Graham S.T. Smith

Divalent cations induce a compaction of intrinsically disordered myelin basic protein.

Central nervous system myelin is a dynamic entity arising from membrane processes extended from oligodendrocytes, which form a tightly-wrapped multilamellar structure around neurons. In mature myelin, the predominant splice isoform of classic MBP is 18.5kDa. In solution, MBP is an extended, intrinsically disordered protein with a large effective protein surface for myriad interactions, and possesses transient and/or induced ordered secondary structure elements for molecular association or recognition. Here, we show by nanopore analysis that the divalent cations copper and zinc induce a compaction of the extended protein in vitro, suggestive of a tertiary conformation that may reflect its arrangement in myelin.

Proline substitutions and threonine pseudophosphorylation of the SH3 ligand of 18.5‐kDa myelin basic protein decrease its affinity for the Fyn‐SH3 domain and alter process development and protein localization in oligodendrocytes

The developmentally regulated myelin basic proteins (MBPs), which arise from the golli (gene of oligodendrocyte lineage) complex, are highly positively charged, intrinsically disordered, multifunctional proteins having several alternatively spliced isoforms and posttranslational modifications, and they play key roles in myelin compaction. The classic 18.5‐kDa MBP isoform has a proline‐rich region comprising amino acids 92–99 (murine sequence –T92PRTPPPS99–) that contains a minimal SH3 ligand domain. We have previously shown that 18.5‐kDa MBP binds to several SH3 domains, including that of Fyn, a member of the Src family of tyrosine kinases involved in a number of signaling pathways during CNS development. To determine the physiological role of this binding as well as the role of phosphorylation of Thr92 and Thr95, in the current study we have produced several MBP variants specifically targeting phosphorylation sites and key structural regions of MBPs SH3 ligand domain. Using isothermal titration calorimetry, we have demonstrated that, compared with the wild‐type protein, these variants have lower affinity for the SH3 domain of Fyn. Moreover, overexpression of N‐terminal‐tagged GFP versions in immortalized oligodendroglial N19 and N20.1 cell cultures results in aberrant elongation of membrane processes and increased branching complexity and inhibits the ability of MBP to decrease Ca2+ influx. Phosphorylation of Thr92 can also cause MBP to traffic to the nucleus, where it may participate in additional protein–protein interactions. Coexpression of MBP with a constitutively active form of Fyn kinase resulted in membrane process elaboration, a phenomenon that was abolished by point amino acid substitutions in MBPs SH3 ligand domain. These results suggest that MBPs SH3 ligand domain plays a key role in intracellular protein interactions in vivo and may be required for proper membrane elaboration of developing oligodendrocytes and, further, that phosphorylation of Thr92 and Thr95 can regulate this function.

Classical 18.5‐and 21.5‐kDa isoforms of myelin basic protein inhibit calcium influx into oligodendroglial cells, in contrast to golli isoforms

The myelin basic protein (MBP) family arises from different transcription start sites of the golli (gene of oligodendrocyte lineage) complex, with further variety generated by differential splicing. The “classical” MBP isoforms are peripheral membrane proteins that facilitate compaction of the mature myelin sheath but also have multiple protein interactions. The early developmental golli isoforms have previously been shown to promote process extension and enhance Ca2+ influx into primary and immortalized oligodendrocyte cell lines. Here, we have performed similar studies with the classical 18.5‐ and 21.5‐kDa isoforms of MBP. In contrast to golli proteins, overexpression of classical MBP isoforms significantly reduces Ca2+ influx in the oligodendrocyte cell line N19 as well as in primary cultures of oligodendroglial progenitor cells. Pharmacological experiments demonstrate that this effect is mediated by voltage‐operated Ca2+ channels (VOCCs) and not by ligand‐gated Ca2+ channels or Ca2+ release from intracellular stores. The pseudo‐deiminated 18.5‐kDa and the full‐length 21.5‐kDa isoforms do not reduce Ca2+ influx as much as the unmodified 18.5‐kDa isoform. However, more efficient membrane localization (of overexpressed, pseudo‐deiminated 18.5‐kDa and 21.5‐kDa isoforms of classical MBP containing the 21‐nt 3′‐untranslated region transit signal) further reduces the Ca2+ response after plasma membrane depolarization, suggesting that binding of classical MBP isoforms to the plasma membrane is important for modulation of Ca2+ homeostasis. Furthermore, we have found that the mature 18.5‐kDa isoform expressed in oligodendrocytes colocalizes with VOCCs, particularly at the leading edge of extending membrane processes. In summary, our findings suggest a key role for classical MBP proteins in regulating voltage‐gated Ca2+ channels at the plasma membrane of oligodendroglial cells and thus also in regulation of multiple developmental stages in this cell lineage.

Structured Functional Domains of Myelin Basic Protein: Cross Talk between Actin Polymerization and Ca2+-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. Solution NMR spectroscopy and a hydrophobic moment analysis of MBPs amino-acid sequence have previously revealed three regions with high propensity to form strongly amphipathic α-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 cytoskeletal proteins, Src homology 3-domain-containing proteins, Ca(2+)-activated calmodulin (Ca(2+)-CaM), and 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) (which are denoted α1, α2, and α3, respectively). We used a variety of biophysical methods (circular dichroism spectroscopy, isothermal titration calorimetry, transmission electron microscopy, fluorimetry, and solution NMR spectroscopy and chemical shift index analysis) to characterize the interactions of these peptides with actin and Ca(2+)-CaM. Our results show that all three peptides can adopt α-helical structure inherently even in aqueous solution. Both α1- and α3-peptides showed strong binding with Ca(2+)-CaM, and both adopted an α-helical conformation upon interaction, but the binding of the α3-peptide appeared to be more dynamic. Only the α1-peptide exhibited actin polymerization and bundling activity, and the addition of Ca(2+)-CaM resulted in depolymerization of actin that had been polymerized by α1. The results of this study proved that there is an N-terminal binding domain in MBP for Ca(2+)-CaM (in addition to the primary site located in the C-terminus), and that it is sufficient for CaM-induced actin depolymerization. These three domains of MBP represent molecular recognition fragments with multiple roles in both membrane- and protein-association.

Phosphorylation of Thellungiella salsuginea Dehydrins TsDHN-1 and TsDHN-2 Facilitates Cation-Induced Conformational Changes and Actin Assembly

Group 2 late embryogenesis abundant (LEA) proteins, also known as dehydrins, are intrinsically disordered proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperatures. These proteins are characterized by the presence of at least one conserved, lysine-rich K-segment and sometimes by one or more serine-rich S-segments that are phosphorylated. Dehydrins may stabilize proteins and membrane structures during environmental stress and can sequester and scavenge metal ions. Here, we investigate how the conformations of two dehydrins from Thellungiella salsuginea, denoted as TsDHN-1 (acidic) and TsDHN-2 (basic), are affected by pH, interactions with cations and membranes, and phosphorylation. Both TsDHN-1 and TsDHN-2 were expressed as SUMO fusion proteins for in vitro phosphorylation by casein kinase II (CKII), and structural analysis by circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy. We show that the polyproline II conformation can be induced in the dehydrins by their environmental conditions, including changes in the concentration of divalent cations such as Ca(2+). The assembly of actin by these dehydrins was assessed by sedimentation assays and viewed by transmission electron and atomic force microscopy. Phosphorylation allowed both dehydrins to polymerize actin filaments. These results support the hypothesis that dehydrins stabilize the cytoskeleton under stress conditions and further that phosphorylation may be an important feature of this stabilization.

Classic 18.5- and 21.5-kDa Myelin Basic Protein Isoforms Associate with Cytoskeletal and SH3-Domain Proteins in the Immortalized N19-Oligodendroglial Cell Line Stimulated by Phorbol Ester and IGF-1

The 18.5-kDa classic myelin basic protein (MBP) is an intrinsically disordered protein arising from the Golli (Genes of Oligodendrocyte Lineage) gene complex and is responsible for compaction of the myelin sheath in the central nervous system. This MBP splice isoform also has a plethora of post-translational modifications including phosphorylation, deimination, methylation, and deamidation, that reduce its overall net charge and alter its protein and lipid associations within oligodendrocytes (OLGs). It was originally thought that MBP was simply a structural component of myelin; however, additional investigations have demonstrated that MBP is multi-functional, having numerous protein-protein interactions with Ca2+-calmodulin, actin, tubulin, and proteins with SH3-domains, and it can tether these proteins to a lipid membrane in vitro. Here, we have examined cytoskeletal interactions of classic 18.5-kDa MBP, in vivo, using early developmental N19-OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). We show that MBP redistributes to distinct ‘membrane-ruffled’ regions of the plasma membrane where it co-localizes with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. Moreover, using phospho-specific antibody staining, we show an increase in phosphorylated Thr98 MBP (human sequence numbering) in membrane-ruffled OLGs. Previously, Thr98 phosphorylation of MBP has been shown to affect its conformation, interactions with other proteins, and tethering of other proteins to the membrane in vitro. Here, MBP and actin were also co-localized in new focal adhesion contacts induced by IGF-1 stimulation in cells grown on laminin-2. This study supports a role for classic MBP isoforms in cytoskeletal and other protein-protein interactions during membrane and cytoskeletal remodeling in OLGs.

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.

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.

The 21.5-kDa isoform of myelin basic protein has a non-traditional PY-nuclear-localization signal.

The predominant 18.5-kDa classic myelin basic protein (MBP) is mainly responsible for compaction of the myelin sheath in the central nervous system, but is multifunctional, having numerous interactions with Ca(2+)-calmodulin, actin, tubulin, and SH3-domains, and can tether these proteins to a lipid membrane in vitro. The full-length 21.5-kDa MBP isoform has an additional 26 residues encoded by exon-II of the classic gene, which causes it to be trafficked to the nucleus of oligodendrocytes (OLGs). We have performed site-directed mutagenesis of selected residues within this segment in red fluorescent protein (RFP)-tagged constructs, which were then transfected into the immortalized N19-OLG cell line to view protein localization using epifluorescence microscopy. We found that 21.5-kDa MBP contains two non-traditional PY-nuclear-localization signals, and that arginine and lysine residues within these motifs were involved in subcellular trafficking of this protein to the nucleus, where it may have functional roles during myelinogenesis.

Zinc induces disorder-to-order transitions in free and membrane-associated Thellungiella salsuginea dehydrins TsDHN-1 and TsDHN-2: a solution CD and solid-state ATR-FTIR study

Dehydrins are intrinsically unstructured proteins that are expressed in plants experiencing extreme environmental conditions such as drought or low temperature. Although their role is not completely understood, it has been suggested that they stabilize proteins and membrane structures during environmental stress and also sequester metals such as zinc. Here, we investigate two dehydrins (denoted as TsDHN-1 and TsDHN-2) from Thellungiella salsuginea. This plant is a crucifer that thrives in the Canadian sub-Arctic (Yukon Territory) where it grows on saline-rich soils and experiences periods of both extreme cold and drought. We show using circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopy that ordered secondary structure is induced and stabilized in these proteins, both in free and vesicle-bound form, by association with zinc. In membrane-associated form, both proteins have an increased proportion of β-strand conformation induced by the cation, in addition to the amphipathic α-helices formed by their constituent K-segments. These results support the hypothesis that dehydrins stabilize plant plasma and organellar membranes in conditions of stress, and further that zinc may be an important co-factor in stabilization. Whereas dehydrins in the cytosol of a plant cell undergoing dehydration or temperature stress form bulk hydrogels and remain primarily disordered, dehydrins with specific membrane- or protein-associations will have induced ordered secondary structures.