Phosphorylation of Spinophilin Modulates Its Interaction with Actin Filaments

Spinophilin is a protein phosphatase 1 (PP1)- and actin-binding protein that modulates excitatory synaptic transmission and dendritic spine morphology. We report that spinophilin is phosphorylated in vitro by protein kinase A (PKA). Phosphorylation of spinophilin was stimulated by treatment of neostriatal neurons with a dopamine D1 receptor agonist or with forskolin, consistent with spinophilin being a substrate for PKA in intact cells. Using tryptic phosphopeptide mapping, site-directed mutagenesis, and microsequencing analysis, we identified two major sites of phosphorylation, Ser-94 and Ser-177, that are located within the actin-binding domain of spinophilin. Phosphorylation of spinophilin by PKA modulated the association between spinophilin and the actin cytoskeleton. Following subcellular fractionation, unphosphorylated spinophilin was enriched in the postsynaptic density, whereas a pool of phosphorylated spinophilin was found in the cytosol. F-actin co-sedimentation and overlay analysis revealed that phosphorylation of spinophilin reduced the stoichiometry of the spinophilin-actin interaction. In contrast, the ability of spinophilin to bind to PP1 remained unchanged. Taken together, our studies suggest that phosphorylation of spinophilin by PKA modulates the anchoring of the spinophilin-PP1 complex within dendritic spines, thereby likely contributing to the efficacy and plasticity of synaptic transmission

Comprehensive mapping of O-GlcNAc modification sites using a chemically cleavable tag

The post-translational modification of serine or threonine residues of proteins with a single N-acetylglucosamine monosaccharide (O-GlcNAcylation) is essential for cell survival and function. However, relatively few O-GlcNAc modification sites have been mapped due to the difficulty of enriching and detecting O-GlcNAcylated peptides from complex samples. Here we describe an improved approach to quantitatively label and enrich O-GlcNAcylated proteins for site identification. Chemoenzymatic labelling followed by copper(I)-catalysed azide–alkyne cycloaddition (CuAAC) installs a new mass spectrometry (MS)-compatible linker designed for facile purification of O-GlcNAcylated proteins from cell lysates. The linker also allows subsequent quantitative release of O-GlcNAcylated proteins for downstream MS analysis. We validate the approach by unambiguously identifying several established O-GlcNAc sites on the proteins α-crystallin and O-GlcNAc transferase (OGT), as well as discovering new, previously unreported sites on OGT. Notably, these novel sites on OGT lie in key functional domains of the protein, underscoring how this site identification method may reveal important biological insights into protein activity and regulation

A sulfated carbohydrate epitope inhibits axon regeneration after injury

Chondroitin sulfate proteoglycans (CSPGs) represent a major barrier to regenerating axons in the central nervous system (CNS), but the structural diversity of their polysaccharides has hampered efforts to dissect the structure-activity relationships underlying their physiological activity. By taking advantage of our ability to chemically synthesize specific oligosaccharides, we demonstrate that a sugar epitope on CSPGs, chondroitin sulfate-E (CS-E), potently inhibits axon growth. Removal of the CS-E motif significantly attenuates the inhibitory activity of CSPGs on axon growth. Furthermore, CS-E functions as a protein recognition element to engage receptors including the transmembrane protein tyrosine phosphatase PTPσ, thereby triggering downstream pathways that inhibit axon growth. Finally, masking the CS-E motif using a CS-E-specific antibody reversed the inhibitory activity of CSPGs and stimulated axon regeneration in vivo. These results demonstrate that a specific sugar epitope within chondroitin sulfate polysaccharides can direct important physiological processes and provide new therapeutic strategies to regenerate axons after CNS injury

Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics

The addition of the monosaccharide beta-N-acetyl-D-glucosamine to proteins (O-GlcNAc glycosylation) is an intracellular, post-translational modification that shares features with phosphorylation. Understanding the cellular mechanisms and signaling pathways that regulate O-GlcNAc glycosylation has been challenging because of the difficulty of detecting and quantifying the modification. Here, we describe a new strategy for monitoring the dynamics of O-GlcNAc glycosylation using quantitative mass spectrometry-based proteomics. Our method, which we have termed quantitative isotopic and chemoenzymatic tagging (QUIC-Tag), combines selective, chemoenzymatic tagging of O-GlcNAc proteins with an efficient isotopic labeling strategy. Using the method, we detect changes in O-GlcNAc glycosylation on several proteins involved in the regulation of transcription and mRNA translocation. We also provide the first evidence that O-GlcNAc glycosylation is dynamically modulated by excitatory stimulation of the brain in vivo. Finally, we use electron-transfer dissociation mass spectrometry to identify exact sites of O-GlcNAc modification. Together, our studies suggest that O-GlcNAc glycosylation occurs reversibly in neurons and, akin to phosphorylation, may have important roles in mediating the communication between neurons

Elucidating glycosaminoglycan–protein–protein interactions using carbohydrate microarray and computational approaches

Glycosaminoglycan polysaccharides play critical roles in many cellular processes, ranging from viral invasion and angiogenesis to spinal cord injury. Their diverse biological activities are derived from an ability to regulate a remarkable number of proteins. However, few methods exist for the rapid identification of glycosaminoglycan–protein interactions and for studying the potential of glycosaminoglycans to assemble multimeric protein complexes. Here, we report a multidisciplinary approach that combines new carbohydrate microarray and computational modeling methodologies to elucidate glycosaminoglycan–protein interactions. The approach was validated through the study of known protein partners for heparan and chondroitin sulfate, including fibroblast growth factor 2 (FGF2) and its receptor FGFR1, the malarial protein VAR2CSA, and tumor necrosis factor-α (TNF-α). We also applied the approach to identify previously undescribed interactions between a specific sulfated epitope on chondroitin sulfate, CS-E, and the neurotrophins, a critical family of growth factors involved in the development, maintenance, and survival of the vertebrate nervous system. Our studies show for the first time that CS is capable of assembling multimeric signaling complexes and modulating neurotrophin signaling pathways. In addition, we identify a contiguous CS-E-binding site by computational modeling that suggests a potential mechanism to explain how CS may promote neurotrophin-tyrosine receptor kinase (Trk) complex formation and neurotrophin signaling. Together, our combined microarray and computational modeling methodologies provide a general, facile means to identify new glycosaminoglycan–protein–protein interactions, as well as a molecular-level understanding of those complexes

Specific glycosaminoglycan chain length and sulfation patterns are required for cell uptake of tau versus α-synuclein and β-amyloid aggregates

Transcellular propagation of protein aggregate “seeds” has been proposed to mediate the progression of neurodegenerative diseases in tauopathies and α-synucleinopathies. We previously reported that tau and α-synuclein aggregates bind heparan sulfate proteoglycans (HSPGs) on the cell surface, promoting cellular uptake and intracellular seeding. However, the specificity and binding mode of these protein aggregates to HSPGs remain unknown. Here, we measured direct interaction with modified heparins to determine the size and sulfation requirements for tau, α-synuclein, and β-amyloid (Aβ) aggregate binding to glycosaminoglycans (GAGs). Varying the GAG length and sulfation patterns, we next conducted competition studies with heparin derivatives in cell-based assays. Tau aggregates required a precise GAG architecture with defined sulfate moieties in the N- and 6-O-positions, whereas the binding of α-synuclein and Aβ aggregates was less stringent. To determine the genes required for aggregate uptake, we used CRISPR/Cas9 to individually knock out the major genes of the HSPG synthesis pathway in HEK293T cells. Knockouts of the extension enzymes exostosin 1 (EXT1), exostosin 2 (EXT2), and exostosin-like 3 (EXTL3), as well as N-sulfotransferase (NDST1) or 6-O-sulfotransferase (HS6ST2) significantly reduced tau uptake, consistent with our biochemical findings, and knockouts of EXT1, EXT2, EXTL3, or NDST1, but not HS6ST2 reduced α-synuclein uptake. In summary, tau aggregates display specific interactions with HSPGs that depend on GAG length and sulfate moiety position, whereas α-synuclein and Aβ aggregates exhibit more flexible interactions with HSPGs. These principles may inform the development of mechanism-based therapies to block transcellular propagation of amyloid protein–based pathologies

Site-Specific GlcNAcylation of Human Erythrocyte Proteins: Potential Biomarker(s) for Diabetes

OBJECTIVE—O-linked N-acetylglucosamine (O-GlcNAc) is upregulated in diabetic tissues and plays a role in insulin resistance and glucose toxicity. Here, we investigated the extent of GlcNAcylation on human erythrocyte proteins and compared site-specific GlcNAcylation on erythrocyte proteins from diabetic and normal individuals

Intermediate mass excess of dilepton production in heavy ion collisions at BEVALAC energies

Dielectron mass spectra are examined for various nuclear reactions recently measured by the DLS collaboration. A detailed description is given of all dilepton channels included in the transport model UrQMD 1.0, i.e. Dalitz decays of $\pi^0,\eta,\omega,\eta'$ mesons and of the $\Delta(1232)$ resonance, direct decays of vector mesons and $pn$ bremsstrahlung. The microscopic calculations reproduce data for light systems fairly well, but tend to underestimate the data in $pp$ at high energies and in $pd$ at low energies. These conventional sources, however, cannot explain the recently reported enhancement for nucleus-nucleus collisions in the mass region 0.15 GeV<$M_{ee}$<0.6 GeV. Chiral scaling and $\omega$ meson broadening in the medium are investigated as a source of this mass excess. They also cannot explain the recent DLS data.Comment: 26 pages, 9 figures, references update

The Effect of Galaxy Interactions on Molecular Gas Properties

© 2018. The American Astronomical Society. All rights reserved.Galaxy interactions are often accompanied by an enhanced star formation rate (SFR). Since molecular gas is essential for star formation, it is vital to establish whether and by how much galaxy interactions affect the molecular gas properties. We investigate the effect of interactions on global molecular gas properties by studying a sample of 58 galaxies in pairs and 154 control galaxies. Molecular gas properties are determined from observations with the JCMT, PMO, and CSO telescopes and supplemented with data from the xCOLD GASS and JINGLE surveys at 12CO(1-0) and 12CO(2-1). The SFR, gas mass (), and gas fraction (f gas) are all enhanced in galaxies in pairs by ∼2.5 times compared to the controls matched in redshift, mass, and effective radius, while the enhancement of star formation efficiency (SFE ≡SFR/) is less than a factor of 2. We also find that the enhancements in SFR, and f gas, increase with decreasing pair separation and are larger in systems with smaller stellar mass ratio. Conversely, the SFE is only enhanced in close pairs (separation <20 kpc) and equal-mass systems; therefore, most galaxies in pairs lie in the same parameter space on the SFR- plane as controls. This is the first time that the dependence of molecular gas properties on merger configurations is probed statistically with a relatively large sample and a carefully selected control sample for individual galaxies. We conclude that galaxy interactions do modify the molecular gas properties, although the strength of the effect is dependent on merger configuration.Peer reviewedFinal Accepted Versio

Mapping far-IR emission from the central kiloparsec of NGC 1097

Using photometry of NGC 1097 from the Herschel PACS (Photodetector Array Camera and Spectrometer) instrument, we study the resolved properties of thermal dust continuum emission from a circumnuclear starburst ring with a radius ~ 900 pc. These observations are the first to resolve the structure of a circumnuclear ring at wavelengths that probe the peak (i.e. lambda ~ 100 micron) of the dust spectral energy distribution. The ring dominates the far-infrared (far-IR) emission from the galaxy - the high angular resolution of PACS allows us to isolate the ring's contribution and we find it is responsible for 75, 60 and 55% of the total flux of NGC 1097 at 70, 100 and 160 micron, respectively. We compare the far-IR structure of the ring to what is seen at other wavelengths and identify a sequence of far-IR bright knots that correspond to those seen in radio and mid-IR images. The mid- and far-IR band ratios in the ring vary by less than +/- 20% azimuthally, indicating modest variation in the radiation field heating the dust on ~ 600 pc scales. We explore various explanations for the azimuthal uniformity in the far-IR colors of the ring including a lack of well-defined age gradients in the young stellar cluster population, a dominant contribution to the far-IR emission from dust heated by older (> 10 Myr) stars and/or a quick smoothing of local enhancements in dust temperature due to the short orbital period of the ring. Finally, we improve previous limits on the far-IR flux from the inner ~ 600 pc of NGC 1097 by an order of magnitude, providing a better estimate of the total bolometric emission arising from the active galactic nucleus and its associated central starburst.Comment: Accepted for publication in the A&A Herschel Special Editio