ABSTRACT
The immunoreceptor tyrosine-based inhibition motif (ITIM)-containing receptor G6b-B is critical for platelet production and activation. Loss of G6b-B results in severe macrothrombocytopenia, myelofibrosis and aberrant platelet function in mice and humans. Using a combination of immunohistochemistry, affinity chromatography and proteomics, we identified the extracellular matrix heparan sulfate (HS) proteoglycan perlecan as a G6b-B binding partner. Subsequent in vitro biochemical studies and a cell-based genetic screen demonstrated that the interaction is specifically mediated by the HS chains of perlecan. Biophysical analysis revealed that heparin forms a high-affinity complex with G6b-B and mediates dimerization. Using platelets from humans and genetically modified mice, we demonstrate that binding of G6b-B to HS and multivalent heparin inhibits platelet and megakaryocyte function by inducing downstream signaling via the tyrosine phosphatases Shp1 and Shp2. Our findings provide novel insights into how G6b-B is regulated and contribute to our understanding of the interaction of megakaryocytes and platelets with glycans.
Subject(s)
Blood Platelets/physiology , Heparitin Sulfate/metabolism , Megakaryocytes/physiology , Receptors, Immunologic/metabolism , Animals , Humans , Mice, Inbred C57BL , Mice, Knockout , Protein Binding , Protein Multimerization , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , Protein Tyrosine Phosphatase, Non-Receptor Type 6/metabolism , Receptors, Immunologic/deficiency , Receptors, Immunologic/genetics , Signal TransductionABSTRACT
There are a number of small-molecule inhibitors targeting the RAS/RAF/MEK/ERK signaling pathway that have either been approved or are in clinical development for oncology across a range of disease indications. The inhibition of ERK1/2 is of significant current interest, as cell lines with acquired resistance to BRAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition in preclinical models. This article reports on our recent work to identify novel, potent, and selective reversible ERK1/2 inhibitors from a low-molecular-weight, modestly active, and highly promiscuous chemical start point, compound 4. To guide and inform the evolution of this series, inhibitor binding mode information from X-ray crystal structures was critical in the rapid exploration of this template to compound 35, which was active when tested in in vivo antitumor efficacy experiments.
Subject(s)
MAP Kinase Signaling System/drug effects , Protein Kinase Inhibitors/pharmacology , Animals , Biological Availability , Cell Line, Tumor , Dogs , Drug Discovery , Humans , Methylation , Protein Kinase Inhibitors/pharmacokineticsABSTRACT
The RAS/RAF/MEK/ERK signaling pathway has been targeted with a number of small molecule inhibitors in oncology clinical development across multiple disease indications. Importantly, cell lines with acquired resistance to B-RAF and MEK inhibitors have been shown to maintain sensitivity to ERK1/2 inhibition by small molecule inhibitors. There are a number of selective, noncovalent ERK1/2 inhibitors reported along with the promiscuous hypothemycin (and related analogues) that act via a covalent mechanism of action. This article reports the identification of multiple series of highly selective covalent ERK1/2 inhibitors informed by structure-based drug design (SBDD). As a starting point for these covalent inhibitors, reported ERK1/2 inhibitors and a chemical series identified via high-throughput screening were exploited. These approaches resulted in the identification of selective covalent tool compounds for potential in vitro and in vivo studies to assess the risks and or benefits of targeting this pathway through such a mechanism of action.
Subject(s)
Drug Design , Mitogen-Activated Protein Kinase 1/chemistry , Mitogen-Activated Protein Kinase 3/chemistry , Protein Kinase Inhibitors/pharmacology , Amino Acid Sequence , Cells, Cultured , Crystallography, X-Ray , Humans , Immunoblotting , Models, Molecular , Molecular Sequence Data , Molecular Structure , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
The interaction of the extra-membranous domain of tetrameric inwardly rectifying Kir2.1 ion channels (Kir2.1NC(4)) with the membrane associated guanylate kinase protein PSD-95 has been studied using Transmission Electron Microscopy in negative stain. Three types of complexes were observed in electron micrographs corresponding to a 1:1 complex, a large self-enclosed tetrad complex and extended chains of linked channel domains. Using models derived from small angle X-ray scattering experiments in which high resolution structures from X-ray crystallographic and Nuclear Magnetic Resonance studies are positioned, the envelopes from single particle analysis can be resolved as a Kir2.1NC(4):PSD-95 complex and a tetrad of this unit (Kir2.1NC(4):PSD-95)(4). The tetrad complex shows the close association of the Kir2.1 cytoplasmic domains and the influence of PSD-95 mediated self-assembly on the clustering of these channels.
Subject(s)
Cytoplasm/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Potassium Channels, Inwardly Rectifying/metabolism , Disks Large Homolog 4 Protein , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Membrane Proteins/chemistry , Microscopy, Electron, Transmission , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Potassium Channels, Inwardly Rectifying/chemistry , Protein Binding , Reproducibility of Results , Scattering, RadiationABSTRACT
The crystal structure at 4.8 angstrom resolution of the reaction center-light harvesting 1 (RC-LH1) core complex from Rhodopseudomonas palustris shows the reaction center surrounded by an oval LH1 complex that consists of 15 pairs of transmembrane helical alpha- and beta-apoproteins and their coordinated bacteriochlorophylls. Complete closure of the RC by the LH1 is prevented by a single transmembrane helix, out of register with the array of inner LH1 alpha-apoproteins. This break, located next to the binding site in the reaction center for the secondary electron acceptor ubiquinone (UQB), may provide a portal through which UQB can transfer electrons to cytochrome b/c1.
Subject(s)
Bacterial Proteins/chemistry , Light-Harvesting Protein Complexes/chemistry , Photosynthetic Reaction Center Complex Proteins/chemistry , Rhodopseudomonas/chemistry , Apoproteins/chemistry , Bacteriochlorophyll A/chemistry , Binding Sites , Crystallization , Crystallography, X-Ray , Macromolecular Substances , Models, Molecular , Protein Conformation , Protein Structure, Secondary , Ubiquinone/chemistryABSTRACT
A typical purple bacterial photosynthetic unit consists of two types of light-harvesting complex (LH1 and LH2) together with a reaction centre. This short review presents a description of the structure of the LH2 complex from Rhodopseudomonas acidophila, which has recently been improved to a resolution of 2.0 A [Papiz et al., J. Mol. Biol. 326 (2003) 1523-1538]. We show how this structure has helped to reveal the details of the various excitation energy transfer events in which it is involved.
Subject(s)
Bacterial Proteins/chemistry , Light-Harvesting Protein Complexes/chemistry , Bacterial Proteins/radiation effects , Light-Harvesting Protein Complexes/radiation effects , Macromolecular Substances , Models, Molecular , Molecular Structure , Photobiology , Rhodopseudomonas/chemistry , Rhodopseudomonas/radiation effects , Spectrophotometry , Static ElectricityABSTRACT
The phospholipid composition of Rhodopseudomonas acidophila strain 10050 grown aerobically or anaerobically in the light was determined. The major phospholipids present in the aerobic cells were phosphatidylethanolamine (PE; 54%), phosphatidylglycerol (PG; 24%) and cardiolipin (diphosphatidylglycerol, DPG) (14%), together with phosphatidylcholine (PC; 5%). On moving the cells to anaerobic photosynthetic growth in the light PE remained the major phospholipid (37-49%), but there was a major change in the proportion of PC, which increased to 31-33%, and corresponding reductions in the contents of PG to 11-16% and DPG to 4-5%. The fatty acid composition of the phospholipids was unusual, compared with other purple non-sulfur photosynthetic bacteria, in that it contained 16:0 (29%), 17:1 (20%) and 19:1 (9%) plus several mainly unsaturated 2-OH fatty acids (9% total) as major components, when grown aerobically in the dark. In contrast when grown photosynthetically under anaerobic conditions there was <2% 17:1 or 19:1 present, while the amounts of 16:1 and 18:1 increased, and 16:0 decreased. The phospholipid composition of the purified light-harvesting complex 2 (LH2) complex was PE (43%), PC (42%) and DPG (15%). Unexpectedly, there was no PG associated with the purified LH2. These findings contrast with previous studies on several other photosynthetic bacteria, which had shown an increase in PG upon photosynthetic growth [Biochem. J. 181 (1979) 339]. The prior hypothesis that phosphatidylglycerol has some specific role to play in the function of light-harvesting complexes cannot be true for Rps. acidophila. It is suggested that specific integral membrane proteins may strongly influence the phospholipid content of the host membranes into which they are inserted.
Subject(s)
Phosphatidylglycerols/chemistry , Phospholipids/chemistry , Rhodopseudomonas/chemistry , Bacterial Proteins/chemistry , Cell Fractionation , Cell Membrane/chemistry , Light , Macromolecular Substances , Photosynthetic Reaction Center Complex Proteins/chemistry , Protein Structure, Quaternary , Rhodopseudomonas/metabolismABSTRACT
In this paper the fluorescence-excitation spectra of individual LH1-RC complexes (Rhodopseudomonas acidophila) at 1.2 K are presented. All spectra show a limited number of broad bands with a characteristic polarization behavior, indicating that the excitations are delocalized over a large number of pigments. A significant variation in the number of bands, their bandwidths, and polarization behavior is observed. Only 30% of the spectra carry a clear signature of delocalized excited states of a circular structure of the pigments. The large spectral variety suggests that besides site heterogeneity also structural heterogeneity determines the optical spectrum of the individual LH1-RC complexes. Further research should reveal if such heterogeneity is a native property of the complex or induced during the experimental procedures.
