Interaction of myelin basic protein with cytoskeletal and signaling proteins in cultured primary oligodendrocytes and N19 oligodendroglial cells.

BACKGROUND: The classic myelin basic protein (MBP) isoforms are intrinsically-disordered proteins of 14-21.5 kDa in size arising from the Golli (Gene in the Oligodendrocyte Lineage) gene complex, and are responsible for formation of the multilayered myelin sheath in the central nervous system. The predominant membrane-associated isoform of MBP is not simply a structural component of compact myelin but is highly post-translationally modified and multi-functional, having interactions with numerous proteins such as Ca2+-calmodulin, and with actin, tubulin, and proteins with SH3-domains, which it can tether to a lipid membrane in vitro. It co-localizes with such proteins in primary oligodendrocytes (OLGs) and in early developmental N19-OLGs transfected with fluorescently-tagged MBP. RESULTS: To provide further evidence for MBP associations with these proteins in vivo, we show here that MBP isoforms are co-immunoprecipitated from detergent extracts of primary OLGs together with actin, tubulin, zonula occludens 1 (ZO-1), cortactin, and Fyn kinase. We also carry out live-cell imaging of N19-OLGs co-transfected with fluorescent MBP and actin, and show that when actin filaments re-assemble after recovery from cytochalasin D treatment, MBP and actin are rapidly enriched and co-localized at certain sites at the plasma membrane and in newly-formed membrane ruffles. The MBP and actin distributions change similarly with time, suggesting a specific and dynamic association. CONCLUSIONS: These results provide more direct evidence for association of the predominant 18.5-kDa MBP isoform with these proteins in primary OLGs and in live cells than previously could be inferred from co-localization observations. This study supports further a role for classic MBP isoforms in protein-protein interactions during membrane and cytoskeletal extension and remodeling in OLGs.

Structural determinants stabilizing the axial channel of ClpP for substrate translocation.

Acyldepsipeptides (ADEPs) antibiotics bind to Escherichia coli ClpP mimicking the interactions that the IGL/F loops in ClpA or ClpX ATPases establish with the hydrophobic pockets surrounding the axial pore of the tetradecamer that the protease forms. ADEP binding induces opening of the gates blocking the axial channel of ClpP and allowing protein substrates to be translocated and hydrolysed in the degradation chamber. To identify the structural determinants stabilizing the open conformation of the axial channel for efficient substrate translocation, we constructed ClpP variants with amino acid substitutions in the N-terminal region that forms the axial gates. We found that adoption of a ß-hairpin loop by this region and the integrity of the hydrophobic cluster at the base of this loop are necessary elements for the axial gate to efficiently translocate protein substrates. Analysis of ClpP variants from Bacillus subtilis suggested that the identified structural requirements of the axial channel for efficient translocation are conserved between Gram-positive and Gram-negative bacteria. These findings provide mechanistic insights into the activation of ClpP by ADEPs as well as the gating mechanism of the protease in the context of the ClpAP and ClpXP complexes.

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 Ca²âº-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.

Influence of membrane surface charge and post-translational modifications to myelin basic protein on its ability to tether the Fyn-SH3 domain to a membrane in vitro.

Myelin basic protein (MBP) is a highly post-translationally modified, multifunctional structural component of central nervous system myelin, adhering to phospholipid membranes and assembling cytoskeletal proteins, and has previously been shown to bind SH3 domains in vitro and tether them to a membrane surface [Polverini, E., et al. (2008) Biochemistry 47, 267-282]. Since molecular modeling shows that the Fyn-SH3 domain has a negative surface charge density even after binding the MBP ligand, we have investigated the influence of negative membrane surface charge and the effects of post-translational modifications to MBP on the interaction of the Fyn-SH3 domain with membrane-associated MBP. Using a sedimentation assay with multilamellar vesicles consisting of neutral phosphatidylcholine (PC) and negatively charged phosphatidylinositol (PI), we demonstrate that increasing the negative surface charge of the membrane by increasing the proportion of PI reduces the amount of Fyn-SH3 domain that binds to membrane-associated MBP, due to electrostatic repulsion. When one of the phosphoinositides, PI(4)P or PI(4,5)P(2) was substituted for PI in equal proportion, none of the Fyn-SH3 domain bound to MBP under the conditions that were used. Post-translational modifications of MBP which reduced its net positive charge, i.e., phosphorylation or arginine deimination, increased the degree of repulsion of Fyn-SH3 from the membrane surface, an effect further modulated by the lipid charge. This study suggests that changes in membrane negative surface charge due to protein or lipid modifications, which could occur during cell signaling, can regulate the binding of the Fyn-SH3 domain to membrane-associated MBP and thus could regulate the activity of Fyn at the oligodendrocyte membrane surface.

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.

Slow aggregation of lysozyme in alkaline pH monitored in real time employing the fluorescence anisotropy of covalently labelled dansyl probe.

The onset of hen egg white lysozyme aggregation on exposure to alkaline pH of 12.2 and subsequent slow growth of soluble lysozyme aggregates (at 298 K) was directly monitored by steady-state and time-resolved fluorescence anisotropy of covalently attached dansyl probe over a period of 24 h. The rotational correlation time accounting for tumbling of lysozyme in solution (40 microM) increased from approximately 3.6 ns (in pH 7) to approximately 40ns on exposure to pH 12.2 over a period of 6 h and remained stable thereafter. The growth of aggregates was strongly concentration dependent, irreversible after 60 min and inhibited by the presence of 0.9 M l-arginine in the medium. The day old aggregates were resistant to denaturation by 6 M guanidine.HCl. Our results reveal slow segmental motion of the dansyl probe in day old aggregates in the absence of L-arginine (0.9 M), but a much faster motion in its presence, when growth of aggregates is halted.