Complexes of iron and cobalt with new tripodal amido-polyphosphine hybrid ligands

Divalent complexes of iron and cobalt with new, monoanionic tripodal amido-polyphosphine ligands have been thoroughly characterized, and XRD analysis reveals geometries that are distinct for this class of ligand

Identification of the Plasticity-Relevant Fucose-α(1−2)-Galactose Proteome from the Mouse Olfactory Bulb

Fucose-α(1−2)-galactose [Fucα(1−2)Gal] sugars have been implicated in the molecular mechanisms that underlie neuronal development, learning, and memory. However, an understanding of their precise roles has been hampered by a lack of information regarding Fucα(1−2)Gal glycoproteins. Here, we report the first proteomic studies of this plasticity-relevant epitope. We identify five classes of putative Fucα(1−2)Gal glycoproteins: cell adhesion molecules, ion channels and solute carriers/transporters, ATP-binding proteins, synaptic vesicle-associated proteins, and mitochondrial proteins. In addition, we show that Fucα(1−2)Gal glycoproteins are enriched in the developing mouse olfactory bulb (OB) and exhibit a distinct spatiotemporal expression that is consistent with the presence of a “glycocode” to help direct olfactory sensory neuron (OSN) axonal pathfinding. We find that expression of Fucα(1−2)Gal sugars in the OB is regulated by the α(1−2)fucosyltransferase FUT1. FUT1-deficient mice exhibit developmental defects, including fewer and smaller glomeruli and a thinner olfactory nerve layer, suggesting that fucosylation contributes to OB development. Our findings significantly expand the number of Fucα(1−2)Gal glycoproteins and provide new insights into the molecular mechanisms by which fucosyl sugars contribute to neuronal processes

Photoactivatable Glycopolymers for the Proteome-Wide Identification of Fucose-α(1-2)-Galactose Binding Proteins

Although fucose-α(1-2)-galactose (Fucα(1-2)Gal)-containing glycans have been implicated in cognitive processes such as learning and memory, their precise molecular mechanisms are poorly understood. Here we employed the use of multivalent glycopolymers to enable the first proteome-wide identification of weak affinity, low abundance Fucα(1-2)Gal glycan-binding proteins (GBPs). Biotin-terminated glycopolymers containing photoactivatable cross-linking groups were designed to capture and enrich GBPs from rat brain lysates. Candidate proteins were tested for their ability to bind Fucα(1-2)Gal, and the functional significance of the interaction was investigated for the synaptic vesicle protein SV2a using a knockout mouse system. The results suggest a role for SV2a-Fucα(1-2)Gal interactions in SV2a trafficking and synaptic vesicle recycling. More broadly, our studies outline a general chemical approach for the systems-level discovery of novel GBPs

Phosphofructokinase 1 Glycosylation Regulates Cell Growth and Metabolism

Cancer cells must satisfy the metabolic demands of rapid cell growth within a continually changing microenvironment. We demonstrated that the dynamic posttranslational modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAcylation) is a key metabolic regulator of glucose metabolism. O-GlcNAcylation was induced at serine 529 of phosphofructokinase 1 (PFK1) in response to hypoxia. Glycosylation inhibited PFK1 activity and redirected glucose flux through the pentose phosphate pathway, thereby conferring a selective growth advantage on cancer cells. Blocking glycosylation of PFK1 at serine 529 reduced cancer cell proliferation in vitro and impaired tumor formation in vivo. These studies reveal a previously uncharacterized mechanism for the regulation of metabolic pathways in cancer and a possible target for therapeutic intervention

Structural Snapshots of a Flexible Cu_2P_2 Core that Accommodates the Oxidation States Cu^ICu^I, Cu^(1.5)Cu^(1.5), and Cu^(II)Cu^(II)

The phosphido-bridged dicopper(I) complex {(PPP)Cu}_2 has been synthesized and structurally characterized ([PPP]^- = bis(2-di-iso-propylphosphinophenyl)phosphide). Cyclic voltammetry of {(PPP)Cu}_2 in THF shows fully reversible oxidations at −1.02 V (Cu^(1.5)Cu^(1.5)/Cu^ICu^I) and −0.423 V (Cu^(II)Cu^(II)/Cu^(1.5)Cu^(1.5)). Chemical oxidation of {(PPP)Cu}_2 by one electron yields the class III mixed-valence species [{(PPP)Cu}_2]^+ (EPR, UV−vis). Structural data establish an unexpectedly large change (0.538 Å) in the Cu•••Cu distance upon oxidation state. Oxidation of {(PPP)Cu}_2 by two electrons yields the dication [{(PPP)Cu}_2]^(2+), an antiferromagnetically coupled dicopper(II) complex. Maintenance of a pseudotetrahedral geometry that is midway between a square plane and an ideal tetrahedron at the copper centers, along with a high degree of flexibility at the phosphide hinges, allows for efficient access to Cu^ICu^I, Cu^(1.5)Cu^(1.5), and Cu^(II)Cu^(II) redox states without the need for ligand exchange, substitution, or redistribution processes

Parallel identification of O-GlcNAc-modified proteins from cell lysates

We report a new strategy for the parallel identification of O-GlcNAc-glycosylated proteins from cell lysates. The approach permits specific proteins of interest to be rapidly interrogated for the modification in any tissue or cell type and can be extended to peptides to facilitate the mapping of glycosylation sites. As an illustration of the approach, we identified four new O-GlcNAc-glycosylated proteins of low cellular abundance (c-Fos, c-Jun, ATF-1, and CBP) and two short regions of glycosylation in the enzyme O-GlcNAc transferase (OGT). The ability to target specific proteins across various tissue or cell types complements emerging proteomic technologies and should advance our understanding of this important posttranslational modification

Activation of the Transcriptional Function of the NF-κB Protein c-Rel by O-GlcNAc Glycosylation

The transcription factor nuclear factor κB (NF-κB) rapidly reprograms gene expression in response to various stimuli, and its activity is regulated by several posttranslational modifications, including phosphorylation, methylation, and acetylation. The addition of O-linked b-N-acetylglucosamine (a process known as O-GlcNAcylation) is an abundant posttranslational modification that is enhanced in conditions such as hyperglycemia and cellular stress. We report that the NF-κB subunit c-Rel is modified and activated by O-GlcNAcylation. We identified serine 350 as the site of O-GlcNAcylation, which was required for the DNA binding and transactivation functions of c-Rel. Blocking the O-GlcNAcylation of this residue abrogated c-Rel–mediated expression of the cytokine-encoding genes IL2, IFNG, and CSF2 in response to T cell receptor (TCR) activation, whereas increasing the extent of O-GlcNAcylation of cellular proteins enhanced the expression of these genes. TCR- or tumor necrosis factor (TNF)–induced expression of other NF-κB target genes, such as NFKBIA (which encodes IkBa) and TNFAIP3 (which encodes A20), occurred independently of the O-GlcNAcylation of c-Rel. Our findings suggest a stimulus-specific role for hyperglycemia-induced O-GlcNAcylation of c-Rel in promoting T cell–mediated autoimmunity in conditions such as type 1 diabetes by enhancing the production of T helper cell cytokines

FAR and NEAR Target Dynamic Visual Acuity: A Functional Assessment of Canal and Otolith Performance

Upon their return to earth, astronauts experience the effects of vestibular adaptation to microgravity. The postflight changes in vestibular information processing can affect postural and locomotor stability and may lead to oscillopsia during activities of daily living. However, it is likely that time spent in microgravity affects canal and otolith function differently. As a result, the isolated rotational stimuli used in traditional tests of canal function may fail to identify vestibular deficits after spaceflight. Also, the functional consequences of deficits that are identified often remain unknown. In a gaze control task, the relative contributions of the canal and otolith organs are modulated with viewing distance. The ability to stabilize gaze during a perturbation, on visual targets placed at different distances from the head may therefore provide independent insight into the function of this systems. Our goal was to develop a functional measure of gaze control that can also offer independent information about the function of the canal and otolith organs

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

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