kDa Myelin Basic Protein Research Articles

Potential Neurodevelopmental Effects of Pediatric Intensive Care Sedation and Analgesia: Repetitive Benzodiazepine and Opioid Exposure Alters Expression of Glial and Synaptic Proteins in Juvenile Rats.

Objectives:Sedatives are suspected contributors to neurologic dysfunction in PICU patients, to whom they are administered during sensitive neurodevelopment. Relevant preclinical modeling has largely used comparatively brief anesthesia in infant age-approximate animals, with insufficient study of repetitive combined drug administration during childhood. We hypothesized that childhood neurodevelopment is selectively vulnerable to repeated treatment with benzodiazepine and opioid. We report a preclinical model of combined midazolam and morphine in early childhood age-approximate rats.Design:Animal model.Setting:Basic science laboratory.Subjects:Male and female Long-Evans rats.Interventions:Injections of morphine + midazolam were administered twice daily from postnatal days 18–22, tapering on postnatal days 23 and 24. Control groups included saline, morphine, or midazolam. To screen for acute neurodevelopmental effects, brain homogenates were analyzed by western blot for synaptophysin, drebrin, glial fibrillary acidic protein, S100 calcium-binding protein B, ionized calcium-binding adaptor molecule 1, and myelin basic proteins. Data analysis used Kruskal-Wallis with Dunn posttest, with a p value of less than 0.05 significance.Measurements and Main Results:Morphine + midazolam and morphine animals gained less weight than saline or midazolam (p ≤ 0.01). Compared with saline, morphine + midazolam expressed significantly higher drebrin levels (p = 0.01), with numerically but not statistically decreased glial fibrillary acidic protein. Similarly, morphine animals exhibited less glial fibrillary acidic protein and more S100 calcium-binding protein B and synaptophysin. Midazolam animals expressed significantly more S100 calcium-binding protein B (p < 0.001) and 17–18.5 kDa myelin basic protein splicing isoform (p = 0.01), with numerically increased synaptophysin, ionized calcium-binding adaptor molecule 1, and 21.5 kDa myelin basic protein, and decreased glial fibrillary acidic protein.Conclusions:Analysis of brain tissue in this novel rodent model of repetitive morphine and midazolam administration showed effects on synaptic, astrocytic, microglial, and myelin proteins. These findings warrant further investigation because they may have implications for critically ill children requiring sedation and analgesia.

And Yet it is Modified-Holding a Candle to the Dark Matter of White Matter.

The multilamellar membrane myelin sheath of the CNS, that enwraps axons to facilitate saltatory conduction in higher vertebrates, is held together by myelin basic protein (MBP). Yet this generalization masks how enigmatic MBP is, much like cosmological "dark matter." First, the casual use of the singular form for "protein" distracts that there are multiple, developmentally regulated "classic" splice isoforms ranging from 14 to 21.5 kDa, each with extensive PTMs. Second, the static image of MBP adhering two cytoplasmic leaflets of the oligodendrocyte membrane together in close apposition, suggests it to be inaccessible to modifying enzymes. And yet it is modified (to paraphrase Galileo's phrase on the earth's motion). In this issue of Proteomics, Sarg etal. apply an integrated CE-MS approach to investigate the PTMs of 18.5 kDa MBP from mouse brains of different ages. They identify new sites and types of modification, as well as confirming previously known PTMs. Innovative tools for unraveling the intricacies of the myelin basic proteome and how it organizes CNS myelin (much like basic histones organize chromatin), will help us understand white matter development and plasticity in health, during ageing, and in demyelinating diseases such as multiple sclerosis.

The proline-rich region of 18.5 kDa myelin basic protein binds to the SH3-domain of Fyn tyrosine kinase with the aid of an upstream segment to form a dynamic complex invitro.

The intrinsically disordered 18.5 kDa classic isoform of MBP (myelin basic protein) interacts with Fyn kinase during oligodendrocyte development and myelination. It does so primarily via a central proline-rich SH3 (Src homology 3) ligand (T92–R104, murine 18.5 kDa MBP sequence numbering) that is part of a molecular switch due to its high degree of conservation and modification by MAP (mitogen-activated protein) and other kinases, especially at residues T92 and T95. Here, we show using co-transfection experiments of an early developmental oligodendroglial cell line (N19) that an MBP segment upstream of the primary ligand is involved in MBP–Fyn–SH3 association in cellula. Using solution NMR spectroscopy in vitro, we define this segment to comprise MBP residues (T62–L68), and demonstrate further that residues (V83–P93) are the predominant SH3-target, assessed by the degree of chemical shift change upon titration. We show by chemical shift index analysis that there is no formation of local poly-proline type II structure in the proline-rich segment upon binding, and by NOE (nuclear Overhauser effect) and relaxation measurements that MBP remains dynamic even while complexed with Fyn–SH3. The association is a new example first of a non-canonical SH3-domain interaction and second of a fuzzy MBP complex.

A DEAD‐box RNA helicase Ddx54 protein in oligodendrocytes is indispensable for myelination in the central nervous system

We recently reported that a new monoclonal antibody, 4F2, which labels oligodendroglial lineage cells, recognizes a DEAD-box RNA helicase Ddx54 and that Ddx54 binds to myelin basic protein (MBP) in brain and cultured oligodendrocytes. To elucidate the biological function of Ddx54, we generated a recombinant adenovirus, Ad-shRNA:Ddx54, expressing a short hairpin RNA to silence endogenous Ddx54 protein. The virus was intraventricularly injected into the brains of mice on postnatal day (PD) 2. The brains at PD 9 were then analyzed by immunohistochemistry. In untreated normal brain sections, as well as control brains that had been injected with Ad-β-Gal, myelination of axons occurred in the corpus callosum with filamentous patterns of immunosignals of myelin-associated glycoprotein (MAG) and MBP. In Ad-shRNA:Ddx54-injected brain, substantial amounts of MAG and MBP immunosignals were present, but MBP immunosignals accumulated in the subplate layer and did not intrude into the emerging white matter. Immunoblot analysis revealed that Ddx54 knockdown caused a significant decrease in the level of 21.5 kDa MBP isoform and Ddx54, but the amount of Olig2; 2',3'-cyclic nucleotide 3' phosphodiesterase; MAG; three MBP isoforms (14, 17.5, and 18 kDa); and QKI-5, QKI-6, and QKI-7 proteins remained unchanged. Transfection of the Ddx54 expression vector into luciferase reporter-introduced neuroepithelial cells resulted in upregulated MBP promoter activity. Immunoprecipitation of Ddx54 protein in MBP-transfected HEK293 cells indicated that Ddx54 may directly interact with MBP mRNA. These results suggest that Ddx54 protein play an important role in central nervous system myelination, presumably in myelin sheath formation after the differentiation of oligodendrocytes.

Solution Nuclear Magnetic Resonance Structure and Molecular Dynamics Simulations of a Murine 18.5 kDa Myelin Basic Protein Segment (S72–S107) in Association with Dodecylphosphocholine Micelles

The 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in adult mammalian myelin, is a multifunctional, intrinsically disordered protein involved in the development and compaction of the myelin sheath in the central nervous system. A highly conserved central segment comprises a membrane-anchoring amphipathic α-helix followed by a proline-rich segment that represents a ligand for SH3 domain-containing proteins. Here, we have determined using solution nuclear magnetic resonance spectroscopy the structure of a 36-residue peptide fragment of MBP (murine 18.5 kDa residues S72-S107, denoted the α2-peptide) comprising these two structural motifs, in association with dodecylphosphocholine (DPC) micelles. The structure was calculated using CS-ROSETTA (version 1.01) because the nuclear Overhauser effect restraints were insufficient for this protein. The experimental studies were complemented by molecular dynamics simulations of a corresponding 24-residue peptide fragment (murine 18.5 kDa residues E80-G103, denoted the MD-peptide), also in association with a DPC micelle in silico. The experimental and theoretical results agreed well with one another, despite the independence of the starting structures and analyses, both showing membrane association via the amphipathic α-helix, and a sharp bend in the vicinity of the Pro93 residue (murine 18.5 kDa sequence numbering). Overall, the conformations elucidated here show how the SH3 ligand is presented to the cytoplasm for interaction with SH3 domain-containing proteins such as Fyn and contribute to our understanding of myelin architecture at the molecular level.

Structured Functional Domains of Myelin Basic Protein: Cross-Talk Between Actin Polymerization and Ca 2+-Dependent Calmodulin Interaction

The 18.5 kDa myelin basic protein (MBP), the most abundant isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the sheath. A hydrophobic moment analysis of MBP's amino acid sequence reveals three regions with high propensity to form strongly amphipathic alpha-helices. These regions, located in the central, N- and C- terminal parts of the protein, have been shown to play a role in the interactions of MBP with other partners, such as SH3-domain binding proteins, actin, Ca2+-activated calmodulin (Ca2+-CaM), and with myelin-mimetic membrane bilayers. Here, we have further characterized the structure-function relationship of these three domains. We constructed three recombinant peptides derived from the 18.5 kDa murine MBP: A22-K56, S72-S107, and S133-S159 (denoted alpha1, alpha2, and alpha3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, solid-state NMR (ssNMR) spectroscopy, isothermal titration calorimetry, electron microscopy, and fluorimetry) to characterize the interactions of these peptides with actin and calmodulin. Our results show that all three peptides can adopt alpha-helical structure inherently. Although both alpha1 and alpha3 peptides showed strong binding with Ca2+-CaM, only alpha1 exhibited actin polymerization and bundling activity. Calmodulin depolymerized actin that was polymerized by alpha1. Comparing fingerprints of Ala, Pro, and Ser (using ssNMR) in a reconstituted alpha1-actin complex, we showed that this peptide might adopt a better-defined structural state but still exhibits structural polymorphism. The results of this study proved that in addition to the primary calmodulin-binding site located in the C-terminal domain of MBP, there is another N-terminal binding domain for Ca2+-CaM. This secondary binding domain appeared to be essential for CaM-induced actin depolymerization. Acknowledgements: Funded by the CIHR (GH), NSERC (GH), and by Fellowships/Studentships from the MSSC (VVB, GSTS).

Modes of SH3-Domain Interactions of 18.5 kDa Myelin Basic Protein IN Vitro and in Oligodendrocytes

The developmentally-regulated myelin basic proteins (MBPs) play key roles in central nervous system (CNS) myelin formation by oligodendrocytes. They are highly positively-charged, intrinsically disordered, multifunctional proteins having several alternatively-spliced isoforms and combinatorial post-translational modifications. The most common 18.5kDa MBP isoform contains a proline-rich region (murine sequence T92PRTPPPS99) which comprises a minimal SH3-ligand. We have previously shown that 18.5 kDa MBP binds to Fyn, a member of the Src family of tyrosine kinases involved in signaling pathways during CNS development. Here, we have produced an isotopically-labeled fragment of MBP which contains the proline-rich region, and are studying the mode of interaction between MBP and the SH3 domain of Fyn through solution NMR spectroscopy and isothermal titration calorimetry (ITC). We have constructed MBP variants pseudo-phosphorylated at T92 and T95, and with P93G and P96G substitutions to disrupt the conformation of the SH3-ligand. Using ITC, these mutant MBPs are shown to have a decreased affinity towards the SH3-domain of Fyn. Solution NMR spectroscopy shows altered chemical shift patterns for the different variants, confirming that this segment is indeed the SH3-target and that the association can be modulated by phosphorylation. Concomitant over-expression of complementary GFP-tagged MBP forms in cultured oligodendrocytes results in aberrant elongation of membrane processes, increased branching complexity, and in some cases, trafficking of MBP to the nucleus. These data as a whole indicate that MBP's SH3-ligand domain plays a key role in intracellular protein interactions and may be required for proper membrane elaboration in myelin.Acknowledgements: Funded by the Canadian Institutes for Health Research (JMB, GH), NSERC Canada (GH), and by Doctoral Studentships from the Multiple Sclerosis Society of Canada (MDA, GSTS).

Myelin basic protein binds microtubules to a membrane surface and to actin filaments in vitro: Effect of phosphorylation and deimination

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and other proteins. It assembles actin filaments and microtubules, can bind actin filaments and SH3-domains to a membrane surface, and may be able to tether them to the oligodendrocyte membrane and participate in signal transduction in oligodendrocytes/myelin. In the present study, we have shown that the 18.5kDa MBP isoform can also bind microtubules to lipid vesicles in vitro. Phosphorylation of MBP at Thr94 and Thr97 (bovine sequence) by MAPK, and deimination of MBP (using a pseudo-deiminated recombinant form), had little detectable effect on its ability to polymerize and bundle microtubules, in contrast to the effect of these modifications on MBP-mediated assembly of actin. However, these modifications dramatically decreased the ability of MBP to tether microtubules to lipid vesicles. MBP and its phosphorylated and pseudo-deiminated variants were also able to bind microtubules to actin filaments. These results suggest that MBP may be able to tether microtubules to the cytoplasmic surface of the oligodendrocyte membrane, and that this binding can be regulated by post-translational modifications to MBP. We further show that MBP appears to be co-localized with actin filaments and microtubules in cultured oligodendrocytes, and also at the interface between actin filaments at the leading edge of membrane processes and microtubules behind them. Thus, MBP may also cross-link microtubules to actin filaments in vivo.

Conformational choreography of a molecular switch region in myelin basic protein—Molecular dynamics shows induced folding and secondary structure type conversion upon threonyl phosphorylation in both aqueous and membrane-associated environments

The 18.5kDa isoform of myelin basic protein is essential to maintaining the close apposition of myelin membranes in central nervous system myelin, but its intrinsic disorder (conformational dependence on environment), a variety of post-translational modifications, and a diversity of protein ligands (e.g., actin and tubulin) all indicate it to be multifunctional. We have performed molecular dynamics simulations of a conserved central segment of 18.5kDa myelin basic protein (residues Glu80–Gly103, murine sequence numbering) in aqueous and membrane-associated environments to ascertain the stability of constituent secondary structure elements (α-helix from Glu80–Val91 and extended poly-proline type II from Thr92–Gly103) and the effects of phosphorylation of residues Thr92 and Thr95, individually and together. In aqueous solution, all four forms of the peptide bent in the middle to form a hydrophobic cluster. The phosphorylated variants were stabilized further by electrostatic interactions and formation of β-structures, in agreement with previous spectroscopic data. In simulations performed with the peptide in association with a dimyristoylphosphatidylcholine bilayer, the amphipathic α-helical segment remained stable and membrane-associated, although the degree of penetration was less in the phosphorylated variants, and the tilt of the α-helix with respect to the plane of the membrane also changed significantly with the modifications. The extended segment adjacent to this α-helix represents a putative SH3-ligand and remained exposed to the cytoplasm (and thus accessible to binding partners). The results of these simulations demonstrate how this segment of the protein can act as a molecular switch: an amphipathic α-helical segment of the protein is membrane-associated and presents a subsequent proline-rich segment to the cytoplasm for interaction with other proteins. Phosphorylation of threonyl residues alters the degree of membrane penetration of the α-helix and the accessibility of the proline-rich ligand and can stabilize a β-bend. A bend in this region of 18.5kDa myelin basic protein suggests that the N- and C-termini of the proteins can interact with different leaflets of the myelin membrane and explain how a single protein can bring them close together.

Secondary Structure and Solvent Accessibility of a Calmodulin-Binding C-Terminal Segment of Membrane-Associated Myelin Basic Protein

Myelin basic protein (MBP), specifically the 18.5 kDa isoform, is a peripheral membrane protein and a major component of mammalian central nervous system myelin. It is an intrinsically disordered and multifunctional protein that binds cytoskeletal and other cytosolic proteins to a membrane surface and thereby acquires ordered structure. These associations are modulated by post-translational modifications of MBP, as well as by interactions of MBP with Ca(2+)-calmodulin (CaM). Enzymatic deimination of usually six arginine residues to citrulline results in a decrease in the net positive charge of the protein from 19 to ≤13. This deiminated form is found in greater amounts in normal children and in adult patients with the demyelinating disease multiple sclerosis. In this paper, we examine the secondary structure of a calmodulin-binding domain, residues A141-L154, when associated with a lipid bilayer in recombinant murine 18.5 kDa forms rmC1 (unmodified) and rmC8 (pseudodeiminated). We demonstrate here by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy that the Y142-L154 segment in membrane-associated rmC1 forms an amphipathic α-helix, with high accessibility to O(2) and low accessibility to NiEDDA. In membrane-associated rmC8, this segment assumed a structure distorted from an α-helix. Spin-labeled residues in rmC1 in solution were more immobilized on binding Ca(2+)-CaM than those in rmC8. Furthermore, rmC8 was dissociated more readily from a lipid bilayer by Ca(2+)-CaM than was rmC1. These results confirm both a predicted induced ordering upon membrane association in a specific segment of 18.5 kDa MBP, and that this segment is a CaM-binding site, with both interactions weakened by deimination of residues outside of this segment. The deiminated form would be more susceptible to regulation of its membrane binding functions by Ca(2+)-CaM than the unmodified form.

Interaction of Myelin Basic Protein with Actin in the Presence of Dodecylphosphocholine Micelles

The 18.5 kDa myelin basic protein (MBP), the most abundant splice isoform in human adult myelin, is a multifunctional, intrinsically disordered protein that maintains compact assembly of the myelin sheath in the central nervous system. Protein deimination and phosphorylation are two key posttranslational modifications whose balance determines local myelin microdomain stability and function. It has previously been shown that MBP in solution causes both polymerization of G-actin to F-actin and bundling of the microfilaments, and binds them to a negatively charged membrane. However, the binding parameters, and the roles of different possible interacting domains of membrane-associated MBP, have not yet been investigated. Here, we compared the interaction of unmodified (rmC1) and pseudodeiminated (rmC8) recombinant murine MBP (full-length charge variants), and of two terminal deletion variants (rmDeltaC and rmDeltaN), with actin in the presence of DPC (dodecylphosphocholine) to mimic a membrane environment. Our results show that although both charge variants polymerized and bundled actin, the maximal polymerization/bundling due to rmC1 occurred at a lower molar ratio compared to rmC8. In the presence of DPC, rmC1 appeared to be more active than rmC8 in its ability to polymerize and bundle actin, and the binding affinity of both charge variants to G-actin became higher. Moreover, of the two deletion variants studied in the presence of DPC, the one lacking the C-terminal domain (rmDeltaC) was more active compared to the variant lacking the N-terminal domain (rmDeltaN) but exhibited weaker binding to actin. Thus, whereas the N-terminal domain of MBP can be more important for the MBP's actin polymerization activity and membrane-association, the C-terminal domain can regulate its interaction with actin.

Fuzzy complexes of myelin basic protein: NMR spectroscopic investigations of a polymorphic organizational linker of the central nervous systemThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular &amp; Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

The classic 18.5 kDa isoform of myelin basic protein (MBP) is central to maintaining the structural homeostasis of the myelin sheath of the central nervous system. It is an intrinsically disordered, promiscuous, multifunctional, peripheral membrane protein, whose conformation adapts to its particular environment. Its study requires the selective and complementary application of diverse approaches, of which solution and solid-state NMR spectroscopy are the most powerful to elucidate site-specific features. We review here several recent solution and solid-state NMR spectroscopic studies of 18.5 kDa MBP, and the induced partial disorder-to-order transitions that it has been demonstrated to undergo when complexed with calmodulin, actin, and phospholipid membranes.

The interaction of zinc with membrane-associated 18.5 kDa myelin basic protein: an attenuated total reflectance-Fourier transform infrared spectroscopic study

Myelin basic protein (MBP) is an essential structural protein required for tight compaction of the myelin sheath of the central nervous system, and belongs to the family of intrinsically disordered proteins. It contains a high proportion of polar and charged amino acids, and has an adaptive conformation depending on its environment and binding surfaces (membranes) or partners (other proteins or small ligands including divalent cations). Zinc is an important stabilizing component of myelin and its concentration is substantially higher than that of any other trace element in the brain. In this study, we investigate the effect of zinc on different variants of 18.5 kDa MBP, including new recombinant forms lacking hexahistidine tags which would interfere with the binding of the cation. Isothermal titration calorimetry showed the dissociation constant to be in the micromolar range for all variants. Circular dichroism spectroscopy showed that there was minimal effect of zinc on the secondary structure on MBP in aqueous solution. When MBP was reconstituted with myelin-mimetic membranes, attenuated total reflectance-Fourier transform infrared spectroscopy revealed that there was a rearrangement of secondary structure components upon addition of zinc that was subtly different for each variant, indicative of a synergistic protein-membrane-cation interaction.

The Classic Basic Protein of Myelin – Conserved Structural Motifs and the Dynamic Molecular Barcode Involved in Membrane Adhesion and Protein-Protein Interactions

The myelin basic protein (MBP) family comprises a variety of developmentally-regulated members arising from different transcription start sites, differential splicing, and post-translational modifications. The "classic" isoforms of MBP include the 18.5 kDa form, which predominates in adult human myelin and facilitates compaction of the mature myelin sheath in the central nervous system, thereby maintaining its structural integrity. In addition to membrane-association, the 18.5 kDa and all other classic isoforms are able to interact with a multitude of proteins, including Ca(2+)-calmodulin, actin, tubulin, and SH3-domain containing proteins, and thus may be signalling linkers during myelin development and remodelling. All proteins in this family are intrinsically disordered, creating a large effective surface to facilitate multiple protein associations, and are post-translationally modified to various degrees by methylation, phosphorylation, and deimination. We have used spectroscopic (fluorescence, CD, EPR, and NMR) approaches to study MBP's conformational adaptability. A highly-conserved central domain presents an amphipathic alpha-helix in association with a phospholipid membrane, and contains a threonyl residue that is phosphorylated by MAP-kinases. In multiple sclerosis, this segment represents a primary immunodominant epitope. This helical structure is adjacent to a proline-rich region that presents a classic SH3-ligand, comprises a second MAP-kinase phosphorylation site, and forms a polyproline type II helix. This domain of the protein is thus essential to proper positioning of a protein-interaction motif, with the local conformation and accessibility being modulated by MAP-kinases. In addition, the C-terminus of 18.5 kDa MBP has been identified by NMR spectroscopy as a Ca(2+)-calmodulin-binding site, and is of note for having a high density of post-translational modifications (protein kinase C phosphorylation, and deimination). For the most part, any classic protein isoform functions as an entropic spring that interacts in its entirety with membranes and cytoskeletal proteins, but the central and C-terminal motifs may represent molecular switches.

Induced Secondary Structure and Polymorphism in an Intrinsically Disordered Structural Linker of the CNS: Solid-State NMR and FTIR Spectroscopy of Myelin Basic Protein Bound to Actin

The 18.5 kDa isoform of myelin basic protein (MBP) is a peripheral membrane protein that maintains the structural integrity of the myelin sheath of the central nervous system by conjoining the cytoplasmic leaflets of oligodendrocytes and by linking the myelin membrane to the underlying cytoskeleton whose assembly it strongly promotes. It is a multifunctional, intrinsically disordered protein that behaves primarily as a structural stabilizer, but with elements of a transient or induced secondary structure that represent binding sites for calmodulin or SH3-domain-containing proteins, inter alia. In this study we used solid-state NMR (SSNMR) and Fourier transform infrared (FTIR) spectroscopy to study the conformation of 18.5 kDa MBP in association with actin microfilaments and bundles. FTIR spectroscopy of fully 13C, 15N-labeled MBP complexed with unlabeled F-actin showed induced folding of both protein partners, viz., some increase in β-sheet content in actin, and increases in both α-helix and β-sheet content in MBP, albeit with considerable extended structure remaining. Solid-state NMR spectroscopy revealed that MBP in MBP-actin assemblies is structurally heterogeneous but gains ordered secondary structure elements (both α-helical and β-sheet), particularly in the terminal fragments and in a central immunodominant epitope. The overall conformational polymorphism of MBP is consistent with its in vivo roles as both a linker (membranes and cytoskeleton) and a putative signaling hub.

Myelin Basic Protein as a “PI(4,5)P2-Modulin”: A New Biological Function for a Major Central Nervous System Protein

The 18.5 kDa isoform of myelin basic protein (MBP) is multifunctional and has previously been shown to have structural and phenomenological similarities with domains of other membrane- and cytoskeleton-associated proteins such as MARCKS (myristoylated alanine-rich C kinase substrate). Here, we have investigated whether 18.5 kDa MBP can sequester phosphatidylinositol-(4,5)-bis-phosphate (PI(4,5)P 2) in membranes, like MARCKS and other "PIPmodulins" do. Using fluorescence-quenching and electron paramagnetic resonance (EPR) spectroscopy, and model membranes containing BODIPY-FL- or proxyl-labeled PI(4,5)P 2, respectively, we have demonstrated that MBP laterally sequesters PI(4,5)P 2. The MBP-PI(4,5)P 2 interactions are electrostatic, partially cholesterol-dependent, and sensitive to phosphorylation, deimination, and Ca (2+)-CaM binding. Confocal microscopy of cultured oligodendrocytes also revealed patched colocalization of MBP and PI(4,5)P 2, indicating the spatial clustering of PI(4,5)P 2 in the plasma membrane. On the basis of these findings as well as the overwhelming convergence of functional properties, modifying enzymes, and interaction partners, we propose that MBP is mechanistically related to GAP-43, MARCKS, and CAP-23. During myelinogenesis, it may mediate calcium and phosphorylation-sensitive plasma membrane availability of PI(4,5)P 2. This regulation of PI(4,5)P 2 availability at the cell cortex may be coupled to the elaboration and outgrowth of the membranous cellular processes by oligodendrocytes.

Solution NMR and CD spectroscopy of an intrinsically disordered, peripheral membrane protein: evaluation of aqueous and membrane-mimetic solvent conditions for studying the conformational adaptability of the 18.5 kDa isoform of myelin basic protein (MBP)

The stability and secondary structure propensity of recombinant murine 18.5 kDa myelin basic protein (rmMBP, 176 residues) was assessed using circular dichroic and nuclear magnetic resonance spectroscopy (1H-15N HSQC experiments) to determine the optimal sample conditions for further NMR studies (i.e., resonance assignments and protein-protein interactions). Six solvent conditions were selected based on their ability to stabilise the protein, and their tractability to currently standard solution NMR methodology. Selected solvent conditions were further characterised as functions of concentration, temperature, and pH. The results of these trials indicated that 30% TFE-d2 in H2O (v/v), pH 6.5 at 300 K, and 100 mM KCl, pH 6.5 at 277 K were the best conditions to use for future solution NMR studies of MBP. Micelles of DPC were found to be inappropriate for backbone resonance assignments of rmMBP in this instance.

Binding of the Proline-Rich Segment of Myelin Basic Protein to SH3 Domains: Spectroscopic, Microarray, and Modeling Studies of Ligand Conformation and Effects of Posttranslational Modifications

Myelin basic protein (MBP) is a multifunctional protein involved in maintaining the stability and integrity of the myelin sheath by a variety of interactions with membranes and with cytoskeletal and other proteins. A central segment of MBP is highly conserved in mammals and consists of a membrane surface-associated amphipathic alpha-helix, immediately followed by a proline-rich segment that we hypothesize is an SH3 ligand. We show by circular dichroic spectroscopy that this proline-rich segment forms a polyproline type II helix in vitro under physiological conditions and that phosphorylation at a constituent threonyl residue has a stabilizing effect on its conformation. Using SH3 domain microarrays, we observe that the unmodified recombinant murine 18.5 kDa MBP isoform (rmC1 component) binds the following SH3 domains: Yes1 > PSD95 > cortactin = PexD = Abl = Fyn = c-Src = Itk in order of decreasing affinity. A quasi-deiminated form of the protein (rmC8) binds the SH3 domains Yes1 > Fyn > cortactin = c-Src > PexD = Abl. Phosphorylation of rmC1 at 1-2 threonines within the proline-rich segment by mitogen-activated protein kinase in vitro has no effect on the binding specificity to the SH3 domains on the array. An SH3 domain of chicken Fyn is also demonstrated to bind to lipid membrane-associated C1, phosphorylated C1, and rmC8. Molecular docking simulations of the interaction of the putative SH3 ligand of classic MBP with the human Fyn SH3 domain indicate that the strength of the interaction is of the same order of magnitude as with calmodulin and that the molecular recognition and association is mediated by some weak CH...pi interactions between the ligand prolyl residues and the aromatic ones of the SH3 binding site. One such interaction is well-conserved and involves the stacking of an MBP-peptide prolyl and an SH3 domain tryptophanyl residue, as in most other SH3-ligand complexes. Lysyl and arginyl residues in the peptide canonically interact via salt bridges and cation-pi interactions with negatively charged and aromatic residues in the SH3 domain binding site. Posttranslational modifications (phosphorylation or methylation) of the ligand cause noticeable shifts in the conformation of the flexible peptide and its side chains but do not predict any major inhibition of the binding beyond somewhat less favorable interactions for peptides with phosphorylated seryl or threonyl residues.

Solid-state NMR spectroscopy of 18.5 kDa myelin basic protein reconstituted with lipid vesicles: Spectroscopic characterisation and spectral assignments of solvent-exposed protein fragments

Myelin basic protein (MBP, 18.5 kDa isoform) is a peripheral membrane protein that is essential for maintaining the structural integrity of the multilamellar myelin sheath of the central nervous system. Reconstitution of the most abundant 18.5 kDa MBP isoform with lipid vesicles yields an aggregated assembly mimicking the protein's natural environment, but which is not amenable to standard solution NMR spectroscopy. On the other hand, the mobility of MBP in such a system is variable, depends on the local strength of the protein–lipid interaction, and in general is of such a time scale that the dipolar interactions are averaged out. Here, we used a combination of solution and solid-state NMR (ssNMR) approaches: J-coupling-driven polarization transfers were combined with magic angle spinning and high-power decoupling to yield high-resolution spectra of the mobile fragments of 18.5 kDa murine MBP in membrane-associated form. To partially circumvent the problem of short transverse relaxation, we implemented three-dimensional constant-time correlation experiments (NCOCX, NCACX, CONCACX, and CAN(CO)CX) that were able to provide interresidue and intraresidue backbone correlations. These experiments resulted in partial spectral assignments for mobile fragments of the protein. Additional nuclear Overhauser effect spectroscopy (NOESY)-based experiments revealed that the mobile fragments were exposed to solvent and were likely located outside the lipid bilayer, or in its hydrophilic portion. Chemical shift index analysis showed that the fragments were largely disordered under these conditions. These combined approaches are applicable to ssNMR investigations of other peripheral membrane proteins reconstituted with lipids.

The Microtubule-Associated Protein Tumor Overexpressed Gene/Cytoskeleton-Associated Protein 5 Is Necessary for Myelin Basic Protein Expression in Oligodendrocytes

Tumor overexpressed gene (TOG) protein, encoded by cytoskeleton-associated protein CKAP5, is a microtubule-associated protein that binds to heterogeneous nuclear ribonucleoprotein (hnRNP) A2. hnRNP A2 is an RNA trafficking factor that associates with myelin basic protein (MBP) mRNA. In oligodendrocytes, TOG, hnRNP A2, and MBP mRNA colocalize in granules that assemble in the perikaryon and are transported to the peripheral network of processes that extends from it. MBP accumulates preferentially in the membrane of the medial and distal portions of these cellular processes. MBP expression was reduced when TOG level was lowered by short-hairpin (sh) RNA. The reduction in TOG did not affect overall cell morphology or the assembly, transport, localization, or number of MBP mRNA-containing granules. Reduced levels of TOG did not affect another oligodendrocyte-specific component, myelin oligodendrocyte glycoprotein, which is expressed at the same time as MBP but translated from mRNA localized in the cell body. Expression in a neural cell line of a green fluorescent protein (GFP)-MBP fusion protein derived from a construct containing GFP and the full-length cDNA for the rat 14 kDa MBP was reduced when TOG level was lowered by shRNA treatment. Expression of GFP, derived from GFP mRNA containing the hnRNP A2 binding element of MBP mRNA, was similarly reduced in cells with low TOG levels. These data indicate that TOG is necessary for efficient translation of MBP mRNA and suggest that this role is mediated by its interaction with hnRNP A2.