The Experts below are selected from a list of 372 Experts worldwide ranked by ideXlab platform
Georg Haase - One of the best experts on this subject based on the ideXlab platform.
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Golgi Fragmentation in ALS Motor Neurons. New Mechanisms Targeting Microtubules, Tethers, and Transport Vesicles.
Frontiers in neuroscience, 2015Co-Authors: Georg Haase, Catherine RabouilleAbstract:Pathological alterations of the Golgi apparatus, such as its fragmentation represent an early pre-clinical feature of many neurodegenerative diseases and have been widely studied in the motor neuron disease amyotrophic lateral sclerosis (ALS). Yet, the underlying molecular mechanisms have remained cryptic. In principle, Golgi fragmentation may result from defects in three major classes of proteins: structural Golgi proteins, cytoskeletal proteins and molecular motors, as well as proteins mediating transport to and through the Golgi. Here, we present the different mechanisms that may underlie Golgi fragmentation in animal and cellular models of ALS linked to mutations in SOD1, TARDBP (TDP-43), VAPB, and C9Orf72 and we propose a novel one based on findings in progressive motor neuronopathy (pmn) mice. These mice are mutated in the TBCE gene encoding the cis-Golgi localized tubulin-binding cofactor E, one of five chaperones that assist in tubulin folding and microtubule polymerization. Loss of TBCE leads to alterations in Golgi microtubules, which in turn impedes on the maintenance of the Golgi architecture. This is due to down-regulation of COPI coat components, dispersion of Golgi tethers and strong accumulation of ER-Golgi SNAREs. These effects are partially rescued by the GTPase ARF1 through recruitment of TBCE to the Golgi. We hypothesize that defects in COPI vesicles, microtubules and their interaction may also underlie Golgi fragmentation in human ALS linked to other mutations, spinal muscular atrophy (SMA), and related motor neuron diseases. We also discuss the functional relevance of pathological Golgi alterations, in particular their potential causative, contributory, or compensatory role in the degeneration of motor neuron cell bodies, axons and synapses.
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Neurobiology of Disease Progressive Motor Neuronopathy: A Critical Role of the Tubulin Chaperone TBCE in Axonal Tubulin Routing from the Golgi Apparatus
2014Co-Authors: Michael K. E. Schaefer, Henning Schmalbruch, Emmanuelle Buhler, C. Lopez, Natalia Martin, Georg HaaseAbstract:Axonal degeneration represents one of the earliest pathological features in motor neuron diseases. We here studied the underlying molecular mechanisms in progressive motor neuronopathy ( pmn) mice mutated in the tubulin-specific chaperone TBCE. We demon-strate that TBCE is a peripheralmembrane-associatedprotein that accumulates at theGolgi apparatus. Inpmnmice, TBCE is destabilized and disappears from the Golgi apparatus of motor neurons, and microtubules are lost in distal axons. The axonal microtubule loss proceeds retrogradely in parallel with the axonal dying back process. These degenerative changes are inhibited in a dose-dependent manner by transgenic TBCE complementation that restores TBCE expression at theGolgi apparatus. In culturedmotor neurons, the pmn mutation, interference RNA-mediated TBCE depletion, and brefeldin A-mediated Golgi disruption all compromise axonal tubulin rout-ing.Weconclude thatmotor axons criticallydependonaxonal tubulin routing fromtheGolgi apparatus, aprocess that involvesTBCEand possibly other tubulin chaperones. Key words:motor neuron disease; ALS; axon degeneration; tubulin chaperone; microtubules; Golgi apparatu
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golgi fragmentation in pmn mice is due to a defective arf1 TBCE cross talk that coordinates copi vesicle formation and tubulin polymerization
Human Molecular Genetics, 2014Co-Authors: Sarah Bellouze, Michael K. E. Schäfer, Dorothée Buttigieg, Gilbert Baillat, Catherine Rabouille, Georg HaaseAbstract:Golgi fragmentation is an early hallmark of many neurodegenerative diseases but its pathophysiological relevance and molecular mechanisms are unclear. We here demonstrate severe and progressive Golgi fragmentation in motor neurons of progressive motor neuronopathy (pmn) mice due to loss of the Golgi-localized tubulin-binding cofactor E (TBCE). Loss of TBCE in mutant pmn and TBCE-depleted motor neuron cultures causes defects in Golgi-derived microtubules, as expected, but surprisingly also reduced levels of COPI subunits, decreased recruitment of tethering factors p115/GM130 and impaired Golgi SNARE-mediated vesicle fusion. Conversely, ARF1, which stimulates COPI vesicle formation, enhances the recruitment of TBCE to the Golgi, increases polymerization of Golgi-derived microtubules and rescues TBCE-linked Golgi fragmentation. These data indicate an ARF1/TBCE-mediated cross-talk that coordinates COPI formation and tubulin polymerization at the Golgi. We conclude that interruption of this cross-talk causes Golgi fragmentation in pmn mice and hypothesize that similar mechanisms operate in human amyotrophic lateral sclerosis and spinal muscular atrophy.
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Golgi fragmentation in pmn mice is due to a defective ARF1/TBCE cross talk that coordinates COPI vesicle formation and tubulin polymerization
Human molecular genetics, 2014Co-Authors: Sarah Bellouze, Michael K. E. Schäfer, Dorothée Buttigieg, Gilbert Baillat, Catherine Rabouille, Georg HaaseAbstract:Golgi fragmentation is an early hallmark of many neurodegenerative diseases but its pathophysiological relevance and molecular mechanisms are unclear. We here demonstrate severe and progressive Golgi fragmentation in motor neurons of progressive motor neuronopathy (pmn) mice due to loss of the Golgi-localized tubulin-binding cofactor E (TBCE). Loss of TBCE in mutant pmn and TBCE-depleted motor neuron cultures causes defects in Golgi-derived microtubules, as expected, but surprisingly also reduced levels of COPI subunits, decreased recruitment of tethering factors p115/GM130 and impaired Golgi SNARE-mediated vesicle fusion. Conversely, ARF1, which stimulates COPI vesicle formation, enhances the recruitment of TBCE to the Golgi, increases polymerization of Golgi-derived microtubules and rescues TBCE-linked Golgi fragmentation. These data indicate an ARF1/TBCE-mediated cross-talk that coordinates COPI formation and tubulin polymerization at the Golgi. We conclude that interruption of this cross-talk causes Golgi fragmentation in pmn mice and hypothesize that similar mechanisms operate in human amyotrophic lateral sclerosis and spinal muscular atrophy.
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Progressive motor neuronopathy: a critical role of the tubulin chaperone TBCE in axonal tubulin routing from the Golgi apparatus.
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2007Co-Authors: Michael K. E. Schaefer, Henning Schmalbruch, Emmanuelle Buhler, C. Lopez, Natalia Martin, Jean-louis Guénet, Georg HaaseAbstract:Axonal degeneration represents one of the earliest pathological features in motor neuron diseases. We here studied the underlying molecular mechanisms in progressive motor neuronopathy (pmn) mice mutated in the tubulin-specific chaperone TBCE. We demonstrate that TBCE is a peripheral membrane-associated protein that accumulates at the Golgi apparatus. In pmn mice, TBCE is destabilized and disappears from the Golgi apparatus of motor neurons, and microtubules are lost in distal axons. The axonal microtubule loss proceeds retrogradely in parallel with the axonal dying back process. These degenerative changes are inhibited in a dose-dependent manner by transgenic TBCE complementation that restores TBCE expression at the Golgi apparatus. In cultured motor neurons, the pmn mutation, interference RNA-mediated TBCE depletion, and brefeldin A-mediated Golgi disruption all compromise axonal tubulin routing. We conclude that motor axons critically depend on axonal tubulin routing from the Golgi apparatus, a process that involves TBCE and possibly other tubulin chaperones.
Juan Carlos Zabala - One of the best experts on this subject based on the ideXlab platform.
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the structure of the TBCE tbcb chaperones and a tubulin complex shows a tubulin dimer dissociation mechanism
Journal of Cell Science, 2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Robert Janowski, Albert Canals, Miquel Coll, Jaime Martinbenito, José M. ValpuestaAbstract:This work was supported by the Spanish Ministry of Science and Innovation [grant numbers CONSOLIDER CSD 2006‐23 to M.C., J.C.Z. and J.M.V., BFU2011‐22588 to M.C., BFU2011‐25090 to J.M.B., BFU2013‐44202 to J.M.V., and BFU2010‐18948 to J.C.Z.]; the Madrid Regional Government [grant number S2013/MIT‐2807 to J.M.V.]; the Generalitat de Catalunya [grant number SGR2009‐1309 to M.C.]; the Instituto de Investigacion Marques de Valdecilla (IDIVAL); the Universidad de Cantabria [grant number 02.VP01.64005 to J.C.Z.]; and the European Commission FP7 Cooperation Project SILVER ‐ GA [grant number 260644 to M.C.]
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The structure of the complex between a-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism
2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Jaime Martín-benito, Robert Janowski, Albert Canals, Miquel Coll, José M. ValpuestaAbstract:This work was supported by the Spanish Ministry of Science and Innovation [grant numbers CONSOLIDER CSD 2006‐23 to M.C., J.C.Z. and J.M.V., BFU2011‐22588 to M.C., BFU2011‐25090 to J.M.B., BFU2013‐44202 to J.M.V., and BFU2010‐18948 to J.C.Z.]; the Madrid Regional Government [grant number S2013/MIT‐2807 to J.M.V.]; the Generalitat de Catalunya [grant number SGR2009‐1309 to M.C.]; the Instituto de Investigacion Marques de Valdecilla (IDIVAL); the Universidad de Cantabria [grant number 02.VP01.64005 to J.C.Z.]; and the European Commission FP7 Cooperation Project SILVER ‐ GA [grant number 260644 to M.C.]
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the structure of the complex between α tubulin TBCE and tbcb reveals a tubulin dimer dissociation mechanism
Journal of Cell Science, 2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Robert Janowski, Albert Canals, Miquel Coll, Jaime Martinbenito, José M. ValpuestaAbstract:Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-β-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and α-tubulin (αEB) complex, which is formed upon α-tubulin-β-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the αEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the α-tubulin-β-tubulin interface that is caused by a steric interaction between β-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the αEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α-tubulin degradation.
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The structure of the complex between α‐tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism
Journal of cell science, 2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Jaime Martín-benito, Robert Janowski, Albert Canals, Miquel Coll, José M. ValpuestaAbstract:Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-β-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and α-tubulin (αEB) complex, which is formed upon α-tubulin-β-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the αEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the α-tubulin-β-tubulin interface that is caused by a steric interaction between β-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the αEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α-tubulin degradation.
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Expert in tubulins, end binding proteins and microtubules
2014Co-Authors: Juan Carlos Zabala, Mónica L. Fanarraga, Jesús Avila, Raquel Castaño, Juan Carlos Villegas, João Gonçalves, Helena Soares, Marco Marenchino, Ramón Campos-olivas, Guillermo MontoyaAbstract:Abstract: Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that interact and dissociate the tubulin dimer. Here we show how TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO- present in TBCB, which is similar to the EEY/F-COO- element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE-TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated to microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation
José M. Valpuesta - One of the best experts on this subject based on the ideXlab platform.
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the structure of the TBCE tbcb chaperones and a tubulin complex shows a tubulin dimer dissociation mechanism
Journal of Cell Science, 2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Robert Janowski, Albert Canals, Miquel Coll, Jaime Martinbenito, José M. ValpuestaAbstract:This work was supported by the Spanish Ministry of Science and Innovation [grant numbers CONSOLIDER CSD 2006‐23 to M.C., J.C.Z. and J.M.V., BFU2011‐22588 to M.C., BFU2011‐25090 to J.M.B., BFU2013‐44202 to J.M.V., and BFU2010‐18948 to J.C.Z.]; the Madrid Regional Government [grant number S2013/MIT‐2807 to J.M.V.]; the Generalitat de Catalunya [grant number SGR2009‐1309 to M.C.]; the Instituto de Investigacion Marques de Valdecilla (IDIVAL); the Universidad de Cantabria [grant number 02.VP01.64005 to J.C.Z.]; and the European Commission FP7 Cooperation Project SILVER ‐ GA [grant number 260644 to M.C.]
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The structure of the complex between a-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism
2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Jaime Martín-benito, Robert Janowski, Albert Canals, Miquel Coll, José M. ValpuestaAbstract:This work was supported by the Spanish Ministry of Science and Innovation [grant numbers CONSOLIDER CSD 2006‐23 to M.C., J.C.Z. and J.M.V., BFU2011‐22588 to M.C., BFU2011‐25090 to J.M.B., BFU2013‐44202 to J.M.V., and BFU2010‐18948 to J.C.Z.]; the Madrid Regional Government [grant number S2013/MIT‐2807 to J.M.V.]; the Generalitat de Catalunya [grant number SGR2009‐1309 to M.C.]; the Instituto de Investigacion Marques de Valdecilla (IDIVAL); the Universidad de Cantabria [grant number 02.VP01.64005 to J.C.Z.]; and the European Commission FP7 Cooperation Project SILVER ‐ GA [grant number 260644 to M.C.]
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the structure of the complex between α tubulin TBCE and tbcb reveals a tubulin dimer dissociation mechanism
Journal of Cell Science, 2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Robert Janowski, Albert Canals, Miquel Coll, Jaime Martinbenito, José M. ValpuestaAbstract:Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-β-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and α-tubulin (αEB) complex, which is formed upon α-tubulin-β-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the αEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the α-tubulin-β-tubulin interface that is caused by a steric interaction between β-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the αEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α-tubulin degradation.
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The structure of the complex between α‐tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism
Journal of cell science, 2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Jaime Martín-benito, Robert Janowski, Albert Canals, Miquel Coll, José M. ValpuestaAbstract:Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-β-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and α-tubulin (αEB) complex, which is formed upon α-tubulin-β-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the αEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the α-tubulin-β-tubulin interface that is caused by a steric interaction between β-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the αEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α-tubulin degradation.
Gerardo Carranza - One of the best experts on this subject based on the ideXlab platform.
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the structure of the TBCE tbcb chaperones and a tubulin complex shows a tubulin dimer dissociation mechanism
Journal of Cell Science, 2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Robert Janowski, Albert Canals, Miquel Coll, Jaime Martinbenito, José M. ValpuestaAbstract:This work was supported by the Spanish Ministry of Science and Innovation [grant numbers CONSOLIDER CSD 2006‐23 to M.C., J.C.Z. and J.M.V., BFU2011‐22588 to M.C., BFU2011‐25090 to J.M.B., BFU2013‐44202 to J.M.V., and BFU2010‐18948 to J.C.Z.]; the Madrid Regional Government [grant number S2013/MIT‐2807 to J.M.V.]; the Generalitat de Catalunya [grant number SGR2009‐1309 to M.C.]; the Instituto de Investigacion Marques de Valdecilla (IDIVAL); the Universidad de Cantabria [grant number 02.VP01.64005 to J.C.Z.]; and the European Commission FP7 Cooperation Project SILVER ‐ GA [grant number 260644 to M.C.]
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the structure of the complex between α tubulin TBCE and tbcb reveals a tubulin dimer dissociation mechanism
Journal of Cell Science, 2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Robert Janowski, Albert Canals, Miquel Coll, Jaime Martinbenito, José M. ValpuestaAbstract:Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-β-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and α-tubulin (αEB) complex, which is formed upon α-tubulin-β-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the αEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the α-tubulin-β-tubulin interface that is caused by a steric interaction between β-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the αEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α-tubulin degradation.
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The structure of the complex between a-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism
2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Jaime Martín-benito, Robert Janowski, Albert Canals, Miquel Coll, José M. ValpuestaAbstract:This work was supported by the Spanish Ministry of Science and Innovation [grant numbers CONSOLIDER CSD 2006‐23 to M.C., J.C.Z. and J.M.V., BFU2011‐22588 to M.C., BFU2011‐25090 to J.M.B., BFU2013‐44202 to J.M.V., and BFU2010‐18948 to J.C.Z.]; the Madrid Regional Government [grant number S2013/MIT‐2807 to J.M.V.]; the Generalitat de Catalunya [grant number SGR2009‐1309 to M.C.]; the Instituto de Investigacion Marques de Valdecilla (IDIVAL); the Universidad de Cantabria [grant number 02.VP01.64005 to J.C.Z.]; and the European Commission FP7 Cooperation Project SILVER ‐ GA [grant number 260644 to M.C.]
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The structure of the complex between α‐tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism
Journal of cell science, 2015Co-Authors: Marina Serna, Gerardo Carranza, Juan Carlos Zabala, Jaime Martín-benito, Robert Janowski, Albert Canals, Miquel Coll, José M. ValpuestaAbstract:Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-β-tubulin dissociation. We used electron microscopy and image processing to determine the three-dimensional structure of the human TBCE, TBCB and α-tubulin (αEB) complex, which is formed upon α-tubulin-β-tubulin heterodimer dissociation by the two chaperones. Docking the atomic structures of domains of these proteins, including the TBCE UBL domain, as we determined by X-ray crystallography, allowed description of the molecular architecture of the αEB complex. We found that heterodimer dissociation is an energy-independent process that takes place through a disruption of the α-tubulin-β-tubulin interface that is caused by a steric interaction between β-tubulin and the TBCE cytoskeleton-associated protein glycine-rich (CAP-Gly) and leucine-rich repeat (LRR) domains. The protruding arrangement of chaperone ubiquitin-like (UBL) domains in the αEB complex suggests that there is a direct interaction of this complex with the proteasome, thus mediating α-tubulin degradation.
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Autoinhibition of TBCB regulates EB1-mediated microtubule dynamics
Cellular and Molecular Life Sciences, 2013Co-Authors: Gerardo Carranza, Mónica L. Fanarraga, Jesús Avila, Raquel Castaño, Juan Carlos Villegas, João Gonçalves, Helena Soares, Marco Marenchino, Ramón Campos-olivas, Guillermo MontoyaAbstract:Tubulin cofactors (TBCs) participate in the folding, dimerization, and dissociation pathways of the tubulin dimer. Among them, TBCB and TBCE are two CAP-Gly domain-containing proteins that together efficiently interact with and dissociate the tubulin dimer. In the study reported here we showed that TBCB localizes at spindle and midzone microtubules during mitosis. Furthermore, the motif DEI/M-COO^− present in TBCB, which is similar to the EEY/F-COO^− element characteristic of EB proteins, CLIP-170, and α-tubulin, is required for TBCE–TBCB heterodimer formation and thus for tubulin dimer dissociation. This motif is responsible for TBCB autoinhibition, and our analysis suggests that TBCB is a monomer in solution. Mutants of TBCB lacking this motif are derepressed and induce microtubule depolymerization through an interaction with EB1 associated with microtubule tips. TBCB is also able to bind to the chaperonin complex CCT containing α-tubulin, suggesting that it could escort tubulin to facilitate its folding and dimerization, recycling or degradation.
Nicholas J Cowan - One of the best experts on this subject based on the ideXlab platform.
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tubulin specific chaperones components of a molecular machine that assembles the α β heterodimer
Methods in Cell Biology, 2013Co-Authors: Guoling Tian, Nicholas J CowanAbstract:Abstract The tubulin heterodimer consists of one α- and one β-tubulin polypeptide. Neither protein can partition to the native state or assemble into polymerization competent heterodimers without the concerted action of a series of chaperone proteins including five tubulin-specific chaperones (TBCs) termed TBCA–TBCE. TBCA and TBCB bind to and stabilize newly synthesized quasi-native β- and α-tubulin polypeptides, respectively, following their generation via multiple rounds of ATP-dependent interaction with the cytosolic chaperonin. There is free exchange of β-tubulin between TBCA and TBCD, and of α-tubulin between TBCB and TBCE, resulting in the formation of TBCD/β and TBCE/α, respectively. The latter two complexes interact, forming a supercomplex (TBCE/α/TBCD/β). Discharge of the native α/β heterodimer occurs via interaction of the supercomplex with TBCC, which results in the triggering of TBC-bound β-tubulin (E-site) GTP hydrolysis. This reaction acts as a switch for disassembly of the supercomplex and the release of E-site GDP-bound heterodimer, which becomes polymerization competent following spontaneous exchange with GTP. The tubulin-specific chaperones thus function together as a tubulin assembly machine, marrying the α- and β-tubulin subunits into a tightly associated heterodimer. The existence of this evolutionarily conserved pathway explains why it has never proved possible to isolate α- or β-tubulin as stable independent entities in the absence of their cognate partners, and implies that each exists and is maintained in the heterodimer in a nonminimal energy state. Here, we describe methods for the purification of recombinant TBCs as biologically active proteins following their expression in a variety of host/vector systems.
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Methods in Cell Biology - Tubulin-Specific Chaperones
Methods in cell biology, 2013Co-Authors: Guoling Tian, Nicholas J CowanAbstract:The tubulin heterodimer consists of one α- and one β-tubulin polypeptide. Neither protein can partition to the native state or assemble into polymerization competent heterodimers without the concerted action of a series of chaperone proteins including five tubulin-specific chaperones (TBCs) termed TBCA-TBCE. TBCA and TBCB bind to and stabilize newly synthesized quasi-native β- and α-tubulin polypeptides, respectively, following their generation via multiple rounds of ATP-dependent interaction with the cytosolic chaperonin. There is free exchange of β-tubulin between TBCA and TBCD, and of α-tubulin between TBCB and TBCE, resulting in the formation of TBCD/β and TBCE/α, respectively. The latter two complexes interact, forming a supercomplex (TBCE/α/TBCD/β). Discharge of the native α/β heterodimer occurs via interaction of the supercomplex with TBCC, which results in the triggering of TBC-bound β-tubulin (E-site) GTP hydrolysis. This reaction acts as a switch for disassembly of the supercomplex and the release of E-site GDP-bound heterodimer, which becomes polymerization competent following spontaneous exchange with GTP. The tubulin-specific chaperones thus function together as a tubulin assembly machine, marrying the α- and β-tubulin subunits into a tightly associated heterodimer. The existence of this evolutionarily conserved pathway explains why it has never proved possible to isolate α- or β-tubulin as stable independent entities in the absence of their cognate partners, and implies that each exists and is maintained in the heterodimer in a nonminimal energy state. Here, we describe methods for the purification of recombinant TBCs as biologically active proteins following their expression in a variety of host/vector systems.
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effect of tbcd and its regulatory interactor arl2 on tubulin and microtubule integrity
Cytoskeleton, 2010Co-Authors: Guoling Tian, Simi Thomas, Nicholas J CowanAbstract:Assembly of the α/β tubulin heterodimer requires the participation of a series of chaperone proteins (TBCA-E) that function downstream of the cytosolic chaperonin, CCT, as a heterodimer assembly machine. TBCD and TBCE are also capable of acting in a reverse reaction in which they disrupt native heterodimers. Homologs of TBCA-E exist in all eukaryotes, and the amino acid sequences of α- and β-tubulin isotypes are rigidly conserved among vertebrates. However, the efficiency with which TBCD effects tubulin disruption in vivo depends on its origin: bovine (but not human) TBCD efficiently destroys tubulin and microtubules upon overexpression in cultured cells. Here we show that recombinant bovine TBCD is produced in HeLa cells as a stoichiometric co-complex with β-tubulin, consistent with its behavior in vitro and in vivo. In contrast, expression of human TBCD using the same host/vector system results in the generation of TBCD that is not complexed with β-tubulin. We show that recombinant human TBCD functions indistinguishably from its non-recombinant bovine counterpart in in vitro CCT-driven folding reactions, in tubulin disruption reactions, and in tubulin GAP assays in which TBCD and TBCC stimulate GTP hydrolysis by β-tubulin at a heterodimer concentration far below that required for polymerization into microtubules. We conclude that bovine and human TBCD have functionally identical roles in de novo tubulin heterodimer assembly, and show that the inability of human TBCD to disrupt microtubule integrity upon overexpression in vivo can be overcome by siRNA-mediated suppression of expression of the TBCD regulator Arl2 (ADP Ribosylation Factor-like Protein 2).
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Cryptic out-of-frame translational initiation of TBCE rescues tubulin formation in compound heterozygous HRD.
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Guoling Tian, Melissa C. Huang, Ruti Parvari, George A. Diaz, Nicholas J CowanAbstract:Microtubules are indispensable dynamic structures that contribute to many essential biological functions. Assembly of the native α/β tubulin heterodimer, the subunit that polymerizes to form microtubules, requires the participation of several molecular chaperones, namely prefoldin, the cytosolic chaperonin CCT, and a series of five tubulin-specific chaperones termed cofactors A–E (TBCA–E). Among these, TBCC, TBCD, and TBCE are essential in higher eukaryotes; they function together as a multimolecular machine that assembles quasinative CCT-generated α- and β-tubulin polypeptides into new heterodimers. Deletion and truncation mutations in the gene encoding TBCE have been shown to cause the rare autosomal recessive syndrome known as HRD, a devastating disorder characterized by congenital hypoparathyroidism, mental retardation, facial dysmorphism, and extreme growth failure. Here we identify cryptic translational initiation at each of three out-of-frame AUG codons upstream of the genetic lesion as a unique mechanism that rescues a mutant HRD allele by producing a functional TBCE protein. Our data explain how afflicted individuals, who would otherwise lack the capacity to make functional TBCE, can survive and point to a limiting capacity to fold tubulin heterodimers de novo as a contributing factor to disease pathogenesis.
