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

Department of Molecular and Cellular Biology, University of Guelph, 50 Stone Road East, Guelph, Ontario N1G 2W1, Canada.
Biochemistry (Impact Factor: 3.02). 08/2010; 49(32):6903-15. DOI: 10.1021/bi100308d
Source: PubMed


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.

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Available from: Mumdooh A M Ahmed, Nov 20, 2014
    • "The light scattering at saturation was determined by NDP analysis and was plotted versus the tubulin concentration, with the x-intercepts yielding the critical tubulin concentration (C c ) [21]. To determine the apparent binding constant K d and the stoichiometry (n) between MBP-a1-peptide and microtubule assemblies in the presence and absence of DPC, we employed a sedimentation assay as we did previously to investigate binding between MBP-a-peptides and actin assemblies [22], and calculated from the equation: "
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    ABSTRACT: 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 membrane-attaching, molecular recognition fragments of 18.5-kDa MBP, we identified the N-terminal α1-peptide as sufficient to effect microtubule polymerization, the rate of which was significantly enhanced in the presence of dodecylphosphocholine (DPC) micelles to mimic a lipidic environment. Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · Apr 2015 · Biochemical and Biophysical Research Communications
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    • "To obtain further details on the mechanism of proteasomal MBP degradation, we further made an attempt to intercept MBP before it could reach the proteasome or, alternatively, mimic MBP to compete with it for proteasome binding. To this end, we selected a number of proteins (Table 1) that could potentially interfere with the hydrolysis of MBP by proteasome: (i) actin [30] and CaM [31], which are known to bind MBP in vitro and in vivo; (ii) the anti-MS drug glatiramer acetate (GA), which is structurally similar to MBP [32]; (iii) positively charged and intrinsically disordered histone H1.3 [33]; (iv) mono- and tetra-ubiquitin (Ub, Ub4), which can bind the ubiquitin receptors of the 19S regulator [34, 35]; (v) the slightly acidic globular proteins GST and BSA and basic lysozyme with compact globular structure which were used as controls. The hydrolysis of MBP was monitored in a ubiquitin-free in vitro system containing purified 26S proteasome, ATP, test proteins, and none of the components of the ubiquitination system. "
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    ABSTRACT: We recently showed that myelin basic protein (MBP) is hydrolyzed by 26S proteasome without ubiquitination. The previously suggested concept of charge-mediated interaction between MBP and the proteasome led us to attempt to compensate or mimic its positive charge to inhibit proteasomal degradation. We demonstrated that negatively charged actin and calmodulin (CaM), as well as basic histone H1.3, inhibit MBP hydrolysis by competing with the proteasome and MBP, respectively, for binding their counterpart. Interestingly, glatiramer acetate (GA), which is used to treat multiple sclerosis (MS) and is structurally similar to MBP, inhibits intracellular and in vitro proteasome-mediated MBP degradation. Therefore, the data reported in this study may be important for myelin biogenesis in both the normal state and pathophysiological conditions.
    Full-text · Article · Sep 2014 · BioMed Research International

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