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Jack E. Dixon - One of the best experts on this subject based on the ideXlab platform.
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loss of the inactive Myotubularin related phosphatase mtmr13 leads to a charcot marie tooth 4b2 like peripheral neuropathy in mice
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Fred L Robinson, Ingrid R Niesman, Kristina K Beiswenger, Jack E. DixonAbstract:Charcot–Marie–Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by slowed nerve conduction velocity, axon loss, and distinctive myelin outfolding and infolding. CMT4B is caused by recessive mutations in either Myotubularin-related protein 2 (MTMR2; CMT4B1) or MTMR13 (CMT4B2). Myotubularins are phosphoinositide (PI) 3-phosphatases that dephosphorylate phosphatidylinositol 3-phosphate (PtdIns3P) and PtdIns(3,5)P2, two phosphoinositides that regulate endosomal–lysosomal membrane traffic. Interestingly, nearly half of the metazoan Myotubularins are predicted to be catalytically inactive. Both active and inactive Myotubularins have essential functions in mammals and in Caenorhabditis elegans. MTMR2 and MTMR13 are active and inactive PI 3-phosphatases, respectively, and the two proteins have been shown to directly associate, although the functional significance of this association is not well understood. To establish a mouse model of CMT4B2, we disrupted the Mtmr13 gene. Mtmr13-deficient mice develop a peripheral neuropathy characterized by reduced nerve conduction velocity and myelin outfoldings and infoldings. Dysmyelination is evident in Mtmr13-deficient nerves at 14 days and worsens throughout life. Thus, loss of Mtmr13 in mice leads to a peripheral neuropathy with many of the key features of CMT4B2. Although myelin outfoldings and infoldings occur most frequently at the paranode, our morphological analyses indicate that the ultrastructure of the node of Ranvier and paranode is intact in Mtmr13-deficient nerve fibers. We also found that Mtmr2 levels are decreased by ≈50% in Mtmr13-deficient sciatic nerves, suggesting a mode of Mtmr2 regulation. Mtmr13-deficient mice will be an essential tool for studying how the loss of MTMR13 leads to CMT4B2.
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Myotubularin phosphatases policing 3 phosphoinositides
Trends in Cell Biology, 2006Co-Authors: Fred L Robinson, Jack E. DixonAbstract:In eukaryotic cells, phosphatidylinositol is subject to differential phosphorylation, resulting in the production of seven distinct phosphatidylinositol phosphates, often referred to as phosphoinositides (PIs). PIs have numerous distinct roles in cellular regulation and membrane trafficking. Recently, Myotubularin family PI 3-phosphatases have emerged as key regulators of phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate, two PIs that regulate traffic within the endosomal–lysosomal pathway. Mutations in several Myotubularin genes lead to myotubular myopathy and Charcot-Marie-Tooth peripheral neuropathy. Strikingly, nearly half of the members of the human Myotubularin family appear to be catalytically inactive. Several inactive Myotubularins have essential functions in mammals. Recent work in mammalian cells and model organisms is shedding light on the roles of Myotubularins in membrane traffic.
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molecular basis for substrate recognition by mtmr2 a Myotubularin family phosphoinositide phosphatase
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Michael J Begley, Gregory S. Taylor, Melissa A Brock, Partho Ghosh, Virgil L Woods, Jack E. DixonAbstract:Myotubularins, a large family of catalytically active and inactive proteins, belong to a unique subgroup of protein tyrosine phosphatases that use inositol phospholipids, rather than phosphoproteins, as physiological substrates. Here, by integrating crystallographic and deuterium-exchange mass spectrometry studies of human Myotubularin-related protein-2 (MTMR2) in complex with phosphoinositides, we define the molecular basis for this unique substrate specificity. Phosphoinositide substrates bind in a pocket located on a positively charged face of the protein, suggesting an electrostatic mechanism for membrane targeting. A flexible, hydrophobic helix makes extensive interactions with the diacylglycerol moieties of substrates, explaining the specificity for membrane-bound phosphoinositides. An extensive H-bonding network and charge–charge interactions within the active site pocket determine phosphoinositide headgroup specificity. The conservation of these specificity determinants within the active, but not the inactive, Myotubularins provides insight into the functional differences between the active and inactive members.
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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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the phosphoinositide 3 phosphatase mtmr2 associates with mtmr13 a membrane associated pseudophosphatase also mutated in type 4b charcot marie tooth disease
Journal of Biological Chemistry, 2005Co-Authors: Fred L Robinson, Jack E. DixonAbstract:Charcot-Marie-Tooth disease type 4B (CMT4B) is a severe, demyelinating peripheral neuropathy characterized by distinctive, focally folded myelin sheaths. CMT4B is caused by recessively inherited mutations in either Myotubularin-related 2 (MTMR2) or MTMR13 (also called SET-binding factor 2). MTMR2 encodes a member of the Myotubularin family of phosphoinositide-3-phosphatases, which dephosphorylate phosphatidylinositol 3-phosphate (PI(3)P) and bisphosphate PI(3,5)P2. MTMR13 encodes a large, uncharacterized member of the Myotubularin family. The MTMR13 phosphatase domain is catalytically inactive because the essential Cys and Arg residues are absent. Given the genetic association of both MTMR2 and MTMR13 with CMT4B, we investigated the biochemical relationship between these two proteins. We found that the endogenous MTMR2 and MTMR13 proteins are associated in human embryonic kidney 293 cells. MTMR2-MTMR13 association is mediated by coiled-coil sequences present in each protein. We also examined the cellular localization of MTMR2 and MTMR13 using fluorescence microscopy and subcellular fractionation. We found that (i) MTMR13 is a predominantly membrane-associated protein; (ii) MTMR2 and MTMR13 cofractionate in both a light membrane fraction and a cytosolic fraction; and (iii) MTMR13 membrane association is mediated by the segment of the protein which contains the pseudophosphatase domain. This work, which describes the first cellular or biochemical investigation of the MTMR13 pseudophosphatase protein, suggests that MTMR13 functions in association with MTMR2. Loss of MTMR13 function in CMT4B2 patients may lead to alterations in MTMR2 function and subsequent alterations in 3-phosphoinositide signaling. Such a mechanism would explain the strikingly similar phenotypes of patients with recessive mutations in either MTMR2 or MTMR13.
Jean-louis Mandel - One of the best experts on this subject based on the ideXlab platform.
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lack of Myotubularin mtm1 leads to muscle hypotrophy through unbalanced regulation of the autophagy and ubiquitin proteasome pathways
The FASEB Journal, 2013Co-Authors: Lama Alqusairi, Christine Kretz, Jean-louis Mandel, Nadia Messaddeq, Leonela Amoasii, Ivana Prokic, Jocelyn LaporteAbstract:Mutations in the phosphoinositide phos- phatase Myotubularin (MTM1) results in X-linked myo- tubular/centronuclear myopathy (XLMTM), character- ized by a severe decrease in muscle mass and strength in patients and murine models. However, the molecular mechanism involved in the muscle hypotrophy is un- clear. Here we show that the IGF1R/Akt pathway is affected in Mtm1-deficient murine muscles, character- ized by an increase in IGF1 receptor and Akt levels in both the presymptomatic and symptomatic phases. Moreover, up-regulation of atrogenes was observed in the presymptomatic phase of the myopathy, supporting overactivation of the ubiquitin-proteasome pathway. In parallel, the autophagy machinery was affected as indi- cated by the increase in the number of autophagosomes and of autophagy markers, such as LC3 and P62. However, phosphorylation of FOXO3a and mTOR were abnormal at late but not at early stages of the disease, suggesting that Myotubularin acts both up- stream in the IGF1R/Akt pathway and downstream on the balance between the autophagy and ubiquitin-pro- teasome pathways in vivo. Adeno-associated virus-medi- ated delivery of Mtm1 into Mtm1-null muscles rescued muscle mass and normalized the expression levels of IGF1 receptor, the ubiquitin-proteasome pathway, and autophagy markers. These data support the hypothesis that the unbalanced regulation of the ubiquitin protea- some pathway and the autophagy machinery is a pri- mary cause of the XLMTM pathogenesis.—Al-Qusairi, L., Prokic, I., Amoasii, L., Kretz, C., Messaddeq, N., Mandel, J.-L., Laporte, J. Lack of Myotubularin (MTM1) leads to muscle hypotrophy through unbal- anced regulation of the autophagy and ubiquitin-pro- teasome pathways. FASEB J. 27, 000 - 000 (2013). www.fasebj.org
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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, Anne Toussaint, 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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Myotubularins a large disease associated family of cooperating catalytically active and inactive phosphoinositides phosphatases
Human Molecular Genetics, 2003Co-Authors: J F Laporte, Alessandra Bolino, Florence Bedez, Jean-louis MandelAbstract:The Myotubularin family is a large eukaryotic group within the tyrosine/dual-specificity phosphatase super-family (PTP/DSP). Among the 14 human members, three are mutated in genetic diseases: myotubular myopathy and two forms of Charcot-Marie-Tooth neuropathy. We present an analysis of the Myotubularin family in sequenced genomes. The Myotubularin family encompasses catalytically active and inactive phosphatases, and both classes are well conserved from nematode to man. Catalytically active Myotubularins dephosphorylate phosphatidylinositol 3-phosphate (PtdIns3P) and PtdIns3,5P2, leading to the production of PtdIns and PtdIns5P. This activity may be modulated by direct interaction with catalytically inactive Myotubularins. These phosphoinositides are signaling molecules that are notably involved in vacuolar transport and membrane trafficking. Myotubularins are thus proposed to be implicated in these cellular mechanisms, and recent observations on Myotubularins homologues in the nematode Caenorhabditis elegans indicate a role in endocytosis.
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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.
J F Laporte - One of the best experts on this subject based on the ideXlab platform.
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the Myotubularin amphiphysin 2 complex in membrane tubulation and centronuclear myopathies
EMBO Reports, 2013Co-Authors: Barbara Royer, Karim Hnia, Helene Tronchere, Christos Gavriilidis, Valerie Tosch, J F LaporteAbstract:Myotubularin (MTM1) and amphiphysin 2 (BIN1) are two proteins mutated in different forms of centronuclear myopathy, but the functional and pathological relationship between these two proteins was unknown. Here, we identified MTM1 as a novel binding partner of BIN1, both in vitro and endogenously in skeletal muscle. Moreover, MTM1 enhances BIN1-mediated membrane tubulation, depending on binding and phosphoinositide phosphatase activity. BIN1 patient mutations induce a conformational change in BIN1 and alter its binding and regulation by MTM1. In conclusion, we identified the first molecular and functional link between MTM1 and BIN1, supporting a common pathological mechanism in different forms of centronuclear myopathy.
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c p 10 phosphatase inactive Myotubularin rescues x linked centronuclear myotubular myopathy phenotypes in mice
Neuromuscular Disorders, 2012Co-Authors: Leonela Amoasii, Bernard Payrastre, Belinda S Cowling, Arnaud Ferry, Karim Hnia, Helene Tronchere, Bruno Rinaldi, Dimitri Bertazzi, Gaëtan Chicanne, J F LaporteAbstract:Abstract The X-linked centronuclear myopathy, also called myotubular myopathy, is a muscle disorder characterized by neonatal hypotonia and abnormal organelles positioning in skeletal muscle. This myopathy is due to different mutations in the MTM1 gene encoding the phosphoinositide phosphatase Myotubularin. Disease-causing mutations are found all along the protein sequence and not only in the phosphatase catalytic domain. We investigated the link between Myotubularin phosphatase activity and disease phenotypes. We used brewer yeast as a simple cellular model to analyze the in vivo phosphatase activity of different disease mutants. Our results show that mutations responsible for severe forms of myopathy are either active or inactive phosphatases. To further question this finding, we used the murine Myotubularin knock-out model that reproduces faithfully the histopathological findings from human patients. Expression of phosphatase-inactive mutants improves the phenotypes of the knock-out mice comparable to wild-type Myotubularin. This shows that the maintenance of normal skeletal muscles is largely independent from Myotubularin phosphatase activity. Moreover, it could have important implications in the design of therapeutic approaches aiming at manipulating the phosphoinositide level in the different diseases linked to Myotubularin homologues. Finally, this work underlines that removal of enzymes should be used with care to conclude on the physiological importance of their activity.
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Myotubularin phosphoinositide phosphatases cellular functions and disease pathophysiology
Trends in Molecular Medicine, 2012Co-Authors: Karim Hnia, Alessandra Bolino, Ilaria Vaccari, J F LaporteAbstract:The Myotubularin family of phosphoinositide phosphatases includes several members mutated in neuromuscular diseases or associated with metabolic syndrome, obesity, and cancer. Catalytically dead phosphatases regulate their active homologs by heterodimerization and potentially represent key players in the phosphatase–kinase balance. Although the enzymatic specificity for phosphoinositides indicates a role for Myotubularins in endocytosis and membrane trafficking, recent findings in cellular and animal models suggest that Myotubularins regulate additional processes including cell proliferation and differentiation, autophagy, cytokinesis, and cytoskeletal and cell junction dynamics. In this review, we discuss how Myotubularins regulate such diverse processes, emphasizing newly identified functions in a physiological and pathological context. A better understanding of Myotubularin pathophysiology will pave the way towards therapeutic strategies.
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defective membrane remodeling in neuromuscular diseases insights from animal models
PLOS Genetics, 2012Co-Authors: Belinda S Cowling, Anne Toussaint, Jean Muller, J F LaporteAbstract:Proteins involved in membrane remodeling play an essential role in a plethora of cell functions including endocytosis and intracellular transport. Defects in several of them lead to human diseases. Myotubularins, amphiphysins, and dynamins are all proteins implicated in membrane trafficking and/or remodeling. Mutations in Myotubularin, amphiphysin 2 (BIN1), and dynamin 2 lead to different forms of centronuclear myopathy, while mutations in Myotubularin-related proteins cause Charcot-Marie-Tooth neuropathies. In addition to centronuclear myopathy, dynamin 2 is also mutated in a dominant form of Charcot-Marie-Tooth neuropathy. While several proteins from these different families are implicated in similar diseases, mutations in close homologues or in the same protein in the case of dynamin 2 lead to diseases affecting different tissues. This suggests (1) a common molecular pathway underlying these different neuromuscular diseases, and (2) tissue-specific regulation of these proteins. This review discusses the pathophysiology of the related neuromuscular diseases on the basis of animal models developed for proteins of the Myotubularin, amphiphysin, and dynamin families. A better understanding of the common mechanisms between these neuromuscular disorders will lead to more specific health care and therapeutic approaches.
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Myotubularin phosphoinositide phosphatases in human diseases
Current Topics in Microbiology and Immunology, 2012Co-Authors: Leonela Amoasii, Karim Hnia, J F LaporteAbstract:The level and turnover of phosphoinositides (PIs) are tightly controlled by a large set of PI-specific enzymes (PI kinases and phosphatases). Mammalian PI phosphatases are conserved through evolution and among this large family the dual-specificity phosphatase (PTP/DSP) are metal-independent enzymes displaying the amino acid signature Cys-X5-Arg-Thr/Ser (CX5RT/S) in their active site. Such catalytic site characterizes the Myotubularin 3-phosphatases that dephosphorylate PtdIns3P and PtdIns(3,5)P 2 and produce PtdIns5P. Substrates of Myotubularins have been implicated in endocytosis and membrane trafficking while PtdIns5P may have a role in signal transduction. As a paradox, 6 of the 14 members of the Myotubularin family lack enzymatic activity and are considered as dead phosphatases. Several Myotubularins have been genetically linked to human diseases: MTM1 is mutated in the congenital myopathy X-linked centronuclear or myotubular myopathy (XLCNM) and MTMR14 (JUMPY) has been linked to an autosomal form of such disease, while MTMR2 and MTMR13 are mutated in Charcot-Marie-Tooth (CMT) neuropathies. Furthermore, recent evidences from genetic association studies revealed that several other Myotubularins could be associated to chronic disorders such as cancer and obesity, highlighting their importance for human health. Here, we discuss cellular and physiological roles of Myotubularins and their implication in human diseases, and we present potential pathological mechanisms affecting specific tissues in Myotubularin-associated diseases.
Gregory S. Taylor - One of the best experts on this subject based on the ideXlab platform.
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receptor mediated endocytosis 8 is a novel pi 3 p binding protein regulated by Myotubularin related 2
FEBS Letters, 2011Co-Authors: Besa Xhabija, Gregory S. Taylor, Akemi Fujibayashi, Kiyotoshi Sekiguchi, Panayiotis O VacratsisAbstract:Myotubularin related protein 2 (MTMR2) is a member of the Myotubularin family of phosphoinositide lipid phosphatases. Although MTMR2 dephosphorylates the phosphoinositides PI(3)P and PI(3,5)P2, the phosphoinositide binding proteins that are regulated by MTMR2 are poorly characterized. In this study, phosphoinositide affinity chromatography coupled to mass spectrometry identified receptor mediated endocytosis 8 (RME-8) as a novel PI(3)P binding protein. RME-8 co-localized with the PI(3)P marker DsRed-FYVE, while the N-terminal region of RME-8 is required for PI(3)P and PI(3,5)P(2) binding in vitro. Depletion of PI(3)P by MTMR2 S58A or wortmannin treatment attenuated RME-8 endosomal localization and co-localization with EGFR on early endosomes. Our results suggest a model in which the localization of RME-8 to endosomal compartments is spatially mediated by PI(3)P binding and temporally regulated by MTMR2 activity.
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Myotubularin Regulates Akt-dependent Survival Signaling via Phosphatidylinositol 3-Phosphate
Journal of Biological Chemistry, 2011Co-Authors: Gina L Razidlo, Dawn Katafiasz, Gregory S. TaylorAbstract:Myotubularin is a 3-phosphoinositide phosphatase that is mutated in X-linked myotubular myopathy, a severe neonatal disorder in which skeletal muscle development and/or regeneration is impaired. In this report we provide evidence that siRNA-mediated silencing of Myotubularin expression markedly inhibits growth factor-stimulated Akt phosphorylation, leading to activation of caspase-dependent pro-apoptotic signaling in HeLa cells and primary human skeletal muscle myotubes. Myotubularin silencing also inhibits Akt-dependent signaling through the mammalian target of rapamycin complex 1 as assessed by p70 S6-kinase and 4E-BP1 phosphorylation. Similarly, phosphorylation of FoxO transcription factors is also significantly reduced in Myotubularin-deficient cells. Our data further suggest that inhibition of Akt activation and downstream survival signaling in Myotubularin-deficient cells is caused by accumulation of the MTMR substrate lipid phosphatidylinositol 3-phosphate generated from the type II phosphatidylinositol 3-kinase PIK3C2B. Our findings are significant because they suggest that Myotubularin regulates Akt activation via a cellular pool of phosphatidylinositol 3-phosphate that is distinct from that generated by the type III phosphatidylinositol 3-kinase hVps34. Because impaired Akt signaling has been tightly linked to skeletal muscle atrophy, we hypothesize that loss of Akt-dependent growth/survival cues due to impaired Myotubularin function may be a critical factor underlying the severe skeletal muscle atrophy characteristic of muscle fibers in patients with X-linked myotubular myopathy.
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molecular basis for substrate recognition by mtmr2 a Myotubularin family phosphoinositide phosphatase
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Michael J Begley, Gregory S. Taylor, Melissa A Brock, Partho Ghosh, Virgil L Woods, Jack E. DixonAbstract:Myotubularins, a large family of catalytically active and inactive proteins, belong to a unique subgroup of protein tyrosine phosphatases that use inositol phospholipids, rather than phosphoproteins, as physiological substrates. Here, by integrating crystallographic and deuterium-exchange mass spectrometry studies of human Myotubularin-related protein-2 (MTMR2) in complex with phosphoinositides, we define the molecular basis for this unique substrate specificity. Phosphoinositide substrates bind in a pocket located on a positively charged face of the protein, suggesting an electrostatic mechanism for membrane targeting. A flexible, hydrophobic helix makes extensive interactions with the diacylglycerol moieties of substrates, explaining the specificity for membrane-bound phosphoinositides. An extensive H-bonding network and charge–charge interactions within the active site pocket determine phosphoinositide headgroup specificity. The conservation of these specificity determinants within the active, but not the inactive, Myotubularins provides insight into the functional differences between the active and inactive members.
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crystal structure of a phosphoinositide phosphatase mtmr2 insights into myotubular myopathy and charcot marie tooth syndrome
Molecular Cell, 2003Co-Authors: Michael J Begley, Gregory S. Taylor, Jack E. Dixon, Soo-a Kim, Donna M Veine, Jeanne A StuckeyAbstract:Myotubularin-related proteins are a large subfamily of protein tyrosine phosphatases (PTPs) that dephosphorylate D3-phosphorylated inositol lipids. Mutations in members of the Myotubularin family cause the human neuromuscular disorders myotubular myopathy and type 4B Charcot-Marie-Tooth syndrome. The crystal structure of a representative member of this family, MTMR2, reveals a phosphatase domain that is structurally unique among PTPs. A series of mutants are described that exhibit altered enzymatic activity and provide insight into the specificity of Myotubularin phosphatases toward phosphoinositide substrates. The structure also reveals that the GRAM domain, found in Myotubularin family phosphatases and predicted to occur in ∼180 proteins, is part of a larger motif with a pleckstrin homology (PH) domain fold. Finally, the MTMR2 structure will serve as a model for other members of the Myotubularin family and provide a framework for understanding the mechanism whereby mutations in these proteins lead to disease.
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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.
Yasuhiro Mochizuki - One of the best experts on this subject based on the ideXlab platform.
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phosphatidylinositol 3 phosphatase Myotubularin related protein 6 mtmr6 is regulated by small gtpase rab1b in the early secretory and autophagic pathways
Journal of Biological Chemistry, 2013Co-Authors: Yasuhiro Mochizuki, Riuko Ohashi, Makoto Naito, Hiroko Iwanari, Tatsuhiko Kodama, Takeshi Kawamura, Takao HamakuboAbstract:Abstract A large family of Myotubularin phosphatases dephosphorylates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate, which are known to play important roles in vesicular trafficking and autophagy. The family is composed of 16 members, and understanding their regulatory mechanisms is important to understand their functions and related genetic diseases. We prepared anti-Myotubularin-related protein 6 (MTMR6) monoclonal antibody and used it to study the regulatory mechanism of MTMR6. Endogenous MTMR6 was present in the cytoplasm and was condensed in the perinuclear region in a microtubule-dependent manner. MTMR6 preferentially interacted with GDP-bound Rab1B via the GRAM domain and partly overlapped with Rab1B in the pericentrosomal and peri-Golgi regions in normal rat kidney cells. Overexpression of GDP-bound Rab1B and the reduction of Rab1B disrupted the localization of MTMR6, suggesting that Rab1B regulates the localization of MTMR6. The reduction of MTMR6 accelerated the transport of vesicular stomatitis virus glycoprotein in which Rab1B is involved. Furthermore, reduction of MTMR6 or Rab1B inhibited the formation of the tubular omegasome that is induced by overexpression of DFCP1 in autophagy. Our results indicate that the cellular localization of MTMR6 is regulated by Rab1B in the early secretory and autophagic pathways. We propose a new regulatory mechanism of Myotubularin phosphatase by the small GTPase Rab1B.
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phosphatidylinositol 3 phosphatase Myotubularin related protein 6 mtmr6 is regulated by small gtpase rab1b in the early secretory and autophagic pathways
Journal of Biological Chemistry, 2013Co-Authors: Yasuhiro Mochizuki, Riuko Ohashi, Makoto Naito, Hiroko Iwanari, Tatsuhiko Kodama, Takeshi Kawamura, Takao HamakuboAbstract:Abstract A large family of Myotubularin phosphatases dephosphorylates phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate, which are known to play important roles in vesicular trafficking and autophagy. The family is composed of 16 members, and understanding their regulatory mechanisms is important to understand their functions and related genetic diseases. We prepared anti-Myotubularin-related protein 6 (MTMR6) monoclonal antibody and used it to study the regulatory mechanism of MTMR6. Endogenous MTMR6 was present in the cytoplasm and was condensed in the perinuclear region in a microtubule-dependent manner. MTMR6 preferentially interacted with GDP-bound Rab1B via the GRAM domain and partly overlapped with Rab1B in the pericentrosomal and peri-Golgi regions in normal rat kidney cells. Overexpression of GDP-bound Rab1B and the reduction of Rab1B disrupted the localization of MTMR6, suggesting that Rab1B regulates the localization of MTMR6. The reduction of MTMR6 accelerated the transport of vesicular stomatitis virus glycoprotein in which Rab1B is involved. Furthermore, reduction of MTMR6 or Rab1B inhibited the formation of the tubular omegasome that is induced by overexpression of DFCP1 in autophagy. Our results indicate that the cellular localization of MTMR6 is regulated by Rab1B in the early secretory and autophagic pathways. We propose a new regulatory mechanism of Myotubularin phosphatase by the small GTPase Rab1B.
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characterization of Myotubularin related protein 7 and its binding partner Myotubularin related protein 9
Proceedings of the National Academy of Sciences of the United States of America, 2003Co-Authors: Yasuhiro Mochizuki, Philip W MajerusAbstract:Abstract Myotubularin-related protein 7 (MTMR7) is a member of the Myotubularin (MTM) family. The cDNA encoding the mouse MTMR7 contains 1,983 bp, and the predicted protein has a deduced molecular mass of 75.6 kDa. Northern and Western blot analyses showed that MTMR7 is expressed mainly in brain and mouse neuroblastoma N1E-115 cells. Recombinant MTMR7 dephosphorylated the D-3 position of phosphatidylinositol 3-phosphate and inositol 1,3-bisphosphate [Ins(1,3)P2]. The substrate specificity of MTMR7 is different than other MTM proteins in that this enzyme prefers the water-soluble substrate. Immunofluorescence showed that MTMR7 is localized in Golgi-like granules and cytosol, and subcellular fractionation showed both cytoplasmic and membrane localization of MTMR7 in N1E-115 cells. An MTMR7-binding protein was found in an anti-MTMR7 immunoprecipitate from N1E-115 cells and identified as MTM-related protein 9 (MTMR9) by tandem mass spectrometry. The coiled-coil domain of MTMR9 was sufficient for binding to MTMR7. The binding of MTMR9 increased the Ins(1,3)P2 phosphatase activity of MTMR7. Our results show that MTMR7 forms a complex with MTMR9 and dephosphorylates phosphatidylinositol 3-phosphate and Ins(1,3)P2 in neuronal cells.
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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.
