The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform
Klaus Weber - One of the best experts on this subject based on the ideXlab platform.
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Post-translational modifications regulate microtubule function
Nature Reviews Molecular Cell Biology, 2003Co-Authors: Stefan Westermann, Klaus WeberAbstract:The carboxy-terminal tails of α- and β-tubulin are essential for microtubule function. They lie on the outer surface of the microtubule where they can influence the binding of associated proteins. With the exception of acetylation, the post-translational modifications of microtubules — that is, detyrosination/tyrosination, formation of Δ2-tubulin, polyglutamylation and Polyglycylation — are all located in the carboxy-terminal tails. Acetylation of α-tubulin can be abolished without consequences in Tetrahymena , but it seems to have a function in cell motility. Two histone deacetylases, HDAC6 and SIRT2, have been shown to function as tubulin deacetylases. Genetic analysis of Polyglycylation in Tetrahymena demonstrates its essential function in the organization of axonemes, cell motility and cytokinesis. Polyglutamylation can influence the binding of structural and motor microtubule-associated proteins (MAPs) to microtubules. Antibody-injection studies indicate an important role for polyglutamylation in centriole stability. The functional role of the tyrosination cycle of tubulin is still unclear; cells cultured with low activity of the tubulin tyrosine ligase (TTL) enzyme show no obvious defects. TTL-knockout mice, however, die early in development owing to an as-yet-uncharacterized defect. The αβ-tubulin heterodimer, the building block of microtubules, is subject to a large number of post-translational modifications, comparable in diversity to the intensively studied histone modifications. Although these unusual modifications are conserved throughout evolution, their functions have remained almost completely elusive. Recently, however, important advances in the understanding of how tubulin modifications regulate function and organization have been made.
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Posttranslational modifications of trichomonad tubulins; identification of multiple glutamylation sites
FEBS letters, 1998Co-Authors: André Schneider, Uwe Plessmann, Richard Felleisen, Klaus WeberAbstract:The α- and β-tubulins present in cytoskeletons of Tritrichomonas mobilensis are extensively glutamylated. Automated sequencing and mass spectrometry of the carboxyterminal peptides identifies 4 glutamylation sites in α- and 2 sites in β-tubulin. They are marked by asterisks in the terminal sequences GDE*E*E*E*DDG (α) and EGE*E*DEEAEA (β). This is the first report that tubulin glutamylation can occur at multiple sites. Although T. mobilensis has four flagellae the tubulins lack Polyglycylation. Thus glycylation is not necessary for formation or function of axonemal microtubules. α-Tubulin is completely acetylated at lysine 40 and shows no tyrosine cycle. Peptide sequences establish two distinct β-tubulins.
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Posttranslational modifications of alpha- and beta-tubulin in Giardia lamblia, an ancient eukaryote
FEBS Letters, 1997Co-Authors: Klaus Weber, André Schneider, Norbert Müller, Stefan Westermann, Uwe PlessmannAbstract:Abstract Tubulin of Giardia lamblia , a representative of the oldest eukaryotes, was screened for posttranslational modifications. Mass spectrometry of the carboxy-terminal peptides documents a large number of variants. Both α- and β-tubulin show Polyglycylation with up to 20 and 15 extra glycyl residues respectively. Minor variants show a low level of glutamylation without or with glycylation. The glutamylation-specific antibody GT335 detects α- and β-tubulin in immunoblots. The terminal tyrosine is fully retained in α-tubulin, which is completely acetylated at Lys-40. Thus except for the detyrosination/tyrosination cycle all posttranslational modifications known for higher eukaryotes are already present in Giardia .
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Mammalian Sperm Tubulin: An Exceptionally Large Number of Variants Based on Several Posttranslational Modifications
Journal of Protein Chemistry, 1997Co-Authors: Uwe Plessmann, Klaus WeberAbstract:Extraction of demembranated bull sperm flagella by SDS was used to maximize tubulin solubilization. The α- and β-tubulin separated by SDS-PAGE were treated with endoproteinases LysC and AspN, respectively. Carboxy-terminal fragments were isolated by Mono Q chromatography and reversed-phase HPLC. Automated sequencing and mass spectrometry revealed an astonishingly high number of tubulin variants. Many variants were due to polyglutamylation and in particular to Polyglycylation. The number of side-chain glycyl residues ranged from 0 to 28 in α and 0 to 15 in β. Corresponding values for side-chain glutamyl residues were 0–6 in α and 0–3 in β. Additional α variability was based on carboxy-terminal detyrosination and partial loss of the penultimate glutamate. A major glycylation site in α- and β-tubulin was mapped. Some variants seem to display both glycyl and glutamyl side chains.
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Polyglycylation of tubulin in the diplomonad Giardia lamblia, one of the oldest eukaryotes
FEBS Letters, 1996Co-Authors: Klaus Weber, André Schneider, Norbert Müller, Uwe PlessmannAbstract:Abstract We have searched for post-translational modifications in tubulin of the diplomonad Giardia lamblia , which is a representative of the earliest branches in eukaryotic evolution. The carboxyterminal peptide of α-tubulin was isolated and characterized by automated sequencing and mass spectrometry. Some 60% of the peptide is unmodified, while the remainder shows various degrees of Polyglycylation. The number of glycyl residues in the lateral side chain ranges from 2 to 23. All peptide species encountered end with alanine-tyrosine, indicating the absence of a detyrosination/tyrosination cycle. We conclude that tubulin-specific Polyglycylation could be as old as tubulin and axonemal structures.
Edoardo Pozio - One of the best experts on this subject based on the ideXlab platform.
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The Crystal Structure of Giardia duodenalis 14-3-3 in the Apo Form: When Protein Post-Translational Modifications Make the Difference
2016Co-Authors: Annarita Fiorillo, Edoardo Pozio, Daniele Di Marino, Lucia Bertuccini, Allegra Via, Serena Camerini, Andrea Ilari, Marco LalleAbstract:The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the Polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and Polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that Polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of thi
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The Crystal Structure of Giardia duodenalis 14-3-3 in the Apo Form: When Protein Post-Translational Modifications Make the Difference
PloS one, 2014Co-Authors: Annarita Fiorillo, Edoardo Pozio, Daniele Di Marino, Lucia Bertuccini, Allegra Via, Serena Camerini, Andrea Ilari, Marco LalleAbstract:The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the Polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and Polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that Polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.
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Phosophorylation and Polyglycylation of 14-3-3 proteins in Giardia.
2013Co-Authors: Marco Lalle, Edoardo Pozio, Serena Camerini, Flora Leptourgidou, Efthimios M. C. SkoulakisAbstract:A) Multiple Alignments of g14-3-3, LeoII and D14-3-3ε amino-acid sequences showing the peptides derived from trypsin digestion and containing the phosphorylated Thr214 (black boxed) and the Glu246 (grey boxed) of g14-3-3 and the corresponding peptides of LeoII and D14-3-3ε are in bold. Residues are numbered according to published protein sequences. B) Alignment of the C-terminus of g14-3-3, LeoII and D14-3-3ε with α- and β-tubulin of Giardia (GenBankTM/EBI Accession Number AAN46106 and P05304), α- and β-tubulin of Paramecium tetraurelia (GenBankTM/EBI Accession Number CAA67848 and CAE75646), and α- and β-tubulin of Tetrahymena thermophila (GenBankTM/EBI Accession Number P41351 and P41352). The alignment was performed with the ClustalW program and manually refined. The amino acids in grey define the hypothetical Polyglycylation sequence [T/G]X0-1[D/E]X1-3G[D/E]X1-2[E]2-4. Experimentally defined polyglycylated glutamic acid residues are black boxed and highlighted in bold white letters. Putative polyglycylated glutamic acid residues are only black boxed. The underlined glutamic acid of Tetrahymena α-tubulin is predicted to be polyglycylated. C-E) MALDI-MS analysis of affinity purified FLAG-tagged transfected proteins from Giardia trophozoites. MALDI-MS spectra of FLAG-g14-3-3 (C), FLAG-D14-3-3ε (D) and FLAG-LEOII (E), encompassing the MH+ range of 1400–3600. Mono-isotopic masses of relevant peaks are shown. Peptides are indicated by the positions of their N- and C-termini and numbered as in the protein sequence. Peaks shifted from the theoretical MH+ are indicated by arrows as for the 80 Da shift due to phosphorylation. For each protein, analysis of phosphorylation is reported on the left and the analysis of C-terminal Polyglycylation is reported on the right panels. C) For FLAG-g14-3-3, the calculated MH+ peak for peptide 202–219 at 2029.9 is clearly shifted to 2109.93 indicating phosphorylation on Thr214 (202AFDAAITDLDKLTEESYK219). On the right, the peaks corresponding to Polyglycylation of the peptide (230DNLNLWVTDSAGDDNAEEK248) with predicted MH+ = 2047.92 was clearly shifted to 2105.9 indicating multiple glycines on Glu246 as revealed by their number in the lateral chain. The insert shows the lack of the peak corresponding to the unmodified 230-248 peptide. D) For D14-3-3ε-FLAG, the predicted peak at MH+ = 2087.9 for the peptide (197AAFDDAIAELDTLSEESYK21) was shifted by 80 kDa to MH+ = 2167.9, indicating phosphorylation at Ser210. On the right, the peaks corresponding to the unmodified (249EQIQDVEDQDVS260 = 1404.6 MH+) peptide and the shifts to higher MH+ consistent with addition of 7 or 8 glycines (right) and phosphorylation at Ser260 (+80kDa, arrow on the left) are shown. C) For LEOII-FLAG only the peaks corresponding to unmodified (197QAFDDAIAELDTLNEDSYK215 = 2157.98 MH+) and (226DNLTLWTSDTQGDEAEPQEGGDN248 = 2492.03 MH+) peptides were evident.
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MALDI-MS analysis of affinity purified endogenous and transgenic g14-3-3 proteins from Drosophila.
2013Co-Authors: Marco Lalle, Edoardo Pozio, Serena Camerini, Flora Leptourgidou, Efthimios M. C. SkoulakisAbstract:A) Western blot analyses of GST-difopein purified 14-3-3s (1∶10) from wild type flies and His-g14-3-3 -expressing transgenic flies and the relevant controls (WB-C6 for Giardia and w1118 for flies) were separated on 12% SDS-PAGE, blotted and the membranes probed with the indicated antibodies. The AXO49 mAb detecting the Giardia polyglycylated protein did not cross react with the fly isoforms, which are detected with the anti-pan14-3-3 antibody, while the transgenic g14-3-3 protein is detectable with the anti-His in fly lysates. TR denotes trophozoite lysates, while ENC indicates lysates from 12hr encysting parasites. MALDI-MS spectra of the transgenic His-g14-3-3 (B) and the endogenous Drosophila D14-3-3ε (C) and Leo (D) encompassing the MH+ range of 1400-3600. Mono-isotopic masses of relevant peaks are shown. Peptides are indicated by the positions of their NH2- and C-termini and numbered as in the protein sequence. For each protein, the analysis of phosphorylation is reported on the left panels and the analysis of C-terminal Polyglycylation is reported on the right panels. Only peaks corresponding to the unmodified peptides were visible and reported here.
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Giardia Duodenalis 14-3-3 Protein Is Polyglycylated by a Tubulin Tyrosine Ligase-like Member and Deglycylated by Two Metallocarboxypeptidases
The Journal of biological chemistry, 2010Co-Authors: Marco Lalle, Serena Cecchetti, Marco Crescenzi, Serena Camerini, Claudia Blasetti Fantauzzi, Edoardo PozioAbstract:Abstract The flagellated protozoan Giardia duodenalis is a parasite of the upper part of the small intestine of mammals, including humans, and an interesting biological model. Giardia harbors a single 14-3-3 isoform, a multifunctional protein family, that is modified at the C terminus by Polyglycylation, an unusual post-translational modification consisting of the covalent addition of one or multiple glycines on the γ-carboxyl groups of specific glutamic acids. Polyglycylation affects the intracellular localization of g14-3-3, as the shortening of the polyglycine chain is correlated with a partial relocalization of 14-3-3 inside the nuclei during encystation. In this work we demonstrate that the gTTLL3, a member of the tubulin tyrosine ligase-like family, is the enzyme responsible for the 14-3-3 Polyglycylation. We also identify two metallopeptidases of the M20 family, here termed gDIP1 (giardial dipeptidase 1) and gDIP2, as enzymes able to shorten the g14-3-3 polyglycine tail both in vivo and in vitro. Finally, we show that the ectopic expression of gDIP2 alters the g14-3-3 localization and strongly hampers the cyst formation. In conclusion, we have identified a polyglycylase and two deglycylases that act in concert to modulate the stage-dependent glycylation status of the multifunctional regulatory g14-3-3 protein in G. duodenalis.
Jean Rossier - One of the best experts on this subject based on the ideXlab platform.
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Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha- and beta-tubulin and affect the ciliary matrix in Tetrahymena.
Journal of Biological Chemistry, 2005Co-Authors: Virginie Redeker, Nicolette Levilliers, Jacek Gaertig, Dylan Burnette, Jean Rossier, Emilie Vinolo, Danielle Jaillard, Marie-hélène BréAbstract:Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that Polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.
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Mutations of Tubulin Glycylation Sites Reveal Cross-talk between the C Termini of α- and β-Tubulin and Affect the Ciliary Matrix in Tetrahymena
The Journal of biological chemistry, 2004Co-Authors: Virginie Redeker, Dylan T Burnette, Nicolette Levilliers, Jacek Gaertig, Jean Rossier, Emilie Vinolo, Danielle Jaillard, Marie-hélène BréAbstract:Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that Polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.
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Structural characterization by tandem mass spectrometry of the posttranslational Polyglycylation of tubulin.
Biochemistry, 1999Co-Authors: Joelle Vinh, Nicolette Levilliers, Marie-hélène Bré, Virginie Redeker, James I. Langridge, Denis Loyaux, Jean RossierAbstract:Polyglycylation is a posttranslational modification specific to tubulin. This modification was originally identified in highly stable microtubules from Paramecium cilia. As many as 34 posttranslationally added glycine residues have been located in the C-terminal domains of Paramecium alpha- and beta-tubulin. In this study, post source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD MALDI MS) and electrospray ionization on a hybrid quadrupole orthogonal time-of-flight tandem mass spectrometer (ESI Q-TOF MS/MS) were both used to demonstrate that a single molecule of beta-tubulin, from either dynamic cytoplasmic microtubules or stable axonemal microtubules, can be glycylated on each of the last four C-terminal glutamate residues Glu437, Glu438, Glu439, and Glu441 in the sequence 427DATAEEEGEFEEEGEQ442. In both dynamic and stable microtubules the most abundant beta-tubulin isoform contains six posttranslationally added glycine residues: two glycine residues on both Glu437 and Glu438 and one glycine residue on both Glu439 and Glu441. The number and relative abundance of glycylated isoforms of beta-tubulin in both cytoplasmic and axonemal microtubules were compared by MALDI MS.1 The abundance of the major glycylated isoforms in axonemal tubulin decreases regularly with glycylation levels from 6 to 19 whereas it drops abruptly in cytoplasmic tubulin with glycylation levels from 6 to 9. However, the polyglycine chains are similarly distributed on the four C-terminal glutamate residues of cytoplasmic and axonemal tubulin. The Polyglycylation results in bulky C-terminal domains with negatively charged surfaces, all surrounding the microtubular structure.
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Tubulin Polyglycylation: Differential Posttranslational Modification of Dynamic Cytoplasmic and Stable
1998Co-Authors: Axonemal Microtubules In Paramecium, Joelle Vinh, Marie-hélène Bré, Virginie Redeker, Jean Rossier, Nicolette LevilliersAbstract:Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono- and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the �- and �tubulin subunits are modified by only 1–5 and 2–9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence o
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Sequencing branched peptides with CID/PSD MALDI-TOF in the low-picomole range: application to the structural study of the posttranslational Polyglycylation of tubulin.
Analytical chemistry, 1997Co-Authors: Joelle Vinh, Virginie Redeker, Denis Loyaux, Jean RossierAbstract:Sequencing conditions for postsource decay and collision-induced dissociation/postsource decay matrix-assisted laser desorption/ionization time-of-flight mass spectrometry have been optimized to elucidate the structure of Polyglycylation of tubulin. This posttranslational modification involves the linkage of multiple glycine residues through the γ-carboxyl of glutamic acid residues in the carboxyl termini of the protein. Individual α- and β-tubulin polypeptides contain respectively three and four potential glycylation sites. The sample preparation we used was the thin-layer preparation of the target specimen in the presence of α-cyano-4-hydroxycinnamic acid and nitrocellulose. The study of different synthetic polyglycylated peptides fragmentation (modified peptides with the linear sequence 427DATAEEEGEFEEEGEQ441) shows that the peptides fragment regularly to form major fragments of b- and y-type ions with negligible side-chain fragmentation. The rules were applied to the structural elucidation of a Parame...
Marco Lalle - One of the best experts on this subject based on the ideXlab platform.
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The Crystal Structure of Giardia duodenalis 14-3-3 in the Apo Form: When Protein Post-Translational Modifications Make the Difference
2016Co-Authors: Annarita Fiorillo, Edoardo Pozio, Daniele Di Marino, Lucia Bertuccini, Allegra Via, Serena Camerini, Andrea Ilari, Marco LalleAbstract:The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the Polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and Polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that Polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of thi
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The Crystal Structure of Giardia duodenalis 14-3-3 in the Apo Form: When Protein Post-Translational Modifications Make the Difference
PloS one, 2014Co-Authors: Annarita Fiorillo, Edoardo Pozio, Daniele Di Marino, Lucia Bertuccini, Allegra Via, Serena Camerini, Andrea Ilari, Marco LalleAbstract:The 14-3-3s are a family of dimeric evolutionary conserved pSer/pThr binding proteins that play a key role in multiple biological processes by interacting with a plethora of client proteins. Giardia duodenalis is a flagellated protozoan that affects millions of people worldwide causing an acute and chronic diarrheal disease. The single giardial 14-3-3 isoform (g14-3-3), unique in the 14-3-3 family, needs the constitutive phosphorylation of Thr214 and the Polyglycylation of its C-terminus to be fully functional in vivo. Alteration of the phosphorylation and Polyglycylation status affects the parasite differentiation into the cyst stage. To further investigate the role of these post-translational modifications, the crystal structure of the g14-3-3 was solved in the unmodified apo form. Oligomers of g14-3-3 were observed due to domain swapping events at the protein C-terminus. The formation of filaments was supported by TEM. Mutational analysis, in combination with native PAGE and chemical cross-linking, proved that Polyglycylation prevents oligomerization. In silico phosphorylation and molecular dynamics simulations supported a structural role for the phosphorylation of Thr214 in promoting target binding. Our findings highlight unique structural features of g14-3-3 opening novel perspectives on the evolutionary history of this protein family and envisaging the possibility to develop anti-giardial drugs targeting g14-3-3.
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Phosophorylation and Polyglycylation of 14-3-3 proteins in Giardia.
2013Co-Authors: Marco Lalle, Edoardo Pozio, Serena Camerini, Flora Leptourgidou, Efthimios M. C. SkoulakisAbstract:A) Multiple Alignments of g14-3-3, LeoII and D14-3-3ε amino-acid sequences showing the peptides derived from trypsin digestion and containing the phosphorylated Thr214 (black boxed) and the Glu246 (grey boxed) of g14-3-3 and the corresponding peptides of LeoII and D14-3-3ε are in bold. Residues are numbered according to published protein sequences. B) Alignment of the C-terminus of g14-3-3, LeoII and D14-3-3ε with α- and β-tubulin of Giardia (GenBankTM/EBI Accession Number AAN46106 and P05304), α- and β-tubulin of Paramecium tetraurelia (GenBankTM/EBI Accession Number CAA67848 and CAE75646), and α- and β-tubulin of Tetrahymena thermophila (GenBankTM/EBI Accession Number P41351 and P41352). The alignment was performed with the ClustalW program and manually refined. The amino acids in grey define the hypothetical Polyglycylation sequence [T/G]X0-1[D/E]X1-3G[D/E]X1-2[E]2-4. Experimentally defined polyglycylated glutamic acid residues are black boxed and highlighted in bold white letters. Putative polyglycylated glutamic acid residues are only black boxed. The underlined glutamic acid of Tetrahymena α-tubulin is predicted to be polyglycylated. C-E) MALDI-MS analysis of affinity purified FLAG-tagged transfected proteins from Giardia trophozoites. MALDI-MS spectra of FLAG-g14-3-3 (C), FLAG-D14-3-3ε (D) and FLAG-LEOII (E), encompassing the MH+ range of 1400–3600. Mono-isotopic masses of relevant peaks are shown. Peptides are indicated by the positions of their N- and C-termini and numbered as in the protein sequence. Peaks shifted from the theoretical MH+ are indicated by arrows as for the 80 Da shift due to phosphorylation. For each protein, analysis of phosphorylation is reported on the left and the analysis of C-terminal Polyglycylation is reported on the right panels. C) For FLAG-g14-3-3, the calculated MH+ peak for peptide 202–219 at 2029.9 is clearly shifted to 2109.93 indicating phosphorylation on Thr214 (202AFDAAITDLDKLTEESYK219). On the right, the peaks corresponding to Polyglycylation of the peptide (230DNLNLWVTDSAGDDNAEEK248) with predicted MH+ = 2047.92 was clearly shifted to 2105.9 indicating multiple glycines on Glu246 as revealed by their number in the lateral chain. The insert shows the lack of the peak corresponding to the unmodified 230-248 peptide. D) For D14-3-3ε-FLAG, the predicted peak at MH+ = 2087.9 for the peptide (197AAFDDAIAELDTLSEESYK21) was shifted by 80 kDa to MH+ = 2167.9, indicating phosphorylation at Ser210. On the right, the peaks corresponding to the unmodified (249EQIQDVEDQDVS260 = 1404.6 MH+) peptide and the shifts to higher MH+ consistent with addition of 7 or 8 glycines (right) and phosphorylation at Ser260 (+80kDa, arrow on the left) are shown. C) For LEOII-FLAG only the peaks corresponding to unmodified (197QAFDDAIAELDTLNEDSYK215 = 2157.98 MH+) and (226DNLTLWTSDTQGDEAEPQEGGDN248 = 2492.03 MH+) peptides were evident.
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MALDI-MS analysis of affinity purified endogenous and transgenic g14-3-3 proteins from Drosophila.
2013Co-Authors: Marco Lalle, Edoardo Pozio, Serena Camerini, Flora Leptourgidou, Efthimios M. C. SkoulakisAbstract:A) Western blot analyses of GST-difopein purified 14-3-3s (1∶10) from wild type flies and His-g14-3-3 -expressing transgenic flies and the relevant controls (WB-C6 for Giardia and w1118 for flies) were separated on 12% SDS-PAGE, blotted and the membranes probed with the indicated antibodies. The AXO49 mAb detecting the Giardia polyglycylated protein did not cross react with the fly isoforms, which are detected with the anti-pan14-3-3 antibody, while the transgenic g14-3-3 protein is detectable with the anti-His in fly lysates. TR denotes trophozoite lysates, while ENC indicates lysates from 12hr encysting parasites. MALDI-MS spectra of the transgenic His-g14-3-3 (B) and the endogenous Drosophila D14-3-3ε (C) and Leo (D) encompassing the MH+ range of 1400-3600. Mono-isotopic masses of relevant peaks are shown. Peptides are indicated by the positions of their NH2- and C-termini and numbered as in the protein sequence. For each protein, the analysis of phosphorylation is reported on the left panels and the analysis of C-terminal Polyglycylation is reported on the right panels. Only peaks corresponding to the unmodified peptides were visible and reported here.
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Giardia Duodenalis 14-3-3 Protein Is Polyglycylated by a Tubulin Tyrosine Ligase-like Member and Deglycylated by Two Metallocarboxypeptidases
The Journal of biological chemistry, 2010Co-Authors: Marco Lalle, Serena Cecchetti, Marco Crescenzi, Serena Camerini, Claudia Blasetti Fantauzzi, Edoardo PozioAbstract:Abstract The flagellated protozoan Giardia duodenalis is a parasite of the upper part of the small intestine of mammals, including humans, and an interesting biological model. Giardia harbors a single 14-3-3 isoform, a multifunctional protein family, that is modified at the C terminus by Polyglycylation, an unusual post-translational modification consisting of the covalent addition of one or multiple glycines on the γ-carboxyl groups of specific glutamic acids. Polyglycylation affects the intracellular localization of g14-3-3, as the shortening of the polyglycine chain is correlated with a partial relocalization of 14-3-3 inside the nuclei during encystation. In this work we demonstrate that the gTTLL3, a member of the tubulin tyrosine ligase-like family, is the enzyme responsible for the 14-3-3 Polyglycylation. We also identify two metallopeptidases of the M20 family, here termed gDIP1 (giardial dipeptidase 1) and gDIP2, as enzymes able to shorten the g14-3-3 polyglycine tail both in vivo and in vitro. Finally, we show that the ectopic expression of gDIP2 alters the g14-3-3 localization and strongly hampers the cyst formation. In conclusion, we have identified a polyglycylase and two deglycylases that act in concert to modulate the stage-dependent glycylation status of the multifunctional regulatory g14-3-3 protein in G. duodenalis.
Virginie Redeker - One of the best experts on this subject based on the ideXlab platform.
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Mass spectrometry analysis of C-terminal posttranslational modifications of tubulins.
Methods in Cell Biology, 2009Co-Authors: Virginie RedekerAbstract:In mammalian brain and ciliary axonemes from ciliates, alpha- and beta-tubulins exhibit an extraordinary heterogeneity due to a combination of multigene family expression and numerous posttranslational modifications (PTMs). The combination of several PTMs located in the C-terminal tail of tubulins plays a major role in this important polymorphism of tubulin: polyglutamylation, Polyglycylation, detyrosination, tyrosination, removal of the penultimate glutamate residue, and phosphorylation. In order to document the relationship and functions of these PTMs, we have developed a tubulin C-terminal Peptide Mass Fingerprinting (PMF) method. Using simplified microtubule proteins and tubulin C-terminal peptides purifications, direct matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) analysis can generate a complete picture of all tubulin isotype-specific C-terminal peptides together with their respective PTMs. This chapter will illustrate the capability of this approach to compare tubulin isoform compositions and document the changes in PTMs between samples with different tubulin assembly properties or consecutively to inactivation of modification sites or modification enzymes. Complementary MS-based approaches useful to document the structure of the highly heterogeneous posttranslational polymodifications will also be presented.
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Mutations of tubulin glycylation sites reveal cross-talk between the C termini of alpha- and beta-tubulin and affect the ciliary matrix in Tetrahymena.
Journal of Biological Chemistry, 2005Co-Authors: Virginie Redeker, Nicolette Levilliers, Jacek Gaertig, Dylan Burnette, Jean Rossier, Emilie Vinolo, Danielle Jaillard, Marie-hélène BréAbstract:Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that Polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.
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Mutations of Tubulin Glycylation Sites Reveal Cross-talk between the C Termini of α- and β-Tubulin and Affect the Ciliary Matrix in Tetrahymena
The Journal of biological chemistry, 2004Co-Authors: Virginie Redeker, Dylan T Burnette, Nicolette Levilliers, Jacek Gaertig, Jean Rossier, Emilie Vinolo, Danielle Jaillard, Marie-hélène BréAbstract:Two types of polymeric post-translational modifications of alpha/beta-tubulin, glycylation and glutamylation, occur widely in cilia and flagella. Their respective cellular functions are poorly understood. Mass spectrometry and immunoblotting showed that two closely related species, the ciliates Tetrahymena and Paramecium, have dramatically different compositions of tubulin post-translational modifications in structurally identical axonemes. Whereas the axonemal tubulin of Paramecium is highly glycylated and has a very low glutamylation content, the axonemal tubulin of Tetrahymena is glycylated and extensively glutamylated. In addition, only the alpha-tubulin of Tetrahymena undergoes detyrosination. Mutations of the known glycylation sites in Tetrahymena tubulin affected the level of each polymeric modification type in both the mutated and nonmutated subunits, revealing cross-talk between alpha- and beta-tubulin. Ultrastructural analyses of glycylation site mutants uncovered defects in the doublet B-subfiber of axonemes and revealed an accumulation of dense material in the ciliary matrix, reminiscent of intraflagellar transport particles seen by others in Chlamydomonas. We propose that Polyglycylation and/or polyglutamylation stabilize the B-subfiber of outer doublets and regulate the intraflagellar transport.
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Structural characterization by tandem mass spectrometry of the posttranslational Polyglycylation of tubulin.
Biochemistry, 1999Co-Authors: Joelle Vinh, Nicolette Levilliers, Marie-hélène Bré, Virginie Redeker, James I. Langridge, Denis Loyaux, Jean RossierAbstract:Polyglycylation is a posttranslational modification specific to tubulin. This modification was originally identified in highly stable microtubules from Paramecium cilia. As many as 34 posttranslationally added glycine residues have been located in the C-terminal domains of Paramecium alpha- and beta-tubulin. In this study, post source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD MALDI MS) and electrospray ionization on a hybrid quadrupole orthogonal time-of-flight tandem mass spectrometer (ESI Q-TOF MS/MS) were both used to demonstrate that a single molecule of beta-tubulin, from either dynamic cytoplasmic microtubules or stable axonemal microtubules, can be glycylated on each of the last four C-terminal glutamate residues Glu437, Glu438, Glu439, and Glu441 in the sequence 427DATAEEEGEFEEEGEQ442. In both dynamic and stable microtubules the most abundant beta-tubulin isoform contains six posttranslationally added glycine residues: two glycine residues on both Glu437 and Glu438 and one glycine residue on both Glu439 and Glu441. The number and relative abundance of glycylated isoforms of beta-tubulin in both cytoplasmic and axonemal microtubules were compared by MALDI MS.1 The abundance of the major glycylated isoforms in axonemal tubulin decreases regularly with glycylation levels from 6 to 19 whereas it drops abruptly in cytoplasmic tubulin with glycylation levels from 6 to 9. However, the polyglycine chains are similarly distributed on the four C-terminal glutamate residues of cytoplasmic and axonemal tubulin. The Polyglycylation results in bulky C-terminal domains with negatively charged surfaces, all surrounding the microtubular structure.
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Tubulin Polyglycylation: Differential Posttranslational Modification of Dynamic Cytoplasmic and Stable
1998Co-Authors: Axonemal Microtubules In Paramecium, Joelle Vinh, Marie-hélène Bré, Virginie Redeker, Jean Rossier, Nicolette LevilliersAbstract:Polyglycylation, a posttranslational modification of tubulin, was discovered in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tubulin subunit. The observation of this type of posttranslational modification mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules that correspond to the dynamic microtubules in Paramecium. Two anti-glycylated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are shown here to exhibit different affinities toward mono- and polyglycylated synthetic tubulin peptides. Using immunoblotting and mass spectrometry, we show that cytoplasmic tubulin is glycylated. In contrast to the highly glycylated axonemal tubulin, which is recognized by the two mAbs, cytoplasmic tubulin reacts exclusively with TAP 952, and the �- and �tubulin subunits are modified by only 1–5 and 2–9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distributed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal compartments implies the existence o