Characterization of Tubulin in Mouse Brain Myelin

Analysis of mouse brain myelin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that in the high-molecular-weight range it contained, besides the Wolfgram protein doublet, proteins comigrating with actin and with both subunits of tubulin. The occurrence of these alpha and beta subunits was confirmed by peptide mapping in myelin analyzed by two-dimensional electrophoresis. This tubulin did not arise from an artifactual binding of soluble brain tubulin to the myelin fraction: addition of exogenously labeled tubulin to brain homogenates proved that during myelin isolation by the procedure of Norton and Poduslo (1973) the contaminating tubulin was washed out. On the other hand, the distribution of tubulin isoforms in myelin was investigated by isoelectric focusing and compared with the distribution of the 21 isoforms listed for the whole brain soluble tubulin. It was shown that many isoforms were found in myelin (three isoforms for the alpha subunit and nine for the beta subunit), and that some isoforms were represented both in myelin and in soluble tubulin, but in different relative proportions.     

Isolation of Ca2+ sensitive proteins linked to the membrane fraction of the brain cortex and the spinal cord in pigs

Four Ca2+-sensitive proteins of respective subunit molecular weights 67 kDa, 37 kDa, 36 kDa and 32 kDa were purified from pig brain and spinal cord. Associated to the particulate fraction at millimolar concentrations of free Ca2+, they were solubilized using an EGTA-containing buffer and purified by a selective Ca2+-dependent precipitation. The 36 kDa protein is present in the tissues in a tetrameric form of (2 X 36 kDa + 2 X 13 kDa) and in a monomeric form. These proteins with the 37 kDa protein share the functional properties of the two well-known Ca2+-binding proteins, named calpactin I and calpactin II; they were able to interact with F-actin, brain spectrin (fodrin) and phosphatidylserine-liposomes in a Ca2+-dependent manner. The 67 kDa protein depolymerizes the actin filament in presence of Ca2+, it also binds to tubulin and to the neurofilament subunit NF-70, but not to brain spectrin. The 32 kDa protein does not share any association with F-actin and brain spectrin.     

Degradation of the P0, P1, and Pr, Proteins in Peripheral Nervous System Myelin by Plasmin: Implications Regarding the Role of Macrophages in Demyelinating Diseases

Activated macrophages secrete a variety of neutral proteinases, including plasminogen activator. Since macrophages are implicated in primary demyelination in the peripheral nervous system (PNS) in Guillain-Barré syndrome and experimental allergic neuritis, we have investigated the ability of plasmin and of conditioned media from cultured macrophages, in the presence of plasminogen, to degrade the proteins in bovine and rat PNS myelin. The results indicate that (a) the major glycoprotein P0 and the basic P1 and Pr proteins in PNS myelin are extremely sensitive to plasmin, perhaps more so than is the basic protein in CNS myelin; (b) the initial product of degradation of P0 by plasmin has a molecular weight higher than that of the "X" protein; (c) large degradation products of P0 are relatively insensitive to further degradation; and (d) the neuritogenic P2 protein in PNS myelin is quite resistant to the action of plasmin. Results similar to those with plasmin were obtained with conditioned media from macrophages and macrophage-like cell lines together with plasminogen activator, and the degradation of the PNS myelin proteins, Po and P1, under these conditions was inhibited by p-nitrophenylguanidinobenzoate, an inhibitor of plasmin and plasminogen activator. The results suggest that the macrophage plasminogen activator could participate in inflammatory demyelination in the PNS.     

New observations on the compact myelin proteome

Myelin formation and maintenance depends on the establishment of two structurally and biochemically discernible domains: (a)compact myelin, that is multilamellar stacks of plasma membrane sheets; and (b) cytoplasmic channels that border the compact myelin domains, attach them to the cell body and anchor the myelin sheath to the axonal membrane. To identify proteins involved in the organization of these domains we took advantage of the high lipid content of compact myelin to separate it cleanly from other neural membranes and then used reverse-phase HPLC coupled to Electro-Spray Double Mass Spectrometry('MudPIT') to characterize the proteome of this sample. MudPIT allowed us to sidestep the bias of 2D-PAGE against either highly charged or transmembrane proteins. Thus, of 97 proteins that presented at least two, fully tryptic peptides (a stringent threshold), seven were well known myelin markers, including the mayor CNS myelin proteins: proteolipid protein and myelin basic protein, which are not resolvable by 2D-PAGE. Furthermore, we have confirmed and extended the known compact myelin proteome by 22 proteins and confirmed that CNS and PNS myelinated tracts present Sirtuin 2, a tubulin deacetylase, and Septin7, a small GTPase that is likely to be involved in membrane and cytoplasm partitioning.     

Rationalization of paclitaxel insensitivity of yeast ß-tubulin and human ßIII-tubulin isotype using principal component analysis

ABSTRACT: BACKGROUND: The chemotherapeutic agent paclitaxel arrests cell division by binding to the hetero-dimeric protein tubulin. Subtle differences in tubulin sequences, across eukaryotes and among beta-tubulin isotypes, can have profound impact on paclitaxel-tubulin binding. To capture the experimentally observed paclitaxel-resistance of human betaIII tubulin isotype and yeast beta-tubulin, within a common theoretical framework, we have performed structural principal component analyses of beta-tubulin sequences across eukaryotes. RESULTS: The paclitaxel-resistance of human betaIII tubulin isotype and yeast beta-tubulin uniquely mapped on to the lowest two principal components, defining the paclitaxel-binding site residues of beta-tubulin. The molecular mechanisms behind paclitaxel-resistance, mediated through key residues, were identified from structural consequences of characteristic mutations that confer paclitaxel-resistance. Specifically, Ala277 in betaIII isotype was shown to be crucial for paclitaxel-resistance. CONCLUSIONS: The present analysis captures the origin of two apparently unrelated events, paclitaxel-insensitivity of yeast tubulin and human betaIII tubulin isotype, through two common collective sequence vectors.     

IRS-2 branch of IGF-1 receptor signaling is essential for appropriate timing of myelination

Insulin-like growth factor (IGF)-1 increases proliferation, inhibits apoptosis and promotes differentiation of oligodendrocytes and their precursor cells, indicating an important function for IGF-1 receptor (IGF-1R) signaling in myelin development. The insulin receptor substrates (IRS), IRS-1 and -2 serve as intracellular IGF-1R adaptor proteins and are expressed in neurons, oligodendrocytes and their precursors. To address the role of IRS-2 in myelination, we analyzed myelination in IRS-2 deficient (IRS-2(-/-)) mice and age-matched controls during postnatal development. Interestingly, expression of the most abundant myelin proteins, myelin basic protein and proteolipid protein was reduced in IRS-2(-/-) brains at postnatal day 10 (P10) as compared to controls. myelin basic protein immunostaining in P10-IRS-2(-/-) mice revealed a reduced immunostaining, but an unchanged regional distribution pattern. In cerebral myelin isolates at P10 unaltered relative expression of different myelin proteins was found, indicating quantitatively reduced but not qualitatively altered myelination. Interestingly, up-regulation of IRS-1 expression and increased IGF-1R signaling were observed in IRS-2(-/-) mice at P10-14, indicating a compensatory mechanism to overcome IRS-2 deficiency. Adult IRS-2(-/-) mice showed unaltered myelination and motor function. Furthermore, in neuronal/brain-specific insulin receptor knockout mice myelination was unchanged. Thus, our experiments reveal that IGF-1R/IRS-2 mediated signals are critical for appropriate timing of myelination in vivo.     

Triton X-100 extractions of central nervous system myelin indicate a possible role for the minor myelin proteins in the stability of lamellae

Isolated CNS myelin membranes were extracted with Triton X-100 under conditions previously established for the isolation of cytoskeletal proteins. Treated myelin retained much of its characteristic lamellar structure despite the removal of most of the major myelin basic protein (18.5 kDa) and the proteolipid protein, which together normally constitute 60% of the total myelin protein. The SDS-PAGE profile of this extract residue demonstrated an enrichment in proteins of Mr 30 to 60 kilodaltons (the Wolfgram group). The major myelin proteins were identified by antibodies on Western immunoblots, as were the 23-cyclic nucleotide 3-phosphodiesterase (CNP), actin, tubulin, myelin-associated glycoprotein (MGP) and the 21.5 kDa MBP. The overall behavior of CNP, the 21.5 kDa MBP, MGP and tubulin towards Triton extraction is reminiscent of the behavior of other membrane-skeletal complexes, supporting the idea that these and other minor myelin proteins might be part of heteromolecular complexes with interactions spanning several lamellae of the myelin sheath.     

Purification and Chemical Characterization of a W2 Protein from Brain Myelin

Abstract: Starting from a pellet of beef brain myelin insoluble in chloroform/ methanol (2:1, vol/vol) (Wolfgram protein fraction), a pure W2 protein with apparent molecular weight of 52,000 was isolated by a simple preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis method. A comparative chemical analysis was carried out between purified W2 and a standard tubulin. Glutamic acid and arginine were the N-terminals detected. Similar peptide maps and amino acid composition were also found in both proteins. Immunological cross-reactivity was detected when W2 protein was tested against antitubulin serum. These results suggest that W2 protein could have a tubulin-like protein nature that is associated with the myelin membrane and could play a role in the myelination process.     

Alpha2B-adrenergic receptor interaction with tubulin controls its transport from the endoplasmic reticulum to the cell surface

It is well recognized that the C terminus (CT) plays a crucial role in modulating G protein-coupled receptor (GPCR) transport from the endoplasmic reticulum (ER) to the cell surface. However the molecular mechanisms that govern CT-dependent ER export remain elusive. To address this issue, we used α(2B)-adrenergic receptor (α(2B)-AR) as a model GPCR to search for proteins interacting with the CT. By using peptide-conjugated affinity matrix combined with proteomics and glutathione S-transferase fusion protein pull-down assays, we identified tubulin directly interacting with the α(2B)-AR CT. The interaction domains were mapped to the acidic CT of tubulin and the basic Arg residues in the α(2B)-AR CT, particularly Arg-437, Arg-441, and Arg-446. More importantly, mutation of these Arg residues to disrupt tubulin interaction markedly inhibited α(2B)-AR transport to the cell surface and strongly arrested the receptor in the ER. These data provide the first evidence indicating that the α(2B)-AR C-terminal Arg cluster mediates its association with tubulin to coordinate its ER-to-cell surface traffic and suggest a novel mechanism of GPCR export through physical contact with microtubules.     

Ca2+, calmodulin-dependent regulation of microtubule formation via phosphorylation of microtubule-associated protein 2, tau factor, and tubulin, and comparison with the cyclic AMP-dependent phosphorylation

Abstract: Isolated microtubule-associated protein 2 (MAP2), τ factor, and tubulin were phosphorylated by a purified Ca²⁺, calmodulin-dependent protein kinase (640K enzyme) from rat brain. The phosphorylation of MAP2 and τ factor separately induced the inhibition of microtubule assembly, in accordance with the degree. Tubulin phosphorylation by the 640K enzyme induced the inhibition of microtubule assembly, whereas the effect of tubulin phosphorylation by the catalytic subunit was undetectable. The effects of tubulin and MAPs phosphorylation on microtubule assembly were greater than that of either tubulin or MAPs phosphorylation. Because MAP2, τ factor, and tubulin were also phosphorylated by the catalytic subunit of type-II cyclic AMP-dependent protein kinase from rat brain, the kinetic properties and phosphorylation sites were compared. The amount of phosphate incorporated into each microtubule protein was three to five times higher by the 640K enzyme than by the catalytic subunit. The K _m values of the 640K enzyme for microtubule proteins were four to 24 times lower than those of the catalytic subunit. The peptide mapping analysis showed that the 640K enzyme and the catalytic subunit incorporated phosphate into different sites on MAP2, τ factor, and tubulin. Investigation of phosphoamino acids revealed that only the seryl residue was phosphorylated by the catalytic subunit, whereas both seryl and threonyl residues were phosphorylated by the 640K enzyme. These data suggest that the Ca²⁺, calmodulin system via phosphorylation of MAP2, τ factor, and tubulin by the 640K enzyme is more effective than the cyclic AMP system on the regulation of microtubule assembly.     

Effect of chemical modifications of myelin basic protein on its interaction with lipid interfaces and cell fusion ability

Summary The ability of native and chemically modified myelin basic protein to induce fusion of chicken erythrocytes and to interact with lipids in monolayers at the air-water interface and liposomes was studied. Chemical modifications of myelin basic protein were performed by acetylation and succinylation: the positive charges of the native protein were blocked to an extent of about 90–95%.Cellular aggregation and fusion of erythrocytes into multinucleated cells was induced by the native myelin basic protein. This effect was diminished for both acetylated and succinylated myelin basic protein. Native myelin basic protein penetrated appreciably in sulphatide-containing lipid monolayers while lower penetration occurred in monolayers of neutral lipids. Contrary to this, both chemically modified myelin basic proteins did not show any selectivity to penetrate into interfaces of neutral or negatively charged lipids. The intrinsic fluorescence of the native and chemically modified myelin basic proteins upon interacting with liposomes constituted by dipalmitoylphosphatidycholine, glycosphingolipids, egg phosphatidic acid or dipalmitoylphosphatidyl glycerol was studied. The interaction with liposomes of anionic lipids is accompanied by a blue shift of the maximum of the native protein emission fluorescence spectrum from 346 nm to 335 nm; no shift was observed with liposomes containing neutral lipids. The acetylated and succinylated myelin basic proteins did not show changes of their emission spectra upon interacting with any of the lipids studied. The results obtained in monolayers and the fluorescence shifts indicate a lack of correlation between the ability of the modified proteins to penetrate lipid interfaces and the microenvironment sensed by the tryptophan-containing domain.Abbreviations MBP myelin basic protein - DPPC dipalmitoyl phosphatidylcholine - DPPG dipalmitoyl phosphatidylglycerol - PA phosphatidic acid     

Tubulin Binds to the Cytoplasmic Loop of TRESK Background K+ Channel In Vitro

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K⁺ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell.     

Distribution of Myelin Basic Protein and P2 mRNAs in Rabbit Spinal Cord Oligodendrocytes

Myelin basic protein (MBP) and P2 protein are small positively charged proteins found in oligodendrocytes of rabbit spinal cord. Both proteins become incorporated into compact myelin. We have begun investigations into the mechanisms by which MBP and P2 become incorporated into the myelin membrane. We find that P2, like the MBPs, is synthesized on free polysomes in rabbit spinal cord. Cell fractionation experiments reveal that rabbit MBP mRNAs are preferentially segregated to the peripheral myelinating regions whereas P2 mRNAs are predominantly localized within the perikaryon of the cell. In vitro synthesized rabbit MBP readily associates with membranes added to translation mixtures, whereas P2 protein does not. It is possible that P2 requires a "receptor" molecule, perhaps a membrane-anchored protein, for association with the cytoplasmic face of the myelin membrane.     

Myelin tetraspan family proteins but no non-tetraspan family proteins are present in the ascidian (Ciona intestinalis) genome

Several of the proteins used to form and maintain myelin sheaths in the central nervous system (CNS) and the peripheral nervous system (PNS) are shared among different vertebrate classes. These proteins include one-to-several alternatively spliced myelin basic protein (MBP) isoforms in all sheaths, proteolipid protein (PLP) and DM20 (except in amphibians) in tetrapod CNS sheaths, and one or two protein zero (P0) isoforms in fish CNS and in all vertebrate PNS sheaths. Several other proteins, including 2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNP), myelin and lymphocyte protein (MAL), plasmolipin, and peripheral myelin protein 22 (PMP22; prominent in PNS myelin), are localized to myelin and myelin-associated membranes, though class distributions are less well studied. Databases with known and identified sequences of these proteins from cartilaginous and teleost fishes, amphibians, reptiles, birds, and mammals were prepared and used to search for potential homologs in the basal vertebrate, Ciona intestinalis. Homologs of lipophilin proteins, MAL/plasmolipin, and PMP22 were identified in the Ciona genome. In contrast, no MBP, P0, or CNP homologs were found. These studies provide a framework for understanding how myelin proteins were recruited during evolution and how structural adaptations enabled them to play key roles in myelination.     

The P2 protein of bovine root myelin: partial chemical characterization.

The P2 protein of bovine root myelin has chemical characteristics which differentiate it from other myelin basic proteins. The tryptic peptide map of the bovine P2 protein is distinctly different from the map of either the rabbit PI protein or the bovine CNS myelin basic protein. The tryptic peptides of the P2 protein show no overlap in either map positions or amino acid content with the peptides of the CNS myelin basic protein. Analysis of the individual peptides in the P2 map accounted for all of the amino acids present in the analysis of the whole protein. The P2 protein has a blocked NH₂-terminus, lysine at its COOH-terminus and no hexosamine. CD studies revealed that the P2 protein has a very stable β-structure in aqueous solution at neutral or basic pH and retains much of this structure in acid and in 8 M urea. It is suggested that these structural properties are relevant to the dual role of the P2 protein as a membrane constituent and as an antigen.     

Two types of detergent-insoluble, glycosphingolipid/cholesterol-rich membrane domains from isolated myelin

Two different types of low-density detergent-insoluble glycosphingolipid-enriched membrane domain (DIG) fractions were isolated from myelin by extraction with Triton X-100 (TX-100) in 50 mM sodium phosphate buffer at room temperature (20 degrees C) (procedure 1), in contrast to a single low-density fraction obtained by extraction with TX-100 in Tris buffer containing 150 mM NaCl and 5 mM EDTA at 4 degrees C (procedure 2). Procedure 1 has been used in the past by others for myelin extraction to preserve the cytoskeleton and/or radial component of oligodendrocytes and myelin, whereas procedure 2 is now more commonly used to isolate myelin DIG fractions. The two DIG fractions obtained by procedure 1 gave opaque bands, B1 and B2, at somewhat lower and higher sucrose density respectively than myelin itself. The single DIG fraction obtained by procedure 2 gave a single opaque band at a similar sucrose density to B1. Both B1 and B2 had characteristics of lipid rafts, i.e. high galactosylceramide and cholesterol content and enrichment in GPI-linked 120-kDa neural cell adhesion molecule (NCAM)120, as found by others for the single low-density DIG fraction obtained by procedure 2. However, B2 had most of the myelin GM1 and more of the sulfatide than B1, and they differed significantly in their protein composition. B2 contained 41% of the actin, 100% of the tubulin, and most of the flotillin-1 and caveolin in myelin, whereas B1 contained more NCAM120 and other proteins than B2. The single low-density DIG fraction obtained by procedure 2 contained only low amounts of actin and tubulin. B1 and B2 also had size-isoform selectivity for some proteins, suggesting specific interactions and different functions of the two membrane domains. We propose that B1 may come from non-caveolar raft domains whereas B2 may derive from caveolin-containing raft domains associated with cytoskeletal proteins. Some kinases present were active on myelin basic protein suggesting that the DIGs may come from signaling domains.     

Fe65 interacts with P2X2 subunits at excitatory synapses and modulates receptor function

Ionotropic receptors in the neuronal plasma membrane are organized in macromolecular complexes, which assure their proper localization and regulate signal transduction. P2X receptors, the ionotropic receptors activated by extracellular ATP, have been shown to influence synaptic transmission. Using a yeast two-hybrid approach with the P2X(2) subunit C-terminal domain as bait we isolated the beta-amyloid precursor protein-binding proteins Fe65 and Fe65-like 1 as the first identified proteins interacting with neuronal P2X receptors. We confirmed the direct interaction of Fe65 and the P2X(2) C-terminal domain by glutathione S-transferase pull-down experiments. No interaction was observed between Fe65 and the naturally occurring P2X(2) splice variant P2X(2(b)), indicating that alternative splicing can regulate the receptor complex assembly. We generated two antibodies to Fe65 to determine its subcellular localization using postembedding immunogold labeling electron microscopy. We found labeling for Fe65 at the pre- and postsynaptic specialization of CA1 hippocampal pyramidal cell/Schaffer collateral synapses. By double immunogold labeling, we determined that Fe65 colocalizes with P2X(2) subunits at the postsynaptic specialization of excitatory synapses. Moreover, P2X(2) and Fe65 could be coimmunoprecipitated from brain membrane extracts, demonstrating that the interaction occurs in vivo. The assembly with Fe65 regulates the functional properties of P2X(2) receptors. Thus, the time- and activation-dependent change in ionic selectivity of P2X(2) receptors was inhibited by coexpression of Fe65, suggesting a novel role for Fe65 in regulating P2X receptor function and ATP-mediated synaptic transmission.     

A close structural relationship of rat liver Z-protein to cellular retinoid binding proteins and peripheral nerve myelin P2 protein

Striking sequence homologies were found among the major cytosolic lipid and dye binding protein (Z-protein), cellular retinoid binding proteins and peripheral nerve myelin P2 protein. In addition, the presence of a number of cytosolic lipid binding (or exchange) proteins having similar molecular weights and amino acid compositions suggests a new family of intracellular proteins with high affinities to lipids, possibly diverged from a common ancestor. In view of regulatory roles of these proteins in lipid metabolism and transport, myelin P2 protein may also possess a similar physiological function(s).     

The interaction of myelin basic protein with tubulin and the inhibition of tubulin carboxypeptidase activity

Tubulin carboxypeptidase was found to be inhibited by myelin basic protein in a concentration dependent manner. The inhibition was produced by the interaction between myelin basic protein with the substrate. As a consequence of this interaction, turbid insoluble aggregates were formed at either 5 degrees or 37 degrees C. The turbidity increased by increasing the myelin basic protein concentration and it reached a plateau at a molar ratio of myelin basic protein to tubulin dimer of about 6. At plateau, the molar ration in the insoluble aggregates was about 6. When tubulin was in excess, the formation of the insoluble aggregates was diminished. However, if the excess of tubulin was added after the formation of the aggregates, the turbidity was not significantly affected. Turbidity was diminished by increasing the ionic strength.     

Characterization of a cytoskeletal matrix associated with myelin from rat brain.

Extraction of rat brain myelin in a buffer containing Triton X-100 yielded a soluble fraction and an insoluble residue that was enriched in cytoskeletal elements. Immunoblot analysis of the detergent-soluble fraction and the insoluble cytoskeletal residue showed that all of the tubulin and more than half of the actin were found within the cytoskeletal fraction. The distribution of myelin-specific proteins was also examined, and revealed that 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) I and most of the myelin basic proteins (MBPs) were equally distributed between both fractions. By contrast, the large MBP (21.5 kDa) and CNPase II (50 kDa) were observed to partition almost entirely with the cytoskeletal fraction. Proteolipid protein was found predominantly in the detergent-soluble fraction, as was DM-20 protein. Analysis of the cytoskeletal fraction by sucrose-density-gradient centrifugation demonstrated that a distinct subset of lipids was tightly bound to the cytoskeletal protein residue. The cytoskeleton-associated lipid was considerably enriched in cerebroside and sphingomyelin by comparison with total myelin lipids. These results indicate that a cytoskeletal matrix is associated with multilamellar myelin, and suggest that this structure may play a fundamental role in myelinogenesis.