Niche-dependent inhibition of neural stem cell proliferation and oligodendrogenesis is mediated by the presence of myelin basic protein.

Neural stem and progenitor cells (collectively termed neural precursor cells [NPCs]) are found along the ventricular neuraxis extending from the spinal cord to the forebrain in regionally distinct niches comprised of different cell types, architecture, and cell-cell interactions. An understanding of the factors that regulate NPC behavior is critical for developing therapeutics to repair the injured central nervous system. Herein, we demonstrate that myelin basic protein (MBP), the major cytoplasmic protein constituent of the myelin sheath in oligodendrocytes, can regulate NPC behavior. Under physiological conditions, NPCs are not in contact with intracellular MBP; however, upon injury, MBP is released into the neural parenchyma. We reveal that MBP presented in a spinal cord niche is inhibitory to NPC proliferation. This inhibitory effect is regionally distinct as spinal cord NPCs, but not forebrain-derived NPCs, are inhibited by MBP. We performed coculture and conditioned media experiments that reveal the stem cell niche is a key regulator of MBP's inhibitory actions on NPCs. The inhibition is mediated by a heat-labile protein released by spinal cord niche cells, but not forebrain niche cells. However, forebrain NPCs are also inhibited by the spinal cord derived factor as revealed following in vivo infusion of the spinal cord niche-derived conditioned media. Moreover, we show that MBP inhibits oligodendrogenesis from NPCs. Together, these findings highlight the role of MBP and the regionally distinct microenvironment in regulating NPC behavior which has important implications for stem cell-based regenerative strategies.

Interaction of Myelin Basic Protein with Myelin-like Lipid Monolayers at Air-Water Interface.

Interaction of myelin basic protein (MBP) and the cytoplasmic leaflets of the oligodendrocyte membrane is essential for the formation and compaction of the myelin sheath of the central nervous system and is altered aberrantly and implicated in the pathogenesis of neurodegenerative diseases like multiple sclerosis. To gain more detailed insights into this interaction, the adsorption of MBP to model lipid monolayers of similar composition to the myelin of the central nervous system was studied at the air-water interface with monolayer adsorption experiments. Measuring the surface pressure and the related maximum insertion pressure of MBP for different myelin-like lipid monolayers provided information about the specific role of each of the single lipids in the myelin. Depending on the ratio of negatively charged lipids to uncharged lipids and the distance between charges, the adsorption process was found to be determined by two counteracting effects: (i) protein incorporation, resulting in an increasing surface pressure and (ii) lipid condensation due to electrostatic interaction between the positively charged protein and negatively charged lipids, resulting in a decreasing surface pressure. Although electrostatic interactions led to high insertion pressures, the associated lipid condensation lowered the fluidity of the myelin-like monolayer.

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 et al. 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.

Substitutions mimicking deimination and phosphorylation of 18.5-kDa myelin basic protein exert local structural effects that subtly influence its global folding.

Intrinsically-disordered proteins (IDPs) present a complex interplay of conformational variability and multifunctionality, modulated by environment and post-translational modifications. The 18.5-kDa myelin basic protein (MBP) is essential to the formation of the myelin sheath of the central nervous system and is exemplary in this regard. We have recently demonstrated that the unmodified MBP-C1 component undergoes co-operative global conformational changes in increasing concentrations of trifluoroethanol, emulating the decreasing dielectric environment that the protein encounters upon adsorption to the oligodendrocyte membrane [K.A. Vassall et al., Journal of Molecular Biology, 427, 1977-1992, 2015]. Here, we extended this study to the pseudo-deiminated MBP-C8 charge component, one found in greater proportion in developing myelin and in multiple sclerosis. A similar tri-conformational distribution as for MBP-C1 was observed with slight differences in Gibbs free energy. A more dramatic difference was observed by cathepsin D digestion of the protein in both aqueous and membrane environments, which showed significantly greater accessibility of the F42-F43 cut site of MBP-C8, indicative of a global conformational change. In contrast, this modification caused little change in the protein's density of packing on myelin-mimetic membranes as ascertained by double electron-electron resonance spectroscopy [D.R. Kattnig et al., Biochimica et Biophysica Acta (Biomembranes), 1818, 2636-2647, 2012], or in its affinity for Ca(2+)-CaM. Site-specific threonyl pseudo-phosphorylation at residues T92 and/or T95 did not appreciably affect any of the thermodynamic mechanisms of conformational transitions, susceptibility to cathepsin D, or affinity for Ca(2+)-CaM, despite previously having been shown to affect local structure and disposition on the membrane surface.

Docking and molecular dynamics simulations of the Fyn-SH3 domain with free and phospholipid bilayer-associated 18.5-kDa myelin basic protein (MBP)-Insights into a noncanonical and fuzzy interaction.

The molecular details of the association between the human Fyn-SH3 domain, and the fragment of 18.5-kDa myelin basic protein (MBP) spanning residues S38-S107 (denoted as xα2-peptide, murine sequence numbering), were studied in silico via docking and molecular dynamics over 50-ns trajectories. The results show that interaction between the two proteins is energetically favorable and heavily dependent on the MBP proline-rich region (P93-P98) in both aqueous and membrane environments. In aqueous conditions, the xα2-peptide/Fyn-SH3 complex adopts a "sandwich""-like structure. In the membrane context, the xα2-peptide interacts with the Fyn-SH3 domain via the proline-rich region and the ß-sheets of Fyn-SH3, with the latter wrapping around the proline-rich region in a form of a clip. Moreover, the simulations corroborate prior experimental evidence of the importance of upstream segments beyond the canonical SH3-ligand. This study thus provides a more-detailed glimpse into the context-dependent interaction dynamics and importance of the ß-sheets in Fyn-SH3 and proline-rich region of MBP. Proteins 2017; 85:1336-1350. © 2017 Wiley Periodicals, Inc.

Correlation of geographic distributions of haptoglobin alleles with prevalence of multiple sclerosis (MS) - a narrative literature review.

We have proposed that the myelin damage observed in multiple sclerosis (MS) may be partly mediated through the long-term release and degradation of extracellular hemoglobin (Hb) and the products of its oxidative degradation [Cellular and Molecular Life Sciences, 71, 1789-1798, 2014]. The protein haptoglobin (Hpt) binds extracellular Hb as a first line of defense, and can serve as a vascular antioxidant. Humans have two different Hpt alleles: Hpt1 and Hpt2, giving either homozygous Hpt1-1 or Hpt2-2 phenotypes, or a heterozygous Hpt1-2 phenotype. We questioned whether those geographic regions with higher frequency of the Hpt2 allele (conversely, lower frequency of Hpt1 allele) would correlate with an increased incidence of MS, because different Hpt phenotypes will have variable anti-oxidative potentials in protecting myelin from damage inflicted by extracellular Hb and its degradation products. To test this hypothesis, we undertook a systematic analysis of the literature on reported geographic distributions of Hpt alleles to compare them with data reported in the World Health Organization Atlas of worldwide MS prevalence. We found the frequency of the Hpt1 allele to be low in European and North American countries with a high prevalence of MS, consistent with our hypothesis. However, this correlation was not observed in China and India, countries with the lowest Hpt1 frequencies, yet low reported prevalence of MS. Nevertheless, this work shows the need for continued refinement of geographic patterns of MS prevalence, including data on ethnic or racial origin, and for new clinical studies to probe the observed correlation and evaluate Hpt phenotype as a predictor of disease variability and progression, severity, and/or comorbidity with cardiovascular disorders.

In vitro study of the direct effect of extracellular hemoglobin on myelin components.

There is a relationship between cerebral vasculature and multiple sclerosis (MS) lesions: abnormal accumulations of iron have been found in the walls of dilated veins in MS plaques. The sources of this iron can be varied, but capillary and venous hemorrhages leading to blood extravasation have been recorded, and could result in the release of hemoglobin extracellularly. Extracellular hemoglobin oxidizes quickly and is known to become a reactive molecule that triggers low-density lipoprotein oxidation and plays a pivotal role in atherogenesis. In MS, it could lead to local oxidative stress, inflammation, and tissue damage. Here, we investigated whether extracellular hemoglobin and its breakdown products can cause direct oxidative damage to myelin components in a peroxidative environment such as occurs in inflamed tissue. Oxidation of lipids was assessed by the formation of fluorescent peroxidized lipid-protein covalent adducts, by the increase in conjugated diene and malondialdehyde. Oxidation of proteins was analyzed by the change in protein mass. The results suggest that the globin radical could be a trigger of myelin basic protein oxidative cross-linking, and that heme transferred to the lipids is involved in lipid peroxidation. This study provides new insight into the mechanism by which hemoglobin exerts its pathological oxidative activity towards myelin components. This work supports further research into the vascular pathology in MS, to gain insight into the origin and role of iron deposits in disease pathogenesis, or in stimulation of different comorbidities such as cardiovascular disease.

MyelStones: the executive roles of myelin basic protein in myelin assembly and destabilization in multiple sclerosis.

The classic isoforms of myelin basic protein (MBP, 14-21.5 kDa) are essential to formation of the multilamellar myelin sheath of the mammalian central nervous system (CNS). The predominant 18.5-kDa isoform links together the cytosolic surfaces of oligodendrocytes, but additionally participates in cytoskeletal turnover and membrane extension, Fyn-mediated signalling pathways, sequestration of phosphoinositides and maintenance of calcium homoeostasis. All MBP isoforms are intrinsically disordered proteins (IDPs) that interact via molecular recognition fragments (MoRFs), which thereby undergo local disorder-to-order transitions. Their conformations and associations are modulated by environment and by a dynamic barcode of post-translational modifications, particularly phosphorylation by mitogen-activated and other protein kinases and deimination [a hallmark of demyelination in multiple sclerosis (MS)]. The MBPs are thus to myelin what basic histones are to chromatin. Originally thought to be merely structural proteins forming an inert spool, histones are now known to be dynamic entities involved in epigenetic regulation and diseases such as cancer. Analogously, the MBPs are not mere adhesives of compact myelin, but active participants in oligodendrocyte proliferation and in membrane process extension and stabilization during myelinogenesis. A central segment of these proteins is pivotal in membrane-anchoring and SH3 domain (Src homology 3) interaction. We discuss in the present review advances in our understanding of conformational conversions of this classic basic protein upon membrane association, including new thermodynamic analyses of transitions into different structural ensembles and how a shift in the pattern of its post-translational modifications is associated with the pathogenesis and potentially onset of demyelination in MS.

Myelin basic protein cleaves cell adhesion molecule L1 and promotes neuritogenesis and cell survival.

The cell adhesion molecule L1 is a Lewis(x)-carrying glycoprotein that plays important roles in the developing and adult nervous system. Here we show that myelin basic protein (MBP) binds to L1 in a Lewis(x)-dependent manner. Furthermore, we demonstrate that MBP is released by murine cerebellar neurons as a sumoylated dynamin-containing protein upon L1 stimulation and that this MBP cleaves L1 as a serine protease in the L1 extracellular domain at Arg(687) yielding a transmembrane fragment that promotes neurite outgrowth and neuronal survival in cell culture. L1-induced neurite outgrowth and neuronal survival are reduced in MBP-deficient cerebellar neurons and in wild-type cerebellar neurons in the presence of an MBP antibody or L1 peptide containing the MBP cleavage site. Genetic ablation of MBP in shiverer mice and mutagenesis of the proteolytically active site in MBP or of the MBP cleavage site within L1 as well as serine protease inhibitors and an L1 peptide containing the MBP cleavage site abolish generation of the L1 fragment. Our findings provide evidence for novel functions of MBP in the nervous system.

Identification of multiple phosphorylation sites on maize endosperm starch branching enzyme IIb, a key enzyme in amylopectin biosynthesis.

Starch branching enzyme IIb (SBEIIb) plays a crucial role in amylopectin biosynthesis in maize endosperm by defining the structural and functional properties of storage starch and is regulated by protein phosphorylation. Native and recombinant maize SBEIIb were used as substrates for amyloplast protein kinases to identify phosphorylation sites on the protein. A multidisciplinary approach involving bioinformatics, site-directed mutagenesis, and mass spectrometry identified three phosphorylation sites at Ser residues: Ser(649), Ser(286), and Ser(297). Two Ca(2+)-dependent protein kinase activities were partially purified from amyloplasts, termed K1, responsible for Ser(649) and Ser(286) phosphorylation, and K2, responsible for Ser(649) and Ser(297) phosphorylation. The Ser(286) and Ser(297) phosphorylation sites are conserved in all plant branching enzymes and are located at opposite openings of the 8-stranded parallel ß-barrel of the active site, which is involved with substrate binding and catalysis. Molecular dynamics simulation analysis indicates that phospho-Ser(297) forms a stable salt bridge with Arg(665), part of a conserved Cys-containing domain in plant branching enzymes. Ser(649) conservation appears confined to the enzyme in cereals and is not universal, and is presumably associated with functions specific to seed storage. The implications of SBEIIb phosphorylation are considered in terms of the role of the enzyme and the importance of starch biosynthesis for yield and biotechnological application.

Regulation of cell proliferation by nucleocytoplasmic dynamics of postnatal and embryonic exon-II-containing MBP isoforms.

The only known structural protein required for formation of myelin, produced by oligodendrocytes in the central nervous system, is myelin basic protein (MBP). This peripheral membrane protein has different developmentally-regulated isoforms, generated by alternative splicing. The isoforms are targeted to distinct subcellular locations, which is governed by the presence or absence of exon-Il, although their functional expression is often less clear. Here, we investigated the role of exon-Il-containing MBP isoforms and their link with cell proliferation. Live-cell imaging and FRAP analysis revealed a dynamic nucleocytoplasmic translocation of the exon-II-containing postnatal 21.5-kDa MBP isoform upon mitogenic modulation. Its nuclear export was blocked upon treatment with leptomycin B, an inhibitor of nuclear protein export. Next to the postnatal MBP isoforms, embryonic exon-II-containing MBP (e-MBP) is expressed in primary (immature) oligodendrocytes. The e-MBP isoform is exclusively present in OLN-93 cells, a rat-derived oligodendrocyte progenitor cell line, and interestingly, also in several non-CNS cell lines. As seen for postnatal MBPs, a similar nucleocytoplasmic translocation upon mitogenic modulation was observed for e-MBP. Thus, upon serum deprivation, e-MBP was excluded from the nucleus, whereas re-addition of serum re-established its nuclear localization, with a concomitant increase in proliferation. Knockdown of MBP by shRNA confirmed a role for e-MBP in OLN-93 proliferation, whereas the absence of e-MBP similarly reduced the proliferative capacity of non-CNS cell lines. Thus, exon-Il-containing MBP isoforms may regulate cell proliferation via a mechanism that relies on their dynamic nuclear import and export, which is not restricted to the oligodendrocyte lineage.

Proton detection for signal enhancement in solid-state NMR experiments on mobile species in membrane proteins.

Direct proton detection is becoming an increasingly popular method for enhancing sensitivity in solid-state nuclear magnetic resonance spectroscopy. Generally, these experiments require extensive deuteration of the protein, fast magic angle spinning (MAS), or a combination of both. Here, we implement direct proton detection to selectively observe the mobile entities in fully-protonated membrane proteins at moderate MAS frequencies. We demonstrate this method on two proteins that exhibit different motional regimes. Myelin basic protein is an intrinsically-disordered, peripherally membrane-associated protein that is highly flexible, whereas Anabaena sensory rhodopsin is composed of seven rigid transmembrane α-helices connected by mobile loop regions. In both cases, we observe narrow proton linewidths and, on average, a 10× increase in sensitivity in 2D insensitive nuclear enhancement of polarization transfer-based HSQC experiments when proton detection is compared to carbon detection. We further show that our proton-detected experiments can be easily extended to three dimensions and used to build complete amino acid systems, including sidechain protons, and obtain inter-residue correlations. Additionally, we detect signals which do not correspond to amino acids, but rather to lipids and/or carbohydrates which interact strongly with membrane proteins.

Myelin basic protein is a glial microtubule-associated protein -- characterization of binding domains, kinetics of polymerization, and regulation by phosphorylation and a lipidic environment.

The 18.5-kDa splice isoform of myelin basic protein (MBP) predominates in the adult brain, adhering the cytoplasmic leaflets of the oligodendrocyte membrane together, but also assembling the cytoskeleton at leading edges of membrane processes. Here, we characterized MBP's role as a microtubule-assembly protein (MAP). Using light scattering and sedimentation assays we found that pseudo-phosphorylation of Ser54 (murine 18.5-kDa sequence) significantly enhanced the rate but not the final degree of polymerization. This residue lies within a short KPGSG motif identical to one in tau, a ubiquitous MAP important in neuronal microtubule assembly. Using polypeptide constructs, each comprising one of three major amphipathic α-helical molecular recognition fragments of 18.5-kDa MBP, we identified the N-terminal α1-peptide as sufficient to cause microtubule polymerization, the rate of which was significantly enhanced in the presence of dodecylphosphocholine (DPC) micelles to mimic a lipidic environment.

Hemoglobin as a source of iron overload in multiple sclerosis: does multiple sclerosis share risk factors with vascular disorders?

Although iron is known to be essential for the normal development and health of the central nervous system, abnormal iron deposits are found in and around multiple sclerosis (MS) lesions that themselves are closely associated with the cerebral vasculature. However, the origin of this excess iron is unknown, and it is not clear whether this is one of the primary causative events in the pathogenesis of MS, or simply another consequence of the long-lasting inflammatory conditions. Here, applying a systems biology approach, we propose an additional way for understanding the neurodegenerative component of the disease caused by chronic subclinical extravasation of hemoglobin, in combination with multiple other factors including, but not limited to, dysfunction of different cellular protective mechanisms against extracellular hemoglobin reactivity and oxidative stress. Moreover, such considerations could also shed light on and explain the higher susceptibility of MS patients to a wide range of cardiovascular disorders.

Interactions of Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2 with membranes at cold and ambient temperatures-surface morphology and single-molecule force measurements show phase separation, and reveal tertiary and quaternary associations.

Dehydrins (group 2 late embryogenesis abundant proteins) are intrinsically-disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Their roles include stabilizing cellular proteins and membranes, and sequestering metal ions. Here, we investigate the membrane interactions of the acidic dehydrin TsDHN-1 and the basic dehydrin TsDHN-2 derived from the crucifer Thellungiella salsuginea that thrives in the Canadian sub-Arctic. We show using compression studies with a Langmuir-Blodgett trough that both dehydrins can stabilize lipid monolayers with a lipid composition mimicking the composition of the plant outer mitochondrial membrane, which had previously been shown to induce ordered secondary structures (disorder-to-order transitions) in the proteins. Ellipsometry of the monolayers during compression showed an increase in monolayer thickness upon introducing TsDHN-1 (acidic) at 4°C and TsDHN-2 (basic) at room temperature. Atomic force microscopy of supported lipid bilayers showed temperature-dependent phase transitions and domain formation induced by the proteins. These results support the conjecture that acidic dehydrins interact with and potentially stabilize plant outer mitochondrial membranes in conditions of cold stress. Single-molecule force spectroscopy of both proteins pulled from supported lipid bilayers indicated the induced formation of tertiary conformations in both proteins, and potentially a dimeric association for TsDHN-2.

Regulatory effect of the glial Golli-BG21 protein on the full-length murine small C-terminal domain phosphatase (SCP1, or Golli-interacting protein).

The gene in the oligodendrocyte lineage (golli) encodes a number of proteins essential for myelination, comprising Golli and classic isoforms that are expressed in a developmentally-regulated manner. The Golli-interacting-protein (GIP) was previously discovered in a search for potential interacting partners of the Golli-isoform BG21, and was realised to be an acidic phosphatase belonging to the family of RNA-polymerase-2, small-subunit, C-terminal phosphatases (viz., SCP1). Here, we refer to this protein as mSCP1/GIP. In subsequent in vitro studies of recombinant murine SCP1/GIP, the inability to produce an active full-length version of the protein under native conditions necessitated the study of a truncated form ΔN-rmSCP1/GIP, but with inconclusive results regarding its interaction with BG21 [13]. We have since developed a new SUMO-expression and purification protocol for the preparation of a functional, full-length mGIP/SCP1, with no additional purification tags. Here, the interaction between mSCP1/GIP (with intact N-terminus) and BG21 is shown to be different than for the truncation mutant studied previously. Specifically, this interaction shows a dual effect on the enzymatic activity of mSCP1/GIP by BG21: BG21 enhanced mSCP1/GIP phosphatase activity (Ka = 30 µM), whereas PKCα-phosphorylated BG21 inhibited its activity (Ki = 2.9 µM), suggesting a potential role of BG21 as a molecular switch ("quick-brake mechanism") on mSCP1/GIP. The successful production of an active, full-length mSCP1/GIP thus demonstrates a role for its N-terminus in regulation of phosphatase activity, in events such as the regulation of transcription in oligodendrocytes.

Over-expression in E. coli and purification of functional full-length murine small C-terminal domain phosphatase (SCP1, or Golli-interacting protein).

During myelination in the central nervous system, proteins arising from the gene in the oligodendrocyte lineage (golli) participate in diverse events in signal transduction and gene regulation. One of the interacting partners of the Golli-isoform BG21 was discovered by yeast-2-hybrid means and was denoted the Golli-interacting-protein (GIP). In subsequent in vitro studies of recombinant murine GIP, it was not possible to produce a full-length version of recombinant murine rmGIP in functional form under native conditions, primarily because of solubility issues, necessitating the study of a hexahistidine-tagged, truncated form ΔN-rmGIP. This protein is an acidic phosphatase belonging to the family of RNA-polymerase-2, small-subunit, C-terminal phosphatases (SCP1), and studies of the human ortholog hSCP1 have also been performed on truncated forms. Here, a new SUMO-expression and purification protocol has been developed for the preparation of a functional, full-length mSCP1/GIP (our nomenclature henceforth), with no additional purification tags. Both full-length mSCP1/GIP and the truncated murine form (now denoted ΔN-rmSCP1/GIP) had similar melting temperatures, indicating that the integrity of the catalytic core per se was minimally affected by the N-terminus. Characterization of mSCP1/GIP activity with the artificial substrate p-NPP (p-nitrophenylphosphate) yielded kinetic parameters comparable to those of ΔN-rmSCP1/GIP and the truncated human ortholog ΔN-hSCP1. Similarly, mSCP1/GIP dephosphorylated a more natural CTD-peptide substrate (but not protein kinase C-phosphorylated BG21) with comparable kinetics to ΔN-hSCP1. The successful production of an active, full-length mSCP1/GIP will enable future evaluation of the functional role of its N-terminus in protein-protein interactions (e.g., BG21) that regulate its phosphatase activity.

Lateral self-assembly of 18.5-kDa myelin basic protein (MBP) charge component-C1 on membranes.

Myelin basic protein (MBP), particularly the classic 18.5-kDa isoform, is a major structural protein of the myelin sheath of the central nervous system. It is an intrinsically disordered, peripheral membrane protein that shows structural polymorphism in combination with several overlapping interaction sites. Here, double electron-electron resonance (DEER) spectroscopy, in combination with a simplified, semi-quantitative analysis based on Monte Carlo simulations, is used to determine the distance distribution of murine 18.5-kDa MBP, unmodified charge component-C1, on large unilamellar vesicles of a lipid composition mimicking the cytoplasmic leaflet of myelin. Three singly spin-labeled MBP variants and a mixture of singly-labeled MBP variants are used. The MBPs, each bearing only one spin label, exhibit average intermolecular distances that are significantly shorter than the distances expected when assuming a random distribution at the employed lipid-to-protein ratios, indicating self-assembly on the membrane. The distribution of elliptical pervaded areas (hard ellipses) on a two-dimensional surface can serve as a model of the nonspecific self-assembly process. The corresponding pair correlation functions g(r) are determined from Monte Carlo simulations with variation of various parameters such as the ellipses' aspect ratios. Comparing the g(r) values with the DEER-derived distance distributions, the pervaded volume is best characterized by a nearly elliptical projection onto the membrane, with an aspect ratio of approximately 1.5, and with the longer semi-axis of approximately 1.4nm. The approach of using local information from DEER with low-resolution models derived from Monte Carlo simulations can be applied to study the lateral self-assembly properties of other protein complexes on membranes.

Myelin management by the 18.5-kDa and 21.5-kDa classic myelin basic protein isoforms.

The classic myelin basic protein (MBP) splice isoforms range in nominal molecular mass from 14 to 21.5 kDa, and arise from the gene in the oligodendrocyte lineage (Golli) in maturing oligodendrocytes. The 18.5-kDa isoform that predominates in adult myelin adheres the cytosolic surfaces of oligodendrocyte membranes together, and forms a two-dimensional molecular sieve restricting protein diffusion into compact myelin. However, this protein has additional roles including cytoskeletal assembly and membrane extension, binding to SH3-domains, participation in Fyn-mediated signaling pathways, sequestration of phosphoinositides, and maintenance of calcium homeostasis. Of the diverse post-translational modifications of this isoform, phosphorylation is the most dynamic, and modulates 18.5-kDa MBP's protein-membrane and protein-protein interactions, indicative of a rich repertoire of functions. In developing and mature myelin, phosphorylation can result in microdomain or even nuclear targeting of the protein, supporting the conclusion that 18.5-kDa MBP has significant roles beyond membrane adhesion. The full-length, early-developmental 21.5-kDa splice isoform is predominantly karyophilic due to a non-traditional P-Y nuclear localization signal, with effects such as promotion of oligodendrocyte proliferation. We discuss in vitro and recent in vivo evidence for multifunctionality of these classic basic proteins of myelin, and argue for a systematic evaluation of the temporal and spatial distributions of these protein isoforms, and their modified variants, during oligodendrocyte differentiation.

Nucleus-localized 21.5-kDa myelin basic protein promotes oligodendrocyte proliferation and enhances neurite outgrowth in coculture, unlike the plasma membrane-associated 18.5-kDa isoform.

The classic myelin basic protein (MBP) family of central nervous system (CNS) myelin arises from transcription start site 3 of the Golli (gene of oligodendrocyte lineage) complex and comprises splice isoforms ranging in nominal molecular mass from 14 kDa to (full-length) 21.5 kDa. We have determined here a number of distinct functional differences between the major 18.5-kDa and minor 21.5-kDa isoforms of classic MBP with respect to oligodendrocyte (OLG) proliferation. We have found that, in contrast to 18.5-kDa MBP, 21.5-kDa MBP increases proliferation of early developmental immortalized N19-OLGs by elevating the levels of phosphorylated ERK1/2 and Akt1 kinases and of ribosomal protein S6. Coculture of N2a neuronal cells with N19-OLGs transfected with the 21.5-kDa isoform (or conditioned medium from), but not the 18.5-kDa isoform, caused the N2a cells to have increased neurite outgrowth and process branching complexity. These roles were dependent on subcellular localization of 21.5-kDa MBP to the nucleus and on the exon II-encoded segment, suggesting that the nuclear localization of early minor isoforms of MBP may play a crucial role in regulating and/or initiating myelin and neuronal development in the mammalian CNS.