Dense arrays of indium arsenide (InAs) nanowire materials have been grown by selective-area metal-organic vapor-phase epitaxy (SA-MOVPE) using polystyrene-b-poly(methyl methacrylate) (PS/PMMA) diblock copolymer (DBC) nanopatterning technique, which is a catalyst-free approach. Nanoscale openings were defined in a thin (?10 nm) SiNx layer deposited on a (111)B-oriented GaAs substrate using the DBC process and CF4 reactive ion etching (RIE), which served as a hard mask for the nanowire growth. InAs nanowires with diameters down to ?20 nm and micrometer-scale lengths were achieved with a density of ?5 × 10(10) cm(2). The nanowire structures were characterized by scanning electron microscopy and transmission electron microscopy, which indicate twin defects in a primary zincblende crystal structure and the absence of threading dislocation within the imaged regions.
Human alpha-1-antitrypsin (A1PI) is a plasma protein with the function of protecting lung tissues from proteolytic destruction by enzymes from inflammatory cells. A1PI deficiency is an inherited disorder associated with pulmonary emphysema and a higher risk of chronic obstructive pulmonary disease (COPD). Here we present the structural characterization of a recombinant form of human A1PI (Hu-recA1PI) expressed in the human PER.C6 cell line using an array of analytical and biochemical techniques. Hu-recA1PI had the same primary structure as plasma-derived A1PI (pd-A1PI) except reduced N-terminal heterogeneity. The secondary and tertiary structures were indistinguishable from pd-A1PI. Like pd-A1PI, Hu-recA1PI was modified by N-linked glycosylation on N46, N83, and N246. Unlike pd-A1PI, most glycans on recA1P1 were core fucosylated via ?(1-6) linkage. In addition, significantly higher amounts of tri- and tetraantennary glycans were observed. These differences in glycosylation contributed to the apparent higher molecular weight and lower isoelectric point (pI) of Hu-recA1PI than pd-A1PI. Hu-recA1PI contained both ?(2-3)- and ?(2-6)-linked sialic acids and had very similar sialylation levels as pd-A1PI. Hu-recA1PI glycans were differentially distributed, with N46 containing mostly biantennary glycans, N83 containing primarily tri- and tetraantennary glycans, and N247 containing exclusively biantennary glycans.
Graphene is an increasingly important nanomaterial exhibiting great promise in the area of nanotechnology. In this study, the azide-functionalized graphene derivative was synthesized as the click reagent for preparation of polyvalent DNA-graphene conjugates, which provide an effective and stable platform to construct new functional nano-architectures. Assembled with Au nanoparticles, the prepared Au-DNA-graphene nanocomplex exhibits excellent stability that could prevent the nanocomplex from being destroyed by surfactants. Assembled with DNA tetrahedron-structured probes (TSPs), the nanocomplex displays outstanding sensitive electrochemiluminescence properties, which might be used as a biosensor for DNA detection. Therefore, this DNA-graphene conjugates could be explored as the assembly unit for advanced DNA nano-architectures in the field of DNA nanotechnology.
Dynamic posttranslational modification of serine and threonine residues of nucleocytoplasmic proteins by ?-N-acetylglucosamine (O-GlcNAc) is a regulator of cellular processes such as transcription, signaling, and protein-protein interactions. Like phosphorylation, O-GlcNAc cycles in response to a wide variety of stimuli. Although cycling of O-GlcNAc is catalyzed by only two highly conserved enzymes, O-GlcNAc transferase (OGT), which adds the sugar, and ?-N-acetylglucosaminidase (O-GlcNAcase), which hydrolyzes it, the targeting of these enzymes is highly specific and is controlled by myriad interacting subunits. Here, we demonstrate by multiple specific immunological and enzymatic approaches that histones, the proteins that package DNA within the nucleus, are O-GlcNAcylated in vivo. Histones also are substrates for OGT in vitro. We identify O-GlcNAc sites on histones H2A, H2B, and H4 using mass spectrometry. Finally, we show that histone O-GlcNAcylation changes during mitosis and with heat shock. Taken together, these data show that O-GlcNAc cycles dynamically on histones and can be considered part of the histone code.
Protein synthesis is globally regulated through posttranslational modifications of initiation and elongation factors. Recent high-throughput studies have identified translation factors and ribosomal proteins (RPs) as substrates for the O-GlcNAc modification. Here we determine the extent and abundance of O-GlcNAcylated proteins in translational preparations. O-GlcNAc is present on many proteins that form active polysomes. We identify twenty O-GlcNAcylated core RPs, of which eight are newly reported. We map sites of O-GlcNAc modification on four RPs (L6, L29, L32, and L36). RPS6, a component of the mammalian target of rapamycin (mTOR) signaling pathway, follows different dynamics of O-GlcNAcylation than nutrient-induced phosphorylation. We also show that both O-GlcNAc cycling enzymes OGT and OGAse strongly associate with cytosolic ribosomes. Immunofluorescence experiments demonstrate that OGAse is present uniformly throughout the nucleus, whereas OGT is excluded from the nucleolus. Moreover, nucleolar stress only alters OGAse nuclear staining, but not OGT staining. Lastly, adenovirus-mediated overexpression of OGT, but not of OGAse or GFP control, causes an accumulation of 60S subunits and 80S monosomes. Our results not only establish that O-GlcNAcylation extensively modifies RPs, but also suggest that O-GlcNAc play important roles in regulating translation and ribosome biogenesis.
Like phosphorylation, the addition of O-linked beta-N-acetylglucosamine (O-GlcNAcylation) is a ubiquitous, reversible process that modifies serine and threonine residues on nuclear and cytoplasmic proteins. Overexpression of the enzyme that adds O-GlcNAc to target proteins, O-GlcNAc transferase (OGT), perturbs cytokinesis and promotes polyploidy, but the molecular targets of OGT that are important for its cell cycle functions are unknown. Here, we identify 141 previously unknown O-GlcNAc sites on proteins that function in spindle assembly and cytokinesis. Many of these O-GlcNAcylation sites are either identical to known phosphorylation sites or in close proximity to them. Furthermore, we found that O-GlcNAcylation altered the phosphorylation of key proteins associated with the mitotic spindle and midbody. Forced overexpression of OGT increased the inhibitory phosphorylation of cyclin-dependent kinase 1 (CDK1) and reduced the phosphorylation of CDK1 target proteins. The increased phosphorylation of CDK1 is explained by increased activation of its upstream kinase, MYT1, and by a concomitant reduction in the transcript for the CDK1 phosphatase, CDC25C. OGT overexpression also caused a reduction in both messenger RNA expression and protein abundance of Polo-like kinase 1, which is upstream of both MYT1 and CDC25C. The data not only illustrate the crosstalk between O-GlcNAcylation and phosphorylation of proteins that are regulators of crucial signaling pathways but also uncover a mechanism for the role of O-GlcNAcylation in regulation of cell division.
Numerous cellular processes are regulated by the reversible addition of either phosphate or O-linked beta-N-acetylglucosamine (O-GlcNAc) to nuclear and cytoplasmic proteins. Although sensitive methods exist for the enrichment and identification of protein phosphorylation sites, those for the enrichment of O-GlcNAc-containing peptides are lacking. Reported here is highly efficient methodology for the enrichment and characterization of O-GlcNAc sites from complex samples. In this method, O-GlcNAc-modified peptides are tagged with a novel biotinylation reagent, enriched by affinity chromatography, released from the solid support by photochemical cleavage, and analyzed by electron transfer dissociation mass spectrometry. Using this strategy, eight O-GlcNAc sites were mapped from a tau-enriched sample from rat brain. Sites of GlcNAcylation were characterized on important neuronal proteins such as tau, synucleins, and methyl CpG-binding protein 2.
Similar to phosphorylation, GlcNAcylation (the addition of O-GlcNAc to Ser(Thr) residues on polypeptides) is an abundant, dynamic, and inducible post-translational modification. GlcNAcylated proteins are crucial in regulating virtually all cellular processes, including signaling, cell cycle, and transcription. Here we show that calcium/calmodulin-dependent kinase IV (CaMKIV) is highly GlcNAcylated in vivo. In addition, we show that upon activation of HEK293 cells, hemagglutinin-tagged CaMKIV GlcNAcylation rapidly decreases, in a manner directly opposing its phosphorylation at Thr-200. Correspondingly, there is an increase in CaMKIV interaction with O-GlcNAcase during CaMKIV activation. Furthermore, we identify at least five sites of GlcNAcylation on CaMKIV. Using site-directed mutagenesis, we determine that the GlcNAcylation sites located in the active site of CaMKIV can modulate its phosphorylation at Thr-200 and its activity toward cAMP-response element-binding transcription factor. Our results strongly indicate that the O-GlcNAc modification participates in the regulation of CaMKIV activation and function, possibly coordinating nutritional signals with the immune and nervous systems. This is the first example of an O-GlcNAc/phosphate cycle involving O-GlcNAc transferase/kinase cross-talk.
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.
O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimers disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ?100 ?g of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-?-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-?-1,3-Fuc-?-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe ?-1,3-N-acetylglucosaminyltransferases.
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