TPX2-RHAMM-γTuRC supracomplex fulfills the two essential requirements for the activation of MT nucleation by RanGTP: NEDD1 phosphorylation on S405 by the TPX2-activated Aurora A and the recruitment of the complex onto a TPX2-dependent scaffold. Our data identify TPX2 as the only direct RanGTP target and NEDD1 as the only Aurora A substrate essential for the activation of the RanGTP-dependent MT nucleation pathway.
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characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in xenopus egg extracts
Molecular Biology of the Cell, 2004Co-Authors: Stephane Brunet, Eric Karsenti, Rainer Pepperkok, Torsten Wittmann, Teresa Sardon, Timo Zimmerman, Isabelle VernosAbstract:TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.
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structural basis of aurora a activation by TPX2 at the mitotic spindle
Molecular Cell, 2003Co-Authors: Richard Bayliss, Isabelle Vernos, Teresa Sardon, Elena ContiAbstract:Aurora-A is an oncogenic kinase essential for mitotic spindle assembly. It is activated by phosphorylation and by the microtubule-associated protein TPX2, which also localizes the kinase to spindle microtubules. We have uncovered the molecular mechanism of Aurora-A activation by determining crystal structures of its phosphorylated form both with and without a 43 residue long domain of TPX2 that we identified as fully functional for kinase activation and protection from dephosphorylation. In the absence of TPX2, the Aurora-A activation segment is in an inactive conformation, with the crucial phosphothreonine exposed and accessible for deactivation. Binding of TPX2 triggers no global conformational changes in the kinase but pulls on the activation segment, swinging the phosphothreonine into a buried position and locking the active conformation. The recognition between Aurora-A and TPX2 resembles that between the cAPK catalytic core and its flanking regions, suggesting this molecular mechanism may be a recurring theme in kinase regulation.
Sabine Petry - One of the best experts on this subject based on the ideXlab platform.
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branching microtubule nucleation is controlled by importin mediated inhibition of TPX2 phase separation
bioRxiv, 2020Co-Authors: Mohammad S Safari, Matthew R King, Clifford P Brangwynne, Sabine PetryAbstract:The microtubule-based mitotic spindle is responsible for equally partitioning the genome during each cell division, and its assembly is executed by several microtubule nucleation pathways. In the spindle center, Targeting Protein for XKlp2 (TPX2) promotes branching microtubule nucleation, where new microtubules are nucleated from pre-existing ones. Until the onset of spindle assembly, TPX2 is sequestered by importins-/{beta}, yet the molecular nature of this regulation remains unclear, particularly since TPX2 was recently found to undergo a liquid-liquid phase separation to execute its function. Here we demonstrate that TPX2 interacts with importins-/{beta} with nanomolar affinity as a 1:1:1 mono-dispersed trimer. We identify a new nuclear localization sequence (NLS) on TPX2, which contributes to its high-affinity interaction with importin-. Interestingly, importin-{beta} alone can also associate with TPX2, and does so via dispersed, weak interactions. Interactions of both importin- and importin-{beta} with TPX2 each inhibit its propensity for phase separation, and consequently its ability to orchestrate branching microtubule nucleation. In sum, our study explains how TPX2 is regulated in order to facilitate spindle assembly, and provides novel insight into how a protein phase separation can be inhibited via weak biomolecular interactions. Significance StatementThe discovery that proteins can undergo phase separation is revolutionizing biology. Characterization of dozens of phase separating proteins in vitro over the past several years has mainly focused on how macromolecules undergo liquid-liquid phase separation (LLPS). The next, and possibly bigger challenge is to investigate how LLPS is regulated in the cell, namely how it is inhibited to spatiotemporally control a certain cellular function. Here, we addressed this challenge by identifying how the spindle assembly factor TPX2 is inhibited by importins from undergoing LLPS and thereby turning on spindle assembly.
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biochemical reconstitution of branching microtubule nucleation
eLife, 2020Co-Authors: Raymundo Alfaroaco, Akanksha Thawani, Sabine PetryAbstract:Microtubules are nucleated from specific locations at precise times in the cell cycle. However, the factors that constitute these microtubule nucleation pathways and their mode of action still need to be identified. Using purified Xenopus laevis proteins we biochemically reconstitute branching microtubule nucleation, which is critical for chromosome segregation. We found that besides the microtubule nucleator gamma-tubulin ring complex (γ-TuRC), the branching effectors augmin and TPX2 are required to efficiently nucleate microtubules from pre-existing microtubules. TPX2 has the unexpected capacity to directly recruit γ-TuRC as well as augmin, which in turn targets more γ-TuRC along the microtubule lattice. TPX2 and augmin enable γ-TuRC-dependent microtubule nucleation at preferred branching angles of less than 90 degrees from regularly-spaced patches along microtubules. This work provides a blueprint for other microtubule nucleation pathways and helps explain how microtubules are generated in the spindle.
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phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
Nature Communications, 2020Co-Authors: Matthew R King, Sabine PetryAbstract:Phase separation of substrates and effectors is proposed to enhance biological reaction rates and efficiency. Targeting protein for Xklp2 (TPX2) is an effector of branching microtubule nucleation in spindles and functions with the substrate tubulin by an unknown mechanism. Here we show that TPX2 phase separates into a co-condensate with tubulin, which mediates microtubule nucleation in vitro and in isolated cytosol. TPX2-tubulin co-condensation preferentially occurs on pre-existing microtubules, the site of branching microtubule nucleation, at the endogenous and physiologically relevant concentration of TPX2. Truncation and chimera versions of TPX2 suggest that TPX2-tubulin co-condensation enhances the efficiency of TPX2-mediated branching microtubule nucleation. Finally, the known inhibitor of TPX2, the importin-α/β heterodimer, regulates TPX2 condensation in vitro and, consequently, branching microtubule nucleation activity in isolated cytosol. Our study demonstrates how regulated phase separation can simultaneously enhance reaction efficiency and spatially coordinate microtubule nucleation, which may facilitate rapid and accurate spindle formation.
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biochemical reconstitution of branching microtubule nucleation
bioRxiv, 2019Co-Authors: Raymundo Alfaroaco, Akanksha Thawani, Sabine PetryAbstract:Abstract Microtubules are nucleated from specific locations at precise times in the cell cycle. However, the factors that constitute these microtubule nucleation pathways still need to be identified along with their mode of action. Here, using purified Xenopus laevis proteins we biochemically reconstitute branching microtubule nucleation, a nucleation pathway where microtubules originate from pre-existing microtubules, which is essential for spindle assembly and chromosome segregation. We found that besides the microtubule nucleator gamma-tubulin ring complex (γ-TuRC), the two branching effectors augmin and TPX2 are required to efficiently nucleate branched microtubules. Specifically, TPX2 generates regularly-spaced patches that recruit augmin and γ-TuRC to microtubules, which then nucleate new microtubules at preferred branching angles of less than 90 degrees. Our work demonstrates how γ-TuRC is brought to its nucleation site for branching microtubule nucleation. It provides a blueprint for other microtubule nucleation pathways and for generating a particular microtubule architecture by regulating microtubule nucleation.
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phase separation of TPX2 enhances and spatially coordinates microtubule nucleation
bioRxiv, 2019Co-Authors: Matthew R King, Sabine PetryAbstract:Abstract Phase separation of substrates and effectors is proposed to enhance biological reaction rates and efficiency. TPX2 is an effector of microtubule nucleation in spindles, and functions with the substrate tubulin by an unknown mechanism. Here, we show that TPX2 phase separates into a co-condensate with tubulin, which mediates microtubule nucleation in vitro and in isolated cytosol. TPX2-tubulin co-condensation preferentially occurs on pre-existing microtubules at the endogenous and physiologically relevant concentration of TPX2. Truncation and chimera versions of TPX2 directly demonstrate that TPX2-tubulin co-condensation enhances the efficiency of TPX2-mediated microtubule nucleation. Finally, the known inhibitor of TPX2, the importin-α/β heterodimer, regulates both co-condensation and activity. Our study demonstrates how regulated phase separation can simultaneously enhance reaction efficiency and spatially coordinate microtubule nucleation, which may facilitate rapid and accurate spindle formation.
Aaron C. Groen - One of the best experts on this subject based on the ideXlab platform.
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functional overlap of microtubule assembly factors in chromatin promoted spindle assembly
Molecular Biology of the Cell, 2009Co-Authors: Aaron C. Groen, Thomas J Maresca, Jesse C Gatlin, E D Salmon, Timothy J MitchisonAbstract:Distinct pathways from centrosomes and chromatin are thought to contribute in parallel to microtubule nucleation and stabilization during animal cell mitotic spindle assembly, but their full mechanisms are not known. We investigated the function of three proposed nucleation/stabilization factors, TPX2, γ-tubulin and XMAP215, in chromatin-promoted assembly of anastral spindles in Xenopus laevis egg extract. In addition to conventional depletion-add back experiments, we tested whether factors could substitute for each other, indicative of functional redundancy. All three factors were required for microtubule polymerization and bipolar spindle assembly around chromatin beads. Depletion of TPX2 was partially rescued by the addition of excess XMAP215 or EB1, or inhibiting MCAK (a Kinesin-13). Depletion of either γ-tubulin or XMAP215 was partially rescued by adding back XMAP215, but not by adding any of the other factors. These data reveal functional redundancy between specific assembly factors in the chromatin pathway, suggesting individual proteins or pathways commonly viewed to be essential may not have entirely unique functions.
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the brca1 bard1 heterodimer modulates ran dependent mitotic spindle assembly
Cell, 2006Co-Authors: Vladimir Joukov, Tatyana Prokhorova, Ruth Gerson, Erinn White, Aaron C. Groen, Alison Rodriguez, Johannes C Walter, David M LivingstonAbstract:Summary The heterodimeric tumor-suppressor complex BRCA1/BARD1 exhibits E3 ubiquitin ligase activity and participates in cell proliferation and chromosome stability control by incompletely defined mechanisms. Here we show that, in both mammalian cells and Xenopus egg extracts, BRCA1/BARD1 is required for mitotic spindle-pole assembly and for accumulation of TPX2, a major spindle organizer and Ran target, on spindle poles. This function is centrosome independent, operates downstream of Ran GTPase, and depends upon BRCA1/BARD1 E3 ubiquitin ligase activity. Xenopus BRCA1/BARD1 forms endogenous complexes with three spindle-pole proteins, TPX2, NuMA, and XRHAMM—a known TPX2 partner—and specifically attenuates XRHAMM function. These observations reveal a previously unrecognized function of BRCA1/BARD1 in mitotic spindle assembly that likely contributes to its role in chromosome stability control and tumor suppression.
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xrhamm functions in ran dependent microtubule nucleation and pole formation during anastral spindle assembly
Current Biology, 2004Co-Authors: Aaron C. Groen, Timothy J Mitchison, Lisa A Cameron, Margaret Coughlin, David T Miyamoto, Ryoma OhiAbstract:Abstract Background: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes. Results: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with γ-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization. Conclusions: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and γ-TuRC.
Timothy J Mitchison - One of the best experts on this subject based on the ideXlab platform.
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functional overlap of microtubule assembly factors in chromatin promoted spindle assembly
Molecular Biology of the Cell, 2009Co-Authors: Aaron C. Groen, Thomas J Maresca, Jesse C Gatlin, E D Salmon, Timothy J MitchisonAbstract:Distinct pathways from centrosomes and chromatin are thought to contribute in parallel to microtubule nucleation and stabilization during animal cell mitotic spindle assembly, but their full mechanisms are not known. We investigated the function of three proposed nucleation/stabilization factors, TPX2, γ-tubulin and XMAP215, in chromatin-promoted assembly of anastral spindles in Xenopus laevis egg extract. In addition to conventional depletion-add back experiments, we tested whether factors could substitute for each other, indicative of functional redundancy. All three factors were required for microtubule polymerization and bipolar spindle assembly around chromatin beads. Depletion of TPX2 was partially rescued by the addition of excess XMAP215 or EB1, or inhibiting MCAK (a Kinesin-13). Depletion of either γ-tubulin or XMAP215 was partially rescued by adding back XMAP215, but not by adding any of the other factors. These data reveal functional redundancy between specific assembly factors in the chromatin pathway, suggesting individual proteins or pathways commonly viewed to be essential may not have entirely unique functions.
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xrhamm functions in ran dependent microtubule nucleation and pole formation during anastral spindle assembly
Current Biology, 2004Co-Authors: Aaron C. Groen, Timothy J Mitchison, Lisa A Cameron, Margaret Coughlin, David T Miyamoto, Ryoma OhiAbstract:Abstract Background: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes. Results: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with γ-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization. Conclusions: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and γ-TuRC.
Judith R Homberg - One of the best experts on this subject based on the ideXlab platform.
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enhanced aggressive phenotype of tph2 knockout rats is associated with diminished 5 ht1a receptor sensitivity
Neuropharmacology, 2019Co-Authors: Deborah G A Peeters, Adrian Newmantancredi, S.f. De ,boer, Anneke Terneusen, Mark A Varney, Robbertjan Verkes, Judith R HombergAbstract:Brain serotonin (5-HT) plays a key role in aggressive behaviours and related psychopathologies, but its precise mechanism of action remains elusive. Genetic animal models may provide a tool to elucidate the relationship between aggression and serotonin. The present study showed that tryptophan hydroxylase 2 (Tph2) knockout (KO) rats, which exhibit profoundly diminished extracellular serotonin levels, display increased aggressiveness compared to their Tph2 wildtype (WT) counterparts. However, the level of aggression in Tph2 KO rats did not equal that of feral wild type Groningen (WTG) rats. To investigate whether enhanced 5-HT1A receptor functionality may be present in Tph2 KO rats, we tested the acute anti-aggressive potency of the highly selective 5-HT1A receptor full agonist NLX-112 (a.k.a. befiradol or F13640). Data show that compared to Tph2 WT and WTG rats, the NLX-112 dose-effect curve was shifted to the right in Tph2 KO animals. These results suggest that, unlike previous reports in Tph2 KO mice, Tph2 KO rats have a decreased 5-HT1A receptor sensitivity compared to both Tph2 WT and WTG animals.
