Cap-Gly Proteins at Microtubule Plus Ends: Is EB1 Detyrosination Involved?

Localization of CAP-Gly proteins such as CLIP170 at microtubule+ends results from their dual interaction with α-tubulin and EB1 through their C-terminal amino acids −EEY. Detyrosination (cleavage of the terminal tyrosine) of α-tubulin by tubulin-carboxypeptidase abolishes CLIP170 binding. Can detyrosination affect EB1 and thus regulate the presence of CLIP170 at microtubule+ends as well? We developed specific antibodies to discriminate tyrosinated vs detyrosinated forms of EB1 and detected only tyrosinated EB1 in fibroblasts, astrocytes, and total brain tissue. Over-expressed EB1 was not detyrosinated in cells and chimeric EB1 with the eight C-terminal amino acids of α-tubulin was only barely detyrosinated. Our results indicate that detyrosination regulates CLIPs interaction with α-tubulin, but not with EB1. They highlight the specificity of carboxypeptidase toward tubulin

Searching for the detyrosinating enzyme of C-terminal α-tubulin

Dans les cellules, les microtubules interviennent dans de nombreux événements comme le maintien de l'architecture, la division du matériel génétique, la migration cellulaire ou encore le transport de vésicules et d'organites. Les modifications post-traductionnelles du le C-terminus de la tubuline, bloc de base des microtubules, apparaissent comme très impliquées dans la régulation de ces fonctions car elles régulent le recrutement de leurs nombreux partenaires protéiques. Durant ma thèse, je me suis particulièrement intéressée à une de ces modifications post traductionnelles : le cycle de détyrosination/tyrosination du C-terminus de l'α-tubuline. Ce cycle implique deux enzymes la Tubuline CarboxyPeptidase (TCP), qui clive la tyrosine à l'extrémité de l'α-tubuline, et la Tubuline Tyrosine Ligase (TTL) qui ré-additionne une tyrosine à la tubuline détyrosinée. Des études menées sur ce cycle et notamment la découverte de la TTL ont permis de montrer que la présence d'une tyrosine à l'extrémité C-terminale de l'α-tubuline est indispensable au développement neuronal et que son absence favorise la progression tumorale. La TCP quant à elle est encore inconnue et sa découverte apparaît essentielle afin de pouvoir appréhender le cycle de détyrosination/tyrosination dans sa globalité. Avec pour fil rouge l'identification de la TCP, mon travail s'est déroulé en trois temps. Je me suis tout d'abord intéressée à la protéine EB1. Cette protéine se lie à l'extrémité positive des microtubules où elle recrute de nombreux partenaires microtubulaires. EB1, présente le même C-terminus que l'α-tubuline et notamment une tyrosine terminale indispensable à la liaison des protéines à CAP-Gly. Dans des cellules et des tissus sains j'ai montré qu'EB1 n'existe pas sous forme détyrosinée ce qui souligne la spécificité de la TCP pour l'α-tubuline. Dans un second temps, j'ai participé à l'étude d'une famille de carboxypeptidases cytosoliques impliquées dans la neurodégénérescence, les CCPs, parmi lesquelles nous pensions trouver la TCP. Nous avons montré que quatre de ces enzymes (CCP1, 4, 5, et 6) retirent les glutamates présents de manière latérale au C-terminus de la tubuline. Les CCP1, 4 et 6 peuvent également cliver le dernier glutamate de la tubuline détyrosiné générant de l'α-tubuline où les deux derniers acides aminés ont été clivés (tubuline-Δ2). Aucune de ces carboxypeptidases ne se révélant être la TCP, j'ai mis en place une méthode biochimique dans le but de purifier cette enzyme. Après plusieurs étapes de purification à partir de cerveaux de souris, des préparations enrichies en activité carboxypeptidase ont été obtenues. Les analyses spectrométriques et bioinformatiques de ces préparations ont permis d'isoler des candidats TCP actuellement testés pour leur potentielle activité de détyrosination du C-terminus de l'α-tubuline. Si la TCP n'est pas présente parmi eux, les outils développés lors de cette étude devraient permettre une très prochaine identification de cette enzyme essentielle.Microtubules control many aspects of cellular function. Those polymers of tubulin are involved in numerous events ranging from the maintenance of cell architecture to cell division and migration through the transport of vesicles and organelles. The post-translational modifications of the C-terminus of tubulin appear to be involved in the regulation of microtubule functions by recruiting different effectors at the growing end of microtubules. During my PhD, I focused on one of these post-translational modifications: the detyrosination/tyrosination cycle of the C-terminal α-tubulin. This cycle involves the enzymatic removal of the C-terminal tyrosine of α-tubulin by an uncharacterized tubulin carboxypeptidase (TCP) and the re-addition of a tyrosine residue by the Tubulin-Tyrosine-Ligase (TTL) isolated in 1975. On one hand, tubulin tyrosination is important in neuronal organization whereas TTL suppression in human cancers is associated with tumor aggressiveness. Those defects are in part due to the failure of microtubule partners to bind detyrosinated microtubules. My project was divided into three main parts. I have first studied EB1, a microtubule plus end tracking protein which recruits many proteins at the microtubule plus end. This protein ends with the same amino acids as does α-tubulin. As in tubulin, the tyrosine terminal is important for the binding of EB1 partners. I showed that EB1 does not exist under detyrosinated form underlying the TCP specificity. Then, I collaborated to identify the function of a carboxypeptidase family within which we thought we could find the TCP. Four members of this family are deglutamylating enzymes (CCP1, CCP4, CCP5 and CCP5). Three of them (CCP1, CCP4 and CCP6) can cleave the last glutamate of detyrosinated tubulin to generate tubulin without the last two C-terminus amino acids (Δ2-tubulin). However, none of them was identified as the TCP. I consequently developed a biochemical approach to find this enzyme. Extracts enriched in carboxypeptidase activity after purification steps from mice brain were analyzed by mass spec and bioinformatics. Some candidates are currently tested for their potential C-terminus α-tubulin detyrosinating activity. The tools developed here should allow for pending identification of the TCP

A la recherche de l'enzyme de détyrosination du C-terminus de l'a-tubuline

Dans les cellules, les microtubules interviennent dans de nombreux événements comme le maintien de l'architecture, la division du matériel génétique, la migration cellulaire ou encore le transport de vésicules et d'organites. Les modifications post-traductionnelles du le C-terminus de la tubuline, bloc de base des microtubules, apparaissent comme très impliquées dans la régulation de ces fonctions car elles régulent le recrutement de leurs nombreux partenaires protéiques. Durant ma thèse, je me suis particulièrement intéressée à une de ces modifications post traductionnelles : le cycle de détyrosination/tyrosination du C-terminus de l'a-tubuline. Ce cycle implique deux enzymes la Tubuline CarboxyPeptidase (TCP), qui clive la tyrosine à l'extrémité de l'a-tubuline, et la Tubuline Tyrosine Ligase (TTL) qui ré-additionne une tyrosine à la tubuline détyrosinée. Des études menées sur ce cycle et notamment la découverte de la TTL ont permis de montrer que la présence d'une tyrosine à l'extrémité C-terminale de l'a-tubuline est indispensable au développement neuronal et que son absence favorise la progression tumorale. La TCP quant à elle est encore inconnue et sa découverte apparaît essentielle afin de pouvoir appréhender le cycle de détyrosination/tyrosination dans sa globalité. Avec pour fil rouge l'identification de la TCP, mon travail s'est déroulé en trois temps. Je me suis tout d'abord intéressée à la protéine EB1. Cette protéine se lie à l'extrémité positive des microtubules où elle recrute de nombreux partenaires microtubulaires. EB1, présente le même C-terminus que l'a-tubuline et notamment une tyrosine terminale indispensable à la liaison des protéines à CAP-Gly. Dans des cellules et des tissus sains j'ai montré qu'EB1 n'existe pas sous forme détyrosinée ce qui souligne la spécificité de la TCP pour l'a-tubuline. Dans un second temps, j'ai participé à l'étude d'une famille de carboxypeptidases cytosoliques impliquées dans la neurodégénérescence, les CCPs, parmi lesquelles nous pensions trouver la TCP. Nous avons montré que quatre de ces enzymes (CCP1, 4, 5, et 6) retirent les glutamates présents de manière latérale au C-terminus de la tubuline. Les CCP1, 4 et 6 peuvent également cliver le dernier glutamate de la tubuline détyrosiné générant de l'a-tubuline où les deux derniers acides aminés ont été clivés (tubuline- 2). Aucune de ces carboxypeptidases ne se révélant être la TCP, j'ai mis en place une méthode biochimique dans le but de purifier cette enzyme. Après plusieurs étapes de purification à partir de cerveaux de souris, des préparations enrichies en activité carboxypeptidase ont été obtenues. Les analyses spectrométriques et bioinformatiques de ces préparations ont permis d'isoler des candidats TCP actuellement testés pour leur potentielle activité de détyrosination du C-terminus de l'a-tubuline. Si la TCP n'est pas présente parmi eux, les outils développés lors de cette étude devraient permettre une très prochaine identification de cette enzyme essentielle.Microtubules control many aspects of cellular function. Those polymers of tubulin are involved in numerous events ranging from the maintenance of cell architecture to cell division and migration through the transport of vesicles and organelles. The post-translational modifications of the C-terminus of tubulin appear to be involved in the regulation of microtubule functions by recruiting different effectors at the growing end of microtubules. During my PhD, I focused on one of these post-translational modifications: the detyrosination/tyrosination cycle of the C-terminal a-tubulin. This cycle involves the enzymatic removal of the C-terminal tyrosine of a-tubulin by an uncharacterized tubulin carboxypeptidase (TCP) and the re-addition of a tyrosine residue by the Tubulin-Tyrosine-Ligase (TTL) isolated in 1975. On one hand, tubulin tyrosination is important in neuronal organization whereas TTL suppression in human cancers is associated with tumor aggressiveness. Those defects are in part due to the failure of microtubule partners to bind detyrosinated microtubules. My project was divided into three main parts. I have first studied EB1, a microtubule plus end tracking protein which recruits many proteins at the microtubule plus end. This protein ends with the same amino acids as does a-tubulin. As in tubulin, the tyrosine terminal is important for the binding of EB1 partners. I showed that EB1 does not exist under detyrosinated form underlying the TCP specificity. Then, I collaborated to identify the function of a carboxypeptidase family within which we thought we could find the TCP. Four members of this family are deglutamylating enzymes (CCP1, CCP4, CCP5 and CCP5). Three of them (CCP1, CCP4 and CCP6) can cleave the last glutamate of detyrosinated tubulin to generate tubulin without the last two C-terminus amino acids ( 2-tubulin). However, none of them was identified as the TCP. I consequently developed a biochemical approach to find this enzyme. Extracts enriched in carboxypeptidase activity after purification steps from mice brain were analyzed by mass spec and bioinformatics. Some candidates are currently tested for their potential C-terminus a-tubulin detyrosinating activity. The tools developed here should allow for pending identification of the TCP.SAVOIE-SCD - Bib.électronique (730659901) / SudocGRENOBLE1/INP-Bib.électronique (384210012) / SudocGRENOBLE2/3-Bib.électronique (384219901) / SudocSudocFranceF

Study of endogenous EB1 C-termini in fibroblasts and brain from wild type and TTL-deficient mouse.

<p>Western-blot analysis of the indicated control proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033490#pone-0033490-g001" target="_blank">figure 1</a>) or extracts. (A) Immunoprecipitation of endogenous EB1 from wild type (TTL^+/+) or TTL-deficient MEFs using anti-total EB1 antibody, and analysis with anti-Tyr EB1 (1∶15000), anti-detyr EB1 (1∶200), and anti-total EB1 (1∶2000). EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. No detyrosinated EB1 could be detected in the IP fractions. Note that anti-total EB1 antibody being less sensitive than anti-Tyr EB1, EB1 failed to be detected in crude extract (upper panel). (B) Immunodepletion of tyrosinated EB1 with anti-Tyr EB1 (IP 1 to 4) in brain extracts from wild type and TTL-knockout mice, followed by immunoprecipitation of the remaining EB1 with anti-total EB1 (IP5), and analysis with anti-total EB1 (1∶2000). No remaining EB1 could be detected after tyrosinated-EB1 immunodepletion. (C) Tyrosinated and detyrosinated tubulin pools in brain extracts from wild type and TTL-deficient mice were analyzed using anti-α tubulin (1∶10,000), anti-tyrosinated tubulin (YL_1/2, 1∶20,000), and anti-detyrosinated tubulin (L₄, 1∶20,000).</p

Analysis of C-termini of recombinant EB1 forms overexpressed in wild type and TTL-deficient fibroblasts.

<p>Western-blot analysis of the indicated control proteins (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033490#pone-0033490-g001" target="_blank">figure 1</a>) and of fractions of immunoprecipitation experiments carried out on cells transfected with cDNA encoding different EB1 forms. EX: crude extract; SN: supernatant after immunoprecipitation; IP: immunoprecipitated fraction. (A) transfection of fibroblasts (NIH3T3) with plasmids encoding tyrosinated EB1 fused with GFP at the N-terminus (GFP-EB1), followed by immunoprecipitation using anti-GFP antibody and analysis using anti-Tyr EB1 (1∶15000) and anti-deTyr EB1 (1∶200). No detyrosinated GFP-EB1 could be detected. (B) Transfection of fibroblasts with cDNA encoding GFP-EB1 ending with the C-terminus of α-tubulin GEEEGEEY (GFP-EB1-CterTub), followed by immunoprecipitation with anti-GFP antibody and analysis using anti-Tyr Tub (1∶20,000) and anti-deTyr Tub (1∶20,000). NIH3T3 were used as TTL^+/+ cells and MEFs isolated from TTL null mice were used as TTL^−/−. A very low quantity of detyrosinated protein ending with α-tubulin residues was detected (upper band in IP fractions of lower panel).</p

Analysis of developed anti-EB1 antibodies compared to the known anti-tubulin antibodies.

<p>(A) Western-blot analysis of the indicated proteins (15 ng) separated on 10% SDS-PAGE using a commercial anti-EB1 antibody (anti-total EB1, raised against amino-acids 107–268 of mouse EB1), the presently developed antibodies (anti-Tyr EB1 and anti-deTyr EB1), and tubulin antibodies. Detyrosinated EB1 was obtained from recombinant EB1 using carboxypeptidase A. Tyrosinated and detyrosinated tubulin were obtained from purified brain tubulin, using respectively TTL and carboxypeptidase A. Both anti-Tyr EB1 and anti-deTyr EB1 are highly specific. (B) Double immunostaining with anti-total EB1 antibody and anti-Tyr EB1 on fibroblasts after transfection of plasmids allowing expression of either tyrosinated or detyrosinated EB1 with EGFP at the N-terminus. The transfected cells were detected by EGFP signal. Anti-Tyr EB1 is highly specific of tyrosinated form of EB1. (C) Immunostaining of endogenous EB1 in astrocytes with anti-Tyr EB1 and anti-total EB1.</p

A Family of Protein-Deglutamylating Enzymes Associated with Neurodegeneration

SummaryPolyglutamylation is a posttranslational modification that generates glutamate side chains on tubulins and other proteins. Although this modification has been shown to be reversible, little is known about the enzymes catalyzing deglutamylation. Here we describe the enzymatic mechanism of protein deglutamylation by members of the cytosolic carboxypeptidase (CCP) family. Three enzymes (CCP1, CCP4, and CCP6) catalyze the shortening of polyglutamate chains and a fourth (CCP5) specifically removes the branching point glutamates. In addition, CCP1, CCP4, and CCP6 also remove gene-encoded glutamates from the carboxyl termini of proteins. Accordingly, we show that these enzymes convert detyrosinated tubulin into Δ2-tubulin and also modify other substrates, including myosin light chain kinase 1. We further analyze Purkinje cell degeneration (pcd) mice that lack functional CCP1 and show that microtubule hyperglutamylation is directly linked to neurodegeneration. Taken together, our results reveal that controlling the length of the polyglutamate side chains on tubulin is critical for neuronal survival

Vasohibins/SVBP are tubulin carboxypeptidases (TCPs) that regulate neuron differentiation

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