Myelin basic protein (MBP) and two peptides derived from MBP (MBP(1-44) and MBP(152-167)) stimulated Schwann cell (SC) proliferation in a cAMP-mediated process. The two mitogenic regions of MBP did ...not compete with one another for binding to SC suggesting a distinctive SC receptor for each mitogenic peptide. Neutralizing antibodies to the fibroblast growth factor receptor blocked the mitogenic effect of the myelin-related SC mitogen found in the supernatant of myelin-fed macrophages. The binding of 125I-MBP to Schwann cells was specifically inhibited by basic fibroblast growth factor (bFGF) and conversely the binding of 125I-bFGF was competitively inhibited by MBP. These data suggested that the mitogenic effect of one MBP peptide was mediated by a bFGF receptor. The binding of MBP to ganglioside GM1 and the ability of MBP peptides containing homology to the B subunit of cholera toxin (which binds ganglioside GM1) to compete for the binding of a mitogenic peptide (MBP(1-44)) to SC, identified ganglioside GM1 as a second SC receptor. Based on these results, we conclude that MBP(1-44) and MBP(152-167) associate with ganglioside GM1 and the bFGF receptor respectively to stimulate SC mitosis.
Treatment of cultured oligodendrocytes (OLGs) with micromolar quantities of myelin basic protein (MBP) caused a rapid, MBP-dose-dependent cell death. In contrast, a 72-hr incubation of OLGs with MBP ...peptides (1-44, 47-87, 88-151, or 152-167) at comparable concentrations had no effect on cell viability. MBP and MBP peptides (1-44 and 88-151) have been shown to interact with ganglioside GM1 (Tzeng et al.: J Neurochem Res: 42:758-767, 1995). This interaction has been reported to increase calcium influx. Therefore, using the fluorescent dye Indo-1 and an ACAS laser cytometer, we examined the level of intracellular calcium in OLGs after MBP treatment. MBP was shown to provoke a rapid, dramatic, and sustained rise of intracellular calcium in most OLGs. The levels of elevated intracellular calcium were sustained and did not return to baseline even after 10 min. This increase of intracellular calcium was suppressed in the presence of EGTA, indicating that the Ca2+i rise was due to the entry of extracellular calcium. Incubation of cultured OLGs with MBP peptides (1-44 or 88-151) caused a modest and transitory elevation of intracellular calcium ions in a lower percentage of OLGs. The potent OLG cytotoxicity of intact MBP and the loss of potency after proteolysis raise the possibility that MBP proteolysis during demyelination protects OLGs from death.
Previous studies have shown that myelin basic protein (MBP) is mitogenic for Schwann cells (SCs) in the presence of elevated intracellular cAMP. Two mitogenic regions of MBP have been identified: one ...mitogenic region within the first 44 residues of the aminoterminus (1-44) and the other mitogenic region within the terminal 15 residues of the carboxyl end of the molecule (152-167). Unlike the mitogenic effect of a myelin enriched fraction (MEF), the mitogenic effect of MBP was not reduced by the addition of the lysosomal inhibitor, ammonium chloride. These data indicate that MBP causes SC proliferation by direct interaction of MBP with a surface receptor. Using Scatchard analysis of the binding of MBP to SCs, we report that treatment with forskolin does not cause the upregulation of receptors for MBP. Moreover, MBP blocks the cross-linking of 125I-bFGF with two fibroblast growth factor (FGF) receptors having apparent molecular weights of 140 kDa and 120 kDa, respectively. Since neither TGF-beta nor PDGF-BB displaced cell surface bound 125I-MBP, we conclude that MBP binds to the FGF receptor rather than other growth factor receptors. Furthermore, only MBP interacted with ganglioside GM1, whereas MBP did not interact with this ganglioside. These results are consistent with the view that ganglioside GM1 mediates the mitogenic effects of MBP, while the FGF receptor mediates the mitogenic effect of MBP. Intracellular cAMP of SCs was transiently increased after the addition of macrophage conditioned medium, suggesting that macrophages may produce factors in vivo which can transiently elevate intracellular cAMP levels, allowing a wave of SC proliferation in response to MBP-related mitogens.
Despite the increasing number of GPCR structures and recent advances in peptide design, the development of efficient technologies allowing rational design of high-affinity peptide ligands for single ...GPCRs remains an unmet challenge. Here, we develop a computational approach for designing conjugates of lariat-shaped macrocyclized peptides and a small molecule opioid ligand. We demonstrate its feasibility by discovering chemical scaffolds for the kappa-opioid receptor (KOR) with desired pharmacological activities. The designed De Novo Cyclic Peptide (DNCP)-β-naloxamine (NalA) exhibit in vitro potent mixed KOR agonism/mu-opioid receptor (MOR) antagonism, nanomolar binding affinity, selectivity, and efficacy bias at KOR. Proof-of-concept in vivo efficacy studies demonstrate that DNCP-β-NalA(1) induces a potent KOR-mediated antinociception in male mice. The high-resolution cryo-EM structure (2.6 Å) of the DNCP-β-NalA-KOR-Gi1 complex and molecular dynamics simulations are harnessed to validate the computational design model. This reveals a network of residues in ECL2/3 and TM6/7 controlling the intrinsic efficacy of KOR. In general, our computational de novo platform overcomes extensive lead optimization encountered in ultra-large library docking and virtual small molecule screening campaigns and offers innovation for GPCR ligand discovery. This may drive the development of next-generation therapeutics for medical applications such as pain conditions.
Recent advances in targeted protein degradation have enabled chemical hijacking of the ubiquitin–proteasome system to treat disease. The catalytic rate of cereblon (CRBN)-dependent bifunctional ...degradation activating compounds (BiDAC), which recruit CRBN to a chosen target protein, resulting in its ubiquitination and proteasomal degradation, is an important parameter to consider during the drug discovery process. In this work, an in vitro system was developed to measure the kinetics of BRD4 bromodomain 1 (BD1) ubiquitination by fitting an essential activator kinetic model to these data. The affinities between BiDACs, BD1, and CRBN in the binary complex, ternary complex, and full ubiquitination complex were characterized. Together, this work provides a new tool for understanding and optimizing the catalytic and thermodynamic properties of BiDACs.
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