Charcot-Marie-Tooth type 4B demyelinating neuropathy: deciphering the role of MTMR phosphatases

Charcot-Marie-Tooth type 4B (CMT4B) is a severe autosomal recessive neuropathy with demyelination and myelin outfoldings of the nerve. This disorder is genetically heterogeneous, but thus far, mutations in myotubularin-related 2 (MTMR2) and MTMR13 genes have been shown to underlie CMT4B1 and CMT4B2, respectively. MTMR2 and MTMR13 belong to a family of ubiquitously expressed proteins sharing homology with protein tyrosine phosphatases (PTPs). The MTMR family, which has 14 members in humans, comprises catalytically active proteins, such as MTMR2, and catalytically inactive proteins, such as MTMR13. Despite their homology with PTPs, catalytically active MTMR phosphatases dephosphorylate both PtdIns3P and PtdIns(3,5)P2 phosphoinositides. Thus, MTMR2 and MTMR13 may regulate vesicular trafficking in Schwann cells. Loss of these proteins could lead to uncontrolled folding of myelin and, ultimately, to CMT4B. In this review, we discuss recent findings on this interesting protein family with the main focus on MTMR2 and MTMR13 and their involvement in the biology of Schwann cell and CMT4B neuropathies.     

The phosphoinositide-3-phosphatase MTMR2 associates with MTMR13, a membrane-associated pseudophosphatase also mutated in type 4B Charcot-Marie-Tooth disease

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.     

Crystal structure of a phosphoinositide phosphatase, MTMR2: insights into myotubular myopathy and Charcot-Marie-Tooth syndrome

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 approximately 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.     

Mutations in MTMR13, a new pseudophosphatase homologue of MTMR2 and Sbf1, in two families with an autosomal recessive demyelinating form of Charcot-Marie-Tooth disease associated with early-onset glaucoma

Charcot-Marie-Tooth disease (CMT) with autosomal recessive (AR) inheritance is a heterogeneous group of inherited motor and sensory neuropathies. In some families from Japan and Brazil, a demyelinating CMT, mainly characterized by the presence of myelin outfoldings on nerve biopsies, cosegregated as an autosomal recessive trait with early-onset glaucoma. We identified two such large consanguineous families from Tunisia and Morocco with ages at onset ranging from 2 to 15 years. We mapped this syndrome to chromosome 11p15, in a 4.6-cM region overlapping the locus for an isolated demyelinating ARCMT (CMT4B2). In these two families, we identified two different nonsense mutations in the myotubularin-related 13 gene, MTMR13. The MTMR protein family includes proteins with a phosphoinositide phosphatase activity, as well as proteins in which key catalytic residues are missing and that are thus called "pseudophosphatases." MTM1, the first identified member of this family, and MTMR2 are responsible for X-linked myotubular myopathy and Charcot-Marie-Tooth disease type 4B1, an isolated peripheral neuropathy with myelin outfoldings, respectively. Both encode active phosphatases. It is striking to note that mutations in MTMR13 also cause peripheral neuropathy with myelin outfoldings, although it belongs to a pseudophosphatase subgroup, since its closest homologue is MTMR5/Sbf1. This is the first human disease caused by mutation in a pseudophosphatase, emphasizing the important function of these putatively inactive enzymes. MTMR13 may be important for the development of both the peripheral nerves and the trabeculum meshwork, which permits the outflow of the aqueous humor. Both of these tissues have the same embryonic origin.     

Genetic and developmental relationships between two alkaline phosphatases inDrosophila melanogaster

The locus, Aph,for a third-instar larval alkaline phosphatase, 1-Aph, in Drosophila melanogasterhas been placed at 47.3 on the third chromosome. A new and fourth allele, Aph^a,is described. Another alkaline phosphatase, p-Aph, is characteristic of the pupal stage. Developmental studies show that 1-Aph begins to disappear 9.5 hr after prepuparium formation at 25 C, and that p-Aph appears as 1-Aph disappears. Each of the four Aph alleles produces a p-Aph variant distinguishable by electrophoresis. Except for the one produced by Aph^a,the electrophoretic properties of these p-Aph variants parallel those of the 1-Aph's produced by the same allele. Three sources of evidence support the conclusion that p-Aph variation is attributable to the Aphlocus: (1) In experiments where Aphalleles segregate, corresponding segregation of p-Aph variants is observed. (2) The linkage relationships of p-Aph are the same as those of Aph.(3) In experiments capable of detecting with a probability of 0.99 a recombination event between two loci 0.0006 centimorgans apart, no recombination is observed between 1-Aph and p-Aph. It is suggested that the Aphlocus either consists of one cistron which is responsible for both 1-Aph and p-Aph, or that it consists of two cistrons, one for 1-Aph and one for p-Aph. Implications for the structure of these alkaline phosphatases and for the nature of the developmental shift which they exhibit are discussed.Support by a research grant (GM-11777) from the National Institutes of Health. This is paper number 1161 from the Laboratory of Genetics, The University of Wisconsin, Madison, Wisconsin.National Institutes of Health Predoctoral Trainee. Based on a thesis submitted on December 15, 1966 in partial fulfillment of the degree of Master of Science at The University of Wisconsin.     

Genetic interaction between Bmp2 and Bmp4 reveals shared functions during multiple aspects of mouse organogenesis

Vertebrate Bmp2 and Bmp4 diverged from a common ancestral gene and encode closely related proteins. Mice homozygous for null mutations in either gene show early embryonic lethality, thereby precluding analysis of shared functions. In the current studies, we present phenotypic analysis of compound mutant mice heterozygous for a null allele of Bmp2 in combination with null or hypomorphic alleles of Bmp4. Whereas mice lacking a single copy of Bmp2 or Bmp4 are viable and have subtle developmental defects, compound mutants show embryonic and postnatal lethality due to defects in multiple organ systems including the allantois, placental vasculature, ventral body wall, skeleton, eye and heart. Within the heart, BMP2 and BMP4 function coordinately to direct normal lengthening of the outflow tract, proper positioning of the outflow vessels, and septation of the atria, ventricle and atrioventricular canal. Our results identify numerous BMP4-dependent developmental processes that are also very sensitive to BMP2 dosage, thus revealing novel functions of Bmp2.     

The protein phosphatases involved in cellular regulation. Identification of the inhibitor-2 phosphatases in rabbit skeletal muscle

Inhibitor-2, purified by an improved procedure, was used to identify protein phosphatases capable of catalysing its dephosphorylation. The results showed that, under our experimental conditions, protein phosphatases-1, 2A and 2B were the only significant protein phosphatases in rabbit skeletal muscle extracts acting on this substrate. Protein phosphatases-1 and 2A accounted for all the inhibitor-2 phosphatase activity in the absence of Ca2+ (resting muscle), and the potential importance of these enzymes in vivo is discussed. Protein phosphatase-2B, a Ca2+-calmodulin-dependent enzyme, could account for up to 30% of the inhibitor-2 phosphatase activity in contracting muscle. The Km of protein phosphatase-1 for inhibitor-2 (40 nM) was 100-fold lower than the Km for phosphorylase a (4.8 microM). This finding, coupled with the failure of inhibitor-2 to inhibit its own dephosphorylation, suggests that inhibitor-2 is dephosphorylated at one of the two sites on protein phosphatase-1 involved in preventing the dephosphorylation of other substrates. The dephosphorylation of inhibitor-2 by protein phosphatase-1 was also unaffected by inhibitor-1, suggesting that the phosphorylation state of inhibitor-2 is unlikely to be controlled by cyclic AMP in vivo.     

The protein phosphatases involved in cellular regulation. 2. Glycogen metabolism

The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metabolism was investigated in rabbit skeletal muscle and liver. Six 32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-1 were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-1, 2A, 2B and 2C [Ingebritsen, T. S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261] were the only significant enzymes acting on these substrates. The four enzymes can be conveniently separated and identified by a combination of ion-exchange chromatography and gel filtration and by the use of specific inhibitors. Three species of protein phosphatase-2A were resolved on DEAE-cellulose, termed protein phosphatases-2Ao (0.12 M NaCl), 2A1 (0.2 M NaCl) and 2A2 (0.28 M NaCl) that had apparent molecular weights of 210000, 210000 and 150000 respectively. Protein phosphatase-2Ao was a completely inactive enzyme whose activity was only expressed after dissociation to a 34000-Mr(app) catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissociated protein phosphatases 2A1 and 2A2 to more active 34000-Mr(app) catalytic subunits. The catalytic subunits derived from protein phosphatases-2Ao, 2A1 and 2A2 possessed identical substrate specificities, preferentially dephosphorylated the alpha-subunit of phosphorylase kinase, were unaffected by inhibitor-1 and inhibitor-2 and were inhibited by similar concentrations of ATP. The properties of protein phosphatases-2A1 and 2A2 were very similar to those of the catalytic subunits, except that they were less sensitive to inhibition by ATP. Protein phosphatase-2B was eluted from DEAE-cellulose in the same fraction as protein phosphatase-2Ao. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98000. Protein phosphatase-2B was completely inhibited by 100 microM trifluoperazine, which did not affect the activity of protein phosphatase-2Ao or any other protein phosphatase. Freezing and thawing in 0.2 M 2-mercaptoethanol resulted in partial inactivation of protein phosphatase-2B. Protein phosphatase-2C was eluted from DEAE-cellulose at the leading edge of the peak of protein phosphatase-2A1. These activities were completely resolved by gel filtration, since the Mr(app) of protein phosphatase-2C was 46000. Two forms of protein phosphatase-1 can be identified by chromatography on DEAE-cellulose, namely protein phosphatase-1 itself and the Mg X ATP-dependent protein phosphatase. Both these species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2A1. These enzymes did not interfere with measurements of type-2 protein phosphatases, since it was possible to block their activity with inhibitor-2...     

Head-to-tail interaction between amphotericin B and ergosterol occurs in hydrated phospholipid membrane

Amphotericin B (AmB) is thought to exert its antifungal activity by forming an ion-channel assembly in the presence of ergosterol. In the present study we aimed to elucidate the mode of molecular interactions between AmB and ergosterol in hydrated phospholipid bilayers using the rotational echo double resonance (REDOR) spectra. We first performed (13)C{(19)F}REDOR experiments with C14-(19)F-labeled AmB and biosynthetically (13)C-labeled ergosterol and implied that both "head-to-head" and "head-to-tail" orientations occur for AmB-ergosterol interaction in the bilayers. To further confirm the "head-to-tail" pairing, (13)C-labeled ergosterol at the dimethyl terminus (C26/C27) was synthesized and subjected to the REDOR measurements. The spectra unambiguously demonstrated the presence of a "head-to-tail" orientation for AmB-ergosterol pairing. In order to obtain information on the position of the dimethyl terminus of ergosterol in membrane, (13)C{(31)P}REDOR were carried out using the labeled ergosterol and the phosphorus atom of a POPC headgroup. Significant REDOR dephasing was observed at the C26/C27 signal of ergosterol in the presence of AmB, but not in the absence of AmB, clearly indicating that the side-chain terminus of ergosterol in the AmB complex comes close to the bilayer surface.     

Genetic evidence for interaction between eta- and beta-tubulins

The thermosensitive allelic mutations sm19-1 and sm19-2 of Paramecium tetraurelia cause defective basal body duplication: growth at the nonpermissive temperature yields smaller and smaller cells with fewer and fewer basal bodies. Complementation cloning of the SM19 gene identified a new tubulin, eta-tubulin, showing low homology with each of the other five tubulins, α to , characterized in P. tetraurelia. In order to analyze η-tubulin functions, we used a genetic approach to identify interacting molecules. Among a series of extragenic suppressors of the sm19-1 mutation, the su3-1 mutation was characterized as an E288K substitution in the β-PT2 gene coding for a β-tubulin, while the mutation noc^r1 conferring nocodazole resistance and localized in another β-tubulin gene, β-PT3, was shown to enhance the mutant phenotype. The interaction between η-tubulin and microtubules, revealed by genetic data, is supported by two further types of evidence: first, the mutant phenotype is rescued by taxol, which stabilizes microtubules; second, molecular modeling suggests that η-tubulin, like γ- and δ-tubulins, might be a microtubule minus-end capping molecule. The likely function of η-tubulin as part of a complex specifically involved in basal body biogenesis is discussed.     

Genetic interaction between the ero1-1 and leu2 mutations in Saccharomyces cerevisiae

The conditional ero1-1 mutant, deficient in the ER-localized PDI oxidase Ero1p, is blocked in disulfide bond formation under restrictive conditions, such as high temperature, lack of oxygen, or high concentrations of membrane-permeant thiols. Previous studies of the physiological consequences of the ero1-1 mutation were carried out in a leu2 mutant. The ero1-1 leu2 strain does not grow in standard synthetic complete medium at 30 degrees C, a defect that can be remedied by increasing the L-leucine concentration in the medium or by transforming the ero1-1 leu2 strain with the LEU2 wild-type allele. In addition, the LEU2 gene can partially complement the growth impairment at 37 degrees C of the ero1-1 leu2 mutant. The leucine transporter Bap2p exhibits a dramatic decrease in stability in an ero1-1 strain, which may account for the pronounced leucine demand observed in the ero1-1 leu2 mutant.     

A new method for studying the interaction between chlorpromazine and phospholipid bilayer

The spectroscopic and transmission electron microscopy (TEM) studies of interaction between chlorpromazine (CPZ) and dimyristoyl phosphatidylglycerol (DMPG) bilayer by using gold nanoparticles (AuNPs) as probes are reported. The DMPG bilayer-protected AuNPs were prepared by a simple one-step method. The DMPG bilayer tethered on the AuNPs was considered as a biomembrane model. The addition of CPZ affected the surface plasmon resonance (SPR) and morphology of the prepared AuNPs, and this effect was monitored by UV-vis spectroscopy and TEM. The interaction between CPZ and DMPG bialyer was CPZ concentration-dependent, and the possible mechanism was discussed. This simple and facile method may be quite general and work for other surface active drug-biomembrane or protein-biomembrane interactions.     

A redox-dependent interaction between two electron-transfer partners involved in photosynthesis

Ferredoxin:NADP⁺:reductase (FNR) catalyzes one terminal step of the conversion of light energy into chemical energy during photosynthesis. FNR uses two high energy electrons photoproduced by photosystem I (PSI) and conveyed, one by one, by a ferredoxin (Fd), to reduce NADP⁺ to NADPH. The reducing power of NADPH is finally involved in carbon assimilation. The interaction between oxidized FNR and Fd was studied by crystallography at 2.4 Å resolution leading to a three-dimensional picture of an Fd–FNR biologically relevant complex. This complex suggests that FNR and Fd specifically interact prior to each electron transfer and disassemble upon a redox-linked conformational change of the Fd.     

The interaction of phospholipase A2 with phospholipid analogues and inhibitors

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular dynamics simulation of interaction between phospholipid membrane and pyrazine and its derivatives

We have performed a comparative molecular dynamics simulation of the diffusion process of the heterocyclic compound pyrazine and its methylated derivatives into the model membrane phospholipid bilayer. Several structural and dynamical bilayer parameters were measured, and qualitative interrelations between parameter changes and the substituted pyrazine structure were studied. The simulation results support the hypothesis that molecular mechanisms of biological effects of substituted pyrazines involve dissolution of the effector molecule in the membrane bilayer and subsequent changes in bilayer properties. This stage can provide the means for pyrazine molecules to interact with integral membrane proteins, directly or indirectly through the changed lipid environment of the protein.     

Hydrophobic interaction between the monomer of mitochondrial malate dehydrogenase and phospholipid membranes.

Porcine mitochondrial malate dehydrogenase (EC 1.1.1.37) dissociates into subunits on dilution. The enzyme monomer caused large increases in the surface pressure of monolayers of 1:1 phosphatidylserine/phosphatidylcholine at air/water and oil/water interfaces. The monomer increased the permeability of phospholipid vesicles to 22Na+. Both effects were significantly greater than the corresponding effects of ribonuclease A, cytochrome c and the dimeric form of malate dehydrogenase. Changes in the circular-dichroism spectra of the enzyme indicated that conformational changes may be associated with dimer formation or when monomer interacts with lysophosphatidyl-choline. Similar interactions to those described may occur in situ when mitochondrial malate dehydrogenase is transported to the mitochondrial matrix from its site of synthesis on cytosolic ribosomes.