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J F Laporte - One of the best experts on this subject based on the ideXlab platform.
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Myotubularin regulates the function of the late endosome through the gram domain phosphatidylinositol 3 5 bisphosphate interaction
Journal of Biological Chemistry, 2004Co-Authors: Kazuya Tsujita, J F Laporte, Toshiki Itoh, Takeshi Ijuin, Akitsugu Yamamoto, Assia Shisheva, Tadaomi TakenawaAbstract:Myotubularin and related proteins constitute a large and highly conserved family possessing phosphoinositide 3-phosphatase activity, although not all members possess this activity. This family contains a conserved region called the GRAM domain that is found in a variety of proteins associated with membrane-coupled processes and signal transduction. Mutations of Myotubularin are found in X-Linked Myotubular Myopathy, a severe muscle disease. Mutations in the GRAM domain are responsible for this condition, suggesting crucial roles for this region. Here, we show that the GRAM domain of Myotubularin binds to phosphoinositide with the highest affinity to phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P(2)). In patients with Myotubular Myopathy, mutations in the Myotubularin GRAM domain eliminate this binding, indicating that the PtdIns(3,5)P(2) binding ability of the GRAM (glucosyltransferases, Rablike GTPase activators and Myotubularin) domain is crucial for the functions of Myotubularin in vivo. Stimulation of epidermal growth factor recruits Myotubularin to the late endosomal compartment in a manner dependent on the phosphoinositide binding. Overexpression of Myotubularin inhibits epidermal growth factor receptor trafficking from late endosome to lysosome and induces the large endosomal vacuoles. Thus, our data suggest that Myotubularin phosphatase physiologically functions in late endosomal trafficking and vacuolar morphology through interaction with PtdIns(3,5)P(2).
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production of phosphatidylinositol 5 phosphate by the phosphoinositide 3 phosphatase Myotubularin in mammalian cells
Journal of Biological Chemistry, 2004Co-Authors: Helene Tronchere, J F Laporte, Caroline Pendaries, Claire Chaussade, Laurence Liaubet, Luciano Pirola, Bernard PayrastreAbstract:MTM1, the gene encoding Myotubularin (MTM1), is mutated in the X-Linked Myotubular Myopathy (XLMTM), a severe genetic muscular disorder. MTM1 is a phosphoinositide phosphatase hydrolyzing phosphatidylinositol 3-phosphate (PtdIns(3)P) in yeast and in vitro. Because this lipid is implicated in the regulation of vesicular trafficking, we used established cell lines from XLMTM patients to evaluate whether the lack of endogenous MTM1 expression could affect PtdIns(3)P labeling patterns. Our results showed that the vesicular trafficking related to early endosomes was not significantly affected in the XLMTM cell lines compared with control cells. However, in addition to PtdIns(3)P, we found that MTM1 can hydrolyze phosphatidylinositol 3,5-bisphosphate both in vitro and in mammalian cells. Using a mass assay, we demonstrated that the product generated is phosphatidylinositol 5-phosphate (PtdIns(5)P), a recently discovered phosphoinositide, the function of which is still unknown. In L6 myotubes overexpressing MTM1, hyperosmotic shock induced an increase in the mass level of PtdIns(5)P that was reduced by 50% upon overexpression of the MTM1 inactive mutant D278A. These data demonstrate for the first time a role for MTM1 in the production of PtdIns(5)P in mammalian cells, suggesting that the lack of transformation of phosphatidylinositol 3,5-bisphosphate into PtdIns(5)P might be an important component in the etiology of Myotubular Myopathy.
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expression of Myotubularin by an adenoviral vector demonstrates its function as a phosphatidylinositol 3 phosphate ptdins 3 p phosphatase in muscle cell lines involvement of ptdins 3 p in insulin stimulated glucose transport
Molecular Endocrinology, 2003Co-Authors: Claire Chaussade, Francois Blondeau, Bernard Payrastre, Helene Tronchere, Luciano Pirola, Stephanie Bonnafous, Stefano Brenzverca, Fiorella Portis, Sandro Rusconi, J F LaporteAbstract:X-Linked Myotubular Myopathy is a muscle disorder caused by mutations on the Myotubular Myopathy-1 (MTM-1) gene, coding for Myotubularin a 65-kDa polypeptide similar to protein phosphatases. Biochemical and in vivo studies define Myotubularin as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase. To efficiently express Myotubularin in muscle cell lines and adipocytes, we used an adenoviral genome recombinogenic to pcDNA3, and to other widely used expression vectors, to produce adenoviruses expressing wild-type (wt), catalytically inactive C375S, and substrate trap D278A Myotubularin. [32P]Orthophosphate labeling followed by phosphoinositide analysis of differentiated L6 and C2C12 cells expressing Myotubularin demonstrated increased PtdIns(3)P levels upon expression of the C375S and D278A mutants. In keeping with its biochemical function, overexpression of wt Myotubularin as an enhanced green fluorescent protein fusion disrupted the endosomal punctuated staining of the FYVE (Fab1p/YOTB Vac1p/EEA1)...
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identification of Myotubularin as the lipid phosphatase catalytic subunit associated with the 3 phosphatase adapter protein 3 pap
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Harshal Hanumant Nandurkar, Yasuhiro Mochizuki, Philip W Majerus, J F Laporte, Meredith J Layton, Carly Selan, Lisa Corcoran, Kevin K Caldwell, Christina Anne MitchellAbstract:Myotubularin is a dual-specific phosphatase that dephosphorylates phosphatidylinositol 3-phosphate and phosphatidylinositol (3,5)-bisphosphate. Mutations in Myotubularin result in the human disease X-Linked Myotubular Myopathy, characterized by persistence of muscle fibers that retain an immature phenotype. We have previously reported the identification of the 3-phosphatase adapter protein (3-PAP), a catalytically inactive member of the Myotubularin gene family, which coprecipitates lipid phosphatidylinositol 3-phosphate-3-phosphatase activity from lysates of human platelets. We have now identified Myotubularin as the catalytically active 3-phosphatase subunit interacting with 3-PAP. A 65-kDa polypeptide, coprecipitating with endogenous 3-PAP, was purified from SDS/PAGE, subjected to trypsin digestion, and analyzed by collision-induced dissociation tandem MS. Three peptides derived from human Myotubularin were identified. Association between 3-PAP and Myotubularin was confirmed by reciprocal coimmunoprecipitation of both endogenous and recombinant proteins expressed in K562 cells. Recombinant Myotubularin localized to the plasma membrane, causing extensive filopodia formation. However, coexpression of 3-PAP with Myotubularin led to attenuation of the plasma membrane phenotype, associated with Myotubularin relocalization to the cytosol. Collectively these studies indicate 3-PAP functions as an "adapter" for Myotubularin, regulating Myotubularin intracellular location and thereby altering the phenotype resulting from Myotubularin overexpression.
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the lipid phosphatase Myotubularin is essential for skeletal muscle maintenance but not for myogenesis in mice
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Anna Bujbello, Nadia Messaddeq, J F Laporte, Vincent Laugel, Hala Zahreddine, Jeanfrancois Pellissier, Jean-louis MandelAbstract:Myotubularin is a ubiquitously expressed phosphatase that acts on phosphatidylinositol 3-monophosphate [PI(3)P], a lipid implicated in intracellular vesicle trafficking and autophagy. It is encoded by the MTM1 gene, which is mutated in X-Linked Myotubular Myopathy (XLMTM), a muscular disorder characterized by generalized hypotonia and muscle weakness at birth leading to early death of most affected males. The disease was proposed to result from an arrest in myogenesis, as the skeletal muscle from patients contains hypotrophic fibers with centrally located nuclei that resemble fetal myotubes. To understand the physiopathological mechanism of XLMTM, we have generated mice lacking Myotubularin by homologous recombination. These mice are viable, but their lifespan is severely reduced. They develop a generalized and progressive Myopathy starting at around 4 weeks of age, with amyotrophy and accumulation of central nuclei in skeletal muscle fibers leading to death at 6–14 weeks. Contrary to expectations, we show that muscle differentiation in knockout mice occurs normally. We provide evidence that fibers with centralized myonuclei originate mainly from a structural maintenance defect affecting Myotubularin-deficient muscle rather than a regenerative process. In addition, we demonstrate, through a conditional gene-targeting approach, that skeletal muscle is the primary target of murine XLMTM pathology. These mutant mice represent animal models for the human disease and will be a valuable tool for understanding the physiological role of Myotubularin.
Bernard Payrastre - One of the best experts on this subject based on the ideXlab platform.
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Detection of Myotubularin phosphatases activity on phosphoinositides in vitro and ex vivo.
Methods in Molecular Biology, 2008Co-Authors: Holger Maria Rohde, Bernard Payrastre, Helene Tronchere, Jean-francois LaporteAbstract:Phosphoinositides (PPIn) are important regulators of cellular processes like intracellular protein transport, cellular proliferation, apoptosis, and cytoskeletal organization. The amount and localization of these membrane-bound second messengers are regulated through a set of specific phospholipases, lipid kinases, and phosphatases. The elucidation of PPIn-phosphatases and their cellular function has gained much attention because phosphatase dysregulation is often associated with human genetic diseases. Our laboratory has identified the 3'-PPIn-phosphatase Myotubularin 1 (MTM1) mutated in X-Linked Myotubular Myopathy (XLMTM). In addition, a whole family of Myotubularin-related proteins (MTMR1-MTMR13) has been discovered. Some of them display phosphatase activity, whereas for other family members no enzymatic activity could be detected. Nevertheless, these "dead phosphatases" Myotubularins are conserved throughout evolution and probably exert regulatory function by heteromeric interaction with active phosphatase members. It was shown that MTM1 and related phosphatases act on PtdIns3P and PtdIns(3,5)P2; both PPIn species are important regulators of endocytic pathways. We describe two methods to determine phosphatase activity and substrate specificity of Myotubularins. One is an immunoprecipitation-phosphatase assay, testing the activity of Myotubularin immunoprecipitated from overexpressing cells on artificial PPIn. The other method analyzes phosphatase activity indirectly ex vivo in transiently transfected mammalian cells. The presence and subcellular localization of the Myotubularin substrate PtdIns3P were determined using a specific binding domain (2xFYVE) produced recombinantly as a biosensor.
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Phosphoinositide phosphatases in a network of signalling reactions
Pflügers Archiv - European Journal of Physiology, 2007Co-Authors: Daniel Blero, Bernard Payrastre, Stéphane SchurmansAbstract:Phosphoinositide phosphatases dephosphorylate the three positions (D-3, 4 and 5) of the inositol ring of the poly-phosphoinositides. They belong to different families of enzymes. The PtdIns(3,4)P_2 4-phosphatase family, the tumour suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN), SAC1 domain phosphatases and Myotubularins belong to the tyrosine protein phosphatases superfamily. They share the presence of a conserved cysteine residue in the consensus CX_5RT/S. Another family consists of the inositol polyphosphate 5-phosphatase isoenzymes. The importance of these phosphoinositide phosphatases in cell regulation is illustrated by multiple examples of their implications in human diseases such as Lowe syndrome, X-Linked Myotubular Myopathy, cancer, diabetes or bacterial infection.
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production of phosphatidylinositol 5 phosphate by the phosphoinositide 3 phosphatase Myotubularin in mammalian cells
Journal of Biological Chemistry, 2004Co-Authors: Helene Tronchere, J F Laporte, Caroline Pendaries, Claire Chaussade, Laurence Liaubet, Luciano Pirola, Bernard PayrastreAbstract:MTM1, the gene encoding Myotubularin (MTM1), is mutated in the X-Linked Myotubular Myopathy (XLMTM), a severe genetic muscular disorder. MTM1 is a phosphoinositide phosphatase hydrolyzing phosphatidylinositol 3-phosphate (PtdIns(3)P) in yeast and in vitro. Because this lipid is implicated in the regulation of vesicular trafficking, we used established cell lines from XLMTM patients to evaluate whether the lack of endogenous MTM1 expression could affect PtdIns(3)P labeling patterns. Our results showed that the vesicular trafficking related to early endosomes was not significantly affected in the XLMTM cell lines compared with control cells. However, in addition to PtdIns(3)P, we found that MTM1 can hydrolyze phosphatidylinositol 3,5-bisphosphate both in vitro and in mammalian cells. Using a mass assay, we demonstrated that the product generated is phosphatidylinositol 5-phosphate (PtdIns(5)P), a recently discovered phosphoinositide, the function of which is still unknown. In L6 myotubes overexpressing MTM1, hyperosmotic shock induced an increase in the mass level of PtdIns(5)P that was reduced by 50% upon overexpression of the MTM1 inactive mutant D278A. These data demonstrate for the first time a role for MTM1 in the production of PtdIns(5)P in mammalian cells, suggesting that the lack of transformation of phosphatidylinositol 3,5-bisphosphate into PtdIns(5)P might be an important component in the etiology of Myotubular Myopathy.
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expression of Myotubularin by an adenoviral vector demonstrates its function as a phosphatidylinositol 3 phosphate ptdins 3 p phosphatase in muscle cell lines involvement of ptdins 3 p in insulin stimulated glucose transport
Molecular Endocrinology, 2003Co-Authors: Claire Chaussade, Francois Blondeau, Bernard Payrastre, Helene Tronchere, Luciano Pirola, Stephanie Bonnafous, Stefano Brenzverca, Fiorella Portis, Sandro Rusconi, J F LaporteAbstract:X-Linked Myotubular Myopathy is a muscle disorder caused by mutations on the Myotubular Myopathy-1 (MTM-1) gene, coding for Myotubularin a 65-kDa polypeptide similar to protein phosphatases. Biochemical and in vivo studies define Myotubularin as a phosphatidylinositol 3-phosphate [PtdIns(3)P] phosphatase. To efficiently express Myotubularin in muscle cell lines and adipocytes, we used an adenoviral genome recombinogenic to pcDNA3, and to other widely used expression vectors, to produce adenoviruses expressing wild-type (wt), catalytically inactive C375S, and substrate trap D278A Myotubularin. [32P]Orthophosphate labeling followed by phosphoinositide analysis of differentiated L6 and C2C12 cells expressing Myotubularin demonstrated increased PtdIns(3)P levels upon expression of the C375S and D278A mutants. In keeping with its biochemical function, overexpression of wt Myotubularin as an enhanced green fluorescent protein fusion disrupted the endosomal punctuated staining of the FYVE (Fab1p/YOTB Vac1p/EEA1)...
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implication of phosphoinositide phosphatases in genetic diseases the case of Myotubularin
Cellular and Molecular Life Sciences, 2003Co-Authors: Helene Tronchere, Anna Bujbello, Jean-louis Mandel, Bernard PayrastreAbstract:Phosphoinositides play a central role in the control of major eukaryotic cell signaling mechanisms. Accordingly, the list of phosphoinositide-metabolizing enzymes implicated in human diseases has considerably increased these last years. Here we will focus on Myotubularin, the protein mutated in the X-Linked Myotubular Myopathy (XLMTM) and the founding member of a family of 13 related proteins. Recent data demonstrate that Myotubularin and several other members of the family are potent lipid phosphatases showing a marked specificity for phosphatidylinositol 3-phosphate [PtdIns(3)P]. This finding has raised considerable interest as PtdIns(3)P is implicated in vesicular trafficking and sorting through its binding to specific protein domains. The structure of Myotubularin, the molecular mechanisms of its function and its implication in the etiology of XLMTM will be discussed, as well as the potential function and role of the other members of the family.
Jean-louis Mandel - One of the best experts on this subject based on the ideXlab platform.
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t tubule disorganization and defective excitation contraction coupling in muscle fibers lacking Myotubularin lipid phosphatase
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Lama Alqusairi, Christine Kretz, Alan H Beggs, Nadia Messaddeq, Norbert Weiss, Celine Berbey, Despina Sanoudou, Bruno Allard, Jean-louis MandelAbstract:Skeletal muscle contraction is triggered by the excitation-contraction (E-C) coupling machinery residing at the triad, a membrane structure formed by the juxtaposition of T-tubules and sarcoplasmic reticulum (SR) cisternae. The formation and maintenance of this structure is key for muscle function but is not well characterized. We have investigated the mechanisms leading to X-Linked Myotubular Myopathy (XLMTM), a severe congenital disorder due to loss of function mutations in the MTM1 gene, encoding Myotubularin, a phosphoinositide phosphatase thought to have a role in plasma membrane homeostasis and endocytosis. Using a mouse model of the disease, we report that Mtm1-deficient muscle fibers have a decreased number of triads and abnormal longitudinally oriented T-tubules. In addition, SR Ca(2+) release elicited by voltage-clamp depolarizations is strongly depressed in Myotubularin-deficient muscle fibers, with myoplasmic Ca(2+) removal and SR Ca(2+) content essentially unaffected. At the molecular level, Mtm1-deficient myofibers exhibit a 3-fold reduction in type 1 ryanodine receptor (RyR1) protein level. These data reveal a critical role of Myotubularin in the proper organization and function of the E-C coupling machinery and strongly suggest that defective RyR1-mediated SR Ca(2+) release is responsible for the failure of muscle function in Myotubular Myopathy.
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implication of phosphoinositide phosphatases in genetic diseases the case of Myotubularin
Cellular and Molecular Life Sciences, 2003Co-Authors: Helene Tronchere, Anna Bujbello, Jean-louis Mandel, Bernard PayrastreAbstract:Phosphoinositides play a central role in the control of major eukaryotic cell signaling mechanisms. Accordingly, the list of phosphoinositide-metabolizing enzymes implicated in human diseases has considerably increased these last years. Here we will focus on Myotubularin, the protein mutated in the X-Linked Myotubular Myopathy (XLMTM) and the founding member of a family of 13 related proteins. Recent data demonstrate that Myotubularin and several other members of the family are potent lipid phosphatases showing a marked specificity for phosphatidylinositol 3-phosphate [PtdIns(3)P]. This finding has raised considerable interest as PtdIns(3)P is implicated in vesicular trafficking and sorting through its binding to specific protein domains. The structure of Myotubularin, the molecular mechanisms of its function and its implication in the etiology of XLMTM will be discussed, as well as the potential function and role of the other members of the family.
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the lipid phosphatase Myotubularin is essential for skeletal muscle maintenance but not for myogenesis in mice
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Anna Bujbello, Nadia Messaddeq, J F Laporte, Vincent Laugel, Hala Zahreddine, Jeanfrancois Pellissier, Jean-louis MandelAbstract:Myotubularin is a ubiquitously expressed phosphatase that acts on phosphatidylinositol 3-monophosphate [PI(3)P], a lipid implicated in intracellular vesicle trafficking and autophagy. It is encoded by the MTM1 gene, which is mutated in X-Linked Myotubular Myopathy (XLMTM), a muscular disorder characterized by generalized hypotonia and muscle weakness at birth leading to early death of most affected males. The disease was proposed to result from an arrest in myogenesis, as the skeletal muscle from patients contains hypotrophic fibers with centrally located nuclei that resemble fetal myotubes. To understand the physiopathological mechanism of XLMTM, we have generated mice lacking Myotubularin by homologous recombination. These mice are viable, but their lifespan is severely reduced. They develop a generalized and progressive Myopathy starting at around 4 weeks of age, with amyotrophy and accumulation of central nuclei in skeletal muscle fibers leading to death at 6–14 weeks. Contrary to expectations, we show that muscle differentiation in knockout mice occurs normally. We provide evidence that fibers with centralized myonuclei originate mainly from a structural maintenance defect affecting Myotubularin-deficient muscle rather than a regenerative process. In addition, we demonstrate, through a conditional gene-targeting approach, that skeletal muscle is the primary target of murine XLMTM pathology. These mutant mice represent animal models for the human disease and will be a valuable tool for understanding the physiological role of Myotubularin.
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the ptdins3p phosphatase Myotubularin is a cytoplasmic protein that also localizes to rac1 inducible plasma membrane ruffles
Journal of Cell Science, 2002Co-Authors: J F Laporte, Francois Blondeau, Anne Gansmuller, Yves Lutz, Jeanluc Vonesch, Jean-louis MandelAbstract:Myotubularin, the phosphatase mutated in X-Linked Myotubular Myopathy, was shown to dephosphorylate phosphatidylinositol 3-monophosphate (PtdIns3P) and was also reported to interact with nuclear transcriptional regulators from the trithorax family. We have characterized a panel of specific antibodies and investigated the subcellular localization of Myotubularin. Myotubularin is not detected in the nucleus, and localizes mostly as a dense cytoplasmic network. Overexpression of Myotubularin does not detectably affect vesicle trafficking in the mammalian cells investigated, in contrast to previous observations in yeast models. Both mutation of a key aspartate residue of Myotubularin and dominant activation of Rac1 GTPase lead to the recruitment of Myotubularin to specific plasma membrane domains. Localization to Rac1-induced ruffles is dependent on the presence of a domain highly conserved in the Myotubularin family (that we named RID). We thus propose that Myotubularin may dephosphorylate a subpool of PtdIns3P (or another related substrate) at the plasma membrane.
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functional redundancy in the Myotubularin family
Biochemical and Biophysical Research Communications, 2002Co-Authors: J F Laporte, Francois Blondeau, Jean-louis Mandel, Helene Tronchere, Laurence Liaubet, Bernard PayrastreAbstract:Myotubularin-related genes define a novel highly conserved family of eukaryotic proteins of at least 11 human members. The hMTM1 gene that codes for Myotubularin is mutated in X-Linked Myotubular Myopathy, a severe congenital disease. Recently, we and others have characterized Myotubularin as a potent and specific phosphatidylinositol 3-phosphate 3-phosphatase. In the present study we investigated the lipid phosphatase activity and the subcellular localization of two other members of the family, hMTMR2 protein that is mutated in the demyelinating neuropathy Charcot-Marie-Tooth type 4B and the FYVE-finger containing hMTMR3 protein. Our results show that both proteins are potent phosphatidylinositol 3-phosphate 3-phosphatases either in vitro or in yeast where they interfered with vesicular trafficking. Their localization is mainly cytoplasmic, with however strong labeling of Rac-inducible plasma membrane ruffles. The fact that the ubiquitously expressed hMTM1 and hMTMR2 genes are involved in different pathologies indicates that despite their shared enzymatic activity, they are not functionally redundant, at least in certain cell types. This might be explained by subtle differences in expression and/or in recruitment and regulation at their specific site of action.
Jack E. Dixon - One of the best experts on this subject based on the ideXlab platform.
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the structure and regulation of Myotubularin phosphatases
Current Opinion in Structural Biology, 2005Co-Authors: Michael J Begley, Jack E. DixonAbstract:The human neuromuscular diseases X-Linked Myotubular Myopathy and Charcot-Marie-Tooth disease type 4B are caused by mutations in Myotubularin family proteins. The Myotubularins are a unique subfamily of protein tyrosine phosphatases that utilize inositol phospholipids, rather than phosphoproteins, as substrates. Recent structural studies, including the first crystal structure of a Myotubularin family protein, have defined the structural features that are characteristic of the family and revealed the molecular basis of their unique substrate specificity. Interestingly, the Myotubularin family contains a subgroup of proteins that are catalytically inactive. Recent biochemical studies have established that the inactive Myotubularins function as adaptors for the active members and play an important regulatory role within the family.
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regulation of Myotubularin related mtmr 2 phosphatidylinositol phosphatase by mtmr5 a catalytically inactive phosphatase
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Soo-a Kim, Ron Firestein, Michael L Cleary, Panayiotis O Vacratsis, Jack E. DixonAbstract:The Myotubularin (MTM) family constitutes one of the most highly conserved protein-tyrosine phosphatase subfamilies in eukaryotes. MTM1, the archetypal member of this family, is mutated in X-Linked Myotubular Myopathy, whereas mutations in the MTM-related (MTMR)2 gene cause the type 4B1 Charcot-Marie-Tooth disease, a severe hereditary motor and sensory neuropathy. In this study, we identified a protein that specifically interacts with MTMR2 but not MTM1. The interacting protein was shown by mass spectrometry to be MTMR5, a catalytically inactive member of the MTM family. We also demonstrate that MTMR2 interacts with MTMR5 via its coiled-coil domain and that mutations in the coiled-coil domain of either MTMR2 or MTMR5 abrogate this interaction. Through this interaction, MTMR5 increases the enzymatic activity of MTMR2 and dictates its subcellular localization. This article demonstrates an active MTM member being regulated by an inactive family member.
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Myotubularin and MTMR2, Phosphatidylinositol 3-Phosphatases Mutated in Myotubular Myopathy and Type 4B Charcot-Marie-Tooth Disease
The Journal of biological chemistry, 2001Co-Authors: Soo-a Kim, Gregory S. Taylor, Knut Martin Torgersen, Jack E. DixonAbstract:Myotubularin is the archetype of a family of highly conserved protein-tyrosine phosphatase-like enzymes. The Myotubularin gene, MTM1, is mutated in the genetic disorder, X-Linked Myotubular Myopathy. We and others have previously shown that Myotubularin utilizes the lipid second messenger, phosphatidylinositol 3-phosphate (PI(3)P), as a physiologic substrate. We demonstrate here that the Myotubularin-related protein MTMR2, which is mutated in the neurodegenerative disorder, type 4B Charcot-Marie-Tooth disease, is also highly specific for PI(3)P as a substrate. Furthermore, the MTM-related phosphatases MTMR1, MTMR3, and MTMR6 also dephosphorylate PI(3)P, suggesting that activity toward this substrate is common to all Myotubularin family enzymes. A direct comparison of the lipid phosphatase activities of recombinant Myotubularin and MTMR2 demonstrates that their enzymatic properties are indistinguishable, indicating that the lack of functional redundancy between these proteins is likely to be due to factors other than the utilization of different physiologic substrates. To this end, we have analyzed Myotubularin and MTMR2 transcripts during induced differentiation of cultured murine C2C12 myoblasts and find that their expression is divergently regulated. In addition, Myotubularin and MTMR2 enhanced green fluorescent protein fusion proteins exhibit overlapping but distinct patterns of subcellular localization. Finally, we provide evidence that Myotubularin, but not MTMR2, can modulate the levels of endosomal PI(3)P. From these data, we conclude that the developmental expression and subcellular localization of Myotubularin and MTMR2 are differentially regulated, resulting in their utilization of specific cellular pools of PI(3)P.
Anna Bujbello - One of the best experts on this subject based on the ideXlab platform.
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gait characteristics in a canine model of x linked Myotubular Myopathy
Journal of the Neurological Sciences, 2014Co-Authors: Melissa A Goddard, Anna Bujbello, Martin K. Childers, Alan H Beggs, Emily Burlingame, Anthony P Marsh, Valerie E KellyAbstract:article i nfo X-LinkedMyotubularMyopathy(XLMTM)isafatalpediatricdisease whereaffectedboysdisplayprofoundweak- ness of the skeletal muscles. Possible therapies are under development but robust outcome measures in animal models are required for effective translation to human patients. We established a naturally-occurring canine model, where XLMTM dogs display clinical symptoms similar to those observed in humans. The aim of this study was to determine potential endpoints for the assessment of future treatments in this model. Video- based gait analysis was selected, as it is a well-established method of assessing limb function in neuromuscular disease and measures have been correlated to the patient's quality of life. XLMTM dogs (N = 3) and their true littermate wild type controls (N = 3) were assessed at 4-5 time points, beginning at 10 weeks and continuing through 17 weeks. Motion capture and an instrumented carpet were used separately to evaluate spatiotemporal and kinematic changes over time. XLMTM dogs walk more slowly and with shorter stride lengths than wild type dogs, and these differences became greater over time. However, there was no clear difference in angular mea- sures between affected and unaffected dogs. These data demonstrate that spatiotemporal parameters capture functional changes in gait in an XLMTM canine model and support their utility in future therapeutic trials.
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differential muscle hypertrophy is associated with satellite cell numbers and akt pathway activation following activin type iib receptor inhibition in mtm1 p r69c mice
American Journal of Pathology, 2014Co-Authors: Rachel V Edelstein, Michael W Lawlor, Christopher R. Pierson, Marissa G Viola, Hui Meng, Elizabeth J Luna, Alexandra Lerchgaggl, Raymond G Hoffmann, Anna BujbelloAbstract:X-Linked Myotubular Myopathy is a congenital Myopathy caused by deficiency of Myotubularin. Patients often present with severe perinatal weakness, requiring mechanical ventilation to prevent death from respiratory failure. We recently reported that an activin receptor type IIB inhibitor produced hypertrophy of type 2b myofibers and modest increases of strength and life span in the severely myopathic Mtm1δ4 mouse model of X-Linked Myotubular Myopathy. We have now performed a similar study in the less severely symptomatic Mtm1 p.R69C mouse in hopes of finding greater treatment efficacy. Activin receptor type IIB inhibitor treatment of Mtm1 p.R69C animals produced behavioral and histological evidence of hypertrophy in gastrocnemius muscles but not in quadriceps or triceps. The ability of the muscles to respond to activin receptor type IIB inhibitor treatment correlated with treatment-induced increases in satellite cell number and several muscle-specific abnormalities of hypertrophic signaling. Treatment-responsive Mtm1 p.R69C gastrocnemius muscles displayed lower levels of phosphorylated ribosomal protein S6 and higher levels of phosphorylated eukaryotic elongation factor 2 kinase than were observed in Mtm1 p.R69C quadriceps muscle or in muscles from wild-type littermates. Hypertrophy in the Mtm1 p.R69C gastrocnemius muscle was associated with increased levels of phosphorylated ribosomal protein S6. Our findings indicate that muscle-, fiber type-, and mutation-specific factors affect the response to hypertrophic therapies that will be important to assess in future therapeutic trials.
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inhibition of activin receptor type iib increases strength and lifespan in Myotubularin deficient mice
American Journal of Pathology, 2011Co-Authors: Michael W Lawlor, Jennifer Lachey, Benjamin P Read, Rachel V Edelstein, Nicole Yang, Matthew J Stein, Ariana Wermercolan, Anna Bujbello, Christopher R. Pierson, Jasbir SeehraAbstract:X-Linked Myotubular Myopathy (XLMTM) is a congenital disorder caused by deficiency of the lipid phosphatase, Myotubularin. Patients with XLMTM often have severe perinatal weakness that requires mechanical ventilation to prevent death from respiratory failure. Muscle biopsy specimens from patients with XLMTM exhibit small myofibers with central nuclei and central aggregations of organelles in many cells. It was postulated that therapeutically increasing muscle fiber size would cause symptomatic improvement in Myotubularin deficiency. Recent studies have elucidated an important role for the activin-receptor type IIB (ActRIIB) in regulation of muscle growth and have demonstrated that ActRIIB inhibition results in significant muscle hypertrophy. To evaluate whether promoting muscle hypertrophy can attenuate symptoms resulting from Myotubularin deficiency, the effect of ActRIIB-mFC treatment was determined in Myotubularin-deficient (Mtm1δ4) mice. Compared with wild-type mice, untreated Mtm1δ4 mice have decreased body weight, skeletal muscle hypotrophy, and reduced survival. Treatment of Mtm1δ4 mice with ActRIIB-mFC produced a 17% extension of lifespan, with transient increases in weight, forelimb grip strength, and myofiber size. Pathologic analysis of Mtm1δ4 mice during treatment revealed that ActRIIB-mFC produced marked hypertrophy restricted to type 2b myofibers, which suggests that oxidative fibers in Mtm1δ4 animals are incapable of a hypertrophic response in this setting. These results support ActRIIB-mFC as an effective treatment for the weakness observed in Myotubularin deficiency.
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implication of phosphoinositide phosphatases in genetic diseases the case of Myotubularin
Cellular and Molecular Life Sciences, 2003Co-Authors: Helene Tronchere, Anna Bujbello, Jean-louis Mandel, Bernard PayrastreAbstract:Phosphoinositides play a central role in the control of major eukaryotic cell signaling mechanisms. Accordingly, the list of phosphoinositide-metabolizing enzymes implicated in human diseases has considerably increased these last years. Here we will focus on Myotubularin, the protein mutated in the X-Linked Myotubular Myopathy (XLMTM) and the founding member of a family of 13 related proteins. Recent data demonstrate that Myotubularin and several other members of the family are potent lipid phosphatases showing a marked specificity for phosphatidylinositol 3-phosphate [PtdIns(3)P]. This finding has raised considerable interest as PtdIns(3)P is implicated in vesicular trafficking and sorting through its binding to specific protein domains. The structure of Myotubularin, the molecular mechanisms of its function and its implication in the etiology of XLMTM will be discussed, as well as the potential function and role of the other members of the family.
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the lipid phosphatase Myotubularin is essential for skeletal muscle maintenance but not for myogenesis in mice
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Anna Bujbello, Nadia Messaddeq, J F Laporte, Vincent Laugel, Hala Zahreddine, Jeanfrancois Pellissier, Jean-louis MandelAbstract:Myotubularin is a ubiquitously expressed phosphatase that acts on phosphatidylinositol 3-monophosphate [PI(3)P], a lipid implicated in intracellular vesicle trafficking and autophagy. It is encoded by the MTM1 gene, which is mutated in X-Linked Myotubular Myopathy (XLMTM), a muscular disorder characterized by generalized hypotonia and muscle weakness at birth leading to early death of most affected males. The disease was proposed to result from an arrest in myogenesis, as the skeletal muscle from patients contains hypotrophic fibers with centrally located nuclei that resemble fetal myotubes. To understand the physiopathological mechanism of XLMTM, we have generated mice lacking Myotubularin by homologous recombination. These mice are viable, but their lifespan is severely reduced. They develop a generalized and progressive Myopathy starting at around 4 weeks of age, with amyotrophy and accumulation of central nuclei in skeletal muscle fibers leading to death at 6–14 weeks. Contrary to expectations, we show that muscle differentiation in knockout mice occurs normally. We provide evidence that fibers with centralized myonuclei originate mainly from a structural maintenance defect affecting Myotubularin-deficient muscle rather than a regenerative process. In addition, we demonstrate, through a conditional gene-targeting approach, that skeletal muscle is the primary target of murine XLMTM pathology. These mutant mice represent animal models for the human disease and will be a valuable tool for understanding the physiological role of Myotubularin.