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Titanium aluminium nitride (TiAlN) ternary coatings were deposited on glass substrates by means of reactive magnetron sputtering technique, using a Ti-Al alloy metal target (Ti0.5Al0.5). The depositions were performed at various N2 and Ar flux ratios of N2/(Ar + N2) ═ 33, 50, 67, 83%. The structure, morphology, chemical composition and mechanical properties were investigated by X-ray diffraction (XRD), field emission scanning electron microscope (FE-SEM), energy dispersive X-ray spectroscopy (EDS), and nano indenter (MTS System), respectively. The orientation of coatings depends on the flux ratios of N2/(Ar + N2) and substrate temperature. The coatings deposited with N2/(Ar + N2) ratios of 33, 50 at.% consists of pyramid-like column grains separated by porous and voids, which can be attributed to cubic-TiN (220) preferred orientation. The coatings deposited with N2/(Ar + N2) greater than 67% exhibits the phase of hexagonal-AlN and cubic-TiN. The surface of coatings becomes more compact and smoother with the N2/(Ar + N2) ratios increase. The coatings deposited with N2/(Ar+N2) ratio of 83% shows the largest hardness of 21.5 GPa, which is attributed to the preferred (200) orientation. However, this hardness increases significantly with increasing substrate temperature. The coatings deposited at more than 100 °C exhibited the (111) and/or (200) orientation. The amounts of grains grown along the (111) and (200) orientations play a significant role on the mechanical performance of TiAlN coatings. Four independent mechanisms, such as TiAlN stoichiometry and lattice parameter, the (111) preferred growth orientation, and the density increases (elimination of void), were found to contribute to the enhancement of TiAlN mechanical performance.

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Glutamate is the major excitatory neurotransmitter in the mammalian CNS and acts on both ionotropic and metabotropic glutamate receptors (mGluRs). The mGluRs are widely distributed in the CNS and modulate a variety of neuronal processes, including neurotransmitter release and ion channel function. In hippocampus and cortex, mGluR5 is highly expressed and plays an important role in the regulation of synaptic plasticity. Calmodulin (CaM) binding dynamically regulates mGluR5 surface expression; however, the mechanisms linking CaM to mGluR5 trafficking are not clear. Recent studies showed that CaM binding to mGluR7 regulates its trafficking in a phosphorylation-dependent manner by disrupting the binding of protein interacting with C kinase 1. The E3 ligase seven in absentia homolog (Siah)-1A binds to mGluR5 and competes with CaM binding, making it an intriguing molecule to regulate phosphorylation-dependent trafficking of mGluR5. In the present study, we find that CaM competes with Siah-1A for mGluR5 binding in a phosphorylation-dependent manner in rat hippocampal neurons. Specifically, phosphorylation of mGluR5 S901 favors Siah-1A binding by displacing CaM. We identified critical residues regulating Siah-1A binding to mGluR5 and showed that binding is essential for the Siah-1A effects on mGluR5 trafficking. Siah-1A binding decreases mGluR5 surface expression and increases endosomal trafficking and lysosomal degradation of mGluR5. Thus CaM-regulated Siah-1A binding to mGluR5 dynamically regulates mGluR5 trafficking. These findings support a conserved role for CaM in regulating mGluR trafficking by PKC-dependent regulation of receptor-binding proteins.

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Rad9-Rad1-Hus1 (9-1-1) is a checkpoint protein complex playing roles in DNA damage sensing, cell cycle arrest, DNA repair or apoptosis. Human 8-oxoguanine DNA glycosylase (hOGG1) is the major DNA glycosylase responsible for repairing a specific aberrantly oxidized nucleotide, 7,8-dihydro-8-oxoguanine (8-oxoG). In this study, we identified a novel interaction between hOGG1 and human 9-1-1, and investigated the functional consequences of this interaction. Co-immunoprecipitation assays using transiently transfected HEK293 cells demonstrated an interaction between hOGG1 and the 9-1-1 proteins. Subsequently, GST pull-down assays using bacterially expressed and purified hOGG1-His and GST-fused 9-1-1 subunits (GST-hRad9, GST-hRad1, and GST-hHus1) demonstrated that hOGG1 interacted directly with the individual subunits of the human 9-1-1 complex. In vitro excision assay, which employed a DNA duplex containing an 8-oxoG/C mismatch, showed that hRad9, hRad1, and hHus1 enhanced the 8-oxoG excision and beta-elimination activities of hOGG1. In addition, the presence of hRad9, hRad1, and hHus1 enhanced the formation of covalently cross-linked hOGG1-8-oxoG/C duplex complexes, as determined by a trapping assay using NaBH(4). A trimeric human 9-1-1 complex was purified from Escherichia coli cell transformed with hRad9, His-fused hRad1, or His-fused hHus1 expressing vectors. It also showed the similar activity to enhance in vitro hOGG1 glycosylase activity, compared with individual human 9-1-1 subunits. Detection of 8-oxoG in HEK293 cells using flow cytometric and spectrofluorometric analysis revealed that over-expression of hOGG1 or human 9-1-1 reduced the formation of 8-oxoG residues following the H(2)O(2) treatment. The highest 8-oxoG reduction was observed in HEK293 cells over-expressing hOGG1 and all the three subunits of human 9-1-1. These indicate that individual human 9-1-1 subunits and human 9-1-1 complex showed almost the same abilities to enhance the in vitro 8-oxoG excision activity of hOGG1, but that the greatest effect to remove 8-oxoG residues in H(2)O(2)-treated cells was derived from the 9-1-1 complex as a whole.

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Three evolutionarily conserved proteins, Rad9, Hus1, and Rad1, form a heterotrimeric 9-1-1 complex that plays critical roles in cellular responses to DNA damage by activating checkpoints and by recruiting DNA repair enzymes to DNA lesions. We have determined the crystal structure of the human Rad9 (residues 1-272)-Hus1-Rad1 complex at 2.5 A resolution. The 9(1-272)-1-1 complex forms a closed ring, with each subunit having a similar structure. Despite its high level of similarity to proliferating cell nucleus antigen in terms of overall structure, the 9(1-272)-1-1 complex exhibits notable differences in local structures, including interdomain connecting loops, H2 and H3 helices, and loops in the vicinity of the helices of each subunit. These local structural variations provide several unique features to the 9-1-1 heterotrimeric complex-including structures of intermolecular interfaces and the inner surface around the central hole, and different electrostatic potentials at and near the interdomain connecting loops of each 9-1-1 subunit-compared to the proliferating cell nucleus antigen trimer. We propose that these structural features allow the 9-1-1 complex to bind to a damaged DNA during checkpoint control and to serve as a platform for base excision repair. We also show that the 9(1-272)-1-1 complex, but not the full-length 9-1-1 complex, forms a stable complex with the 5' recessed DNA, suggesting that the C-terminal tail of Rad9 is involved in the regulation of the 9-1-1 complex in DNA binding.

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Tumor suppressor programmed cell death protein 4 (PDCD4) inhibits the translation initiation factor eIF4A, an RNA helicase that catalyzes the unwinding of secondary structure at the 5'-untranslated region of mRNAs and controls the initiation of translation. Here, we determined the crystal structure of the human eIF4A and PDCD4 complex. The structure reveals that one molecule of PDCD4 binds to the two eIF4A molecules through the two different binding modes. While the two MA3 domains of PDCD4 bind to one eIF4A molecule, the C-terminal MA3 domain alone of the same PDCD4 also interacts with another eIF4A molecule. The eIF4A-PDCD4 complex structure suggests that the MA3 domain(s) of PDCD4 binds perpendicular to the interface of the two domains of eIF4A, preventing the domain closure of eIF4A and blocking the binding of RNA to eIF4A, both of which are required events in the function of eIF4A helicase. The structure, together with biochemical analyses, reveals insights into the inhibition mechanism of eIF4A by PDCD4 and provides a framework for designing chemicals that target eIF4A.

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A complex of Drosha with DGCR8 (or its homolog Pasha) cleaves primary microRNA (pri-miRNA) substrates into precursor miRNA and initiates the microRNA maturation process. Drosha provides the catalytic site for this cleavage, whereas DGCR8 or Pasha provides a frame for anchoring substrate pri-miRNAs. To clarify the molecular basis underlying recognition of pri-miRNA by DGCR8 and Pasha, we determined the crystal structure of the human DGCR8 core (DGCR8S, residues 493-720). In the structure, the two double-stranded RNA-binding domains (dsRBDs) are arranged with pseudo two-fold symmetry and are tightly packed against the C-terminal helix. The H2 helix in each dsRBD is important for recognition of pri-miRNA substrates. This structure, together with fluorescent resonance energy transfer and mutational analyses, suggests that the DGCR8 core recognizes pri-miRNA in two possible orientations. We propose a model for DGCR8's recognition of pri-miRNA.

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The Drosha-DGCR8 complex initiates microRNA maturation by precise cleavage of the stem loops that are embedded in primary transcripts (pri-miRNAs). Here we propose a model for this process that is based upon evidence from both computational and biochemical analyses. A typical metazoan pri-miRNA consists of a stem of approximately 33 bp, with a terminal loop and flanking segments. The terminal loop is unessential, whereas the flanking ssRNA segments are critical for processing. The cleavage site is determined mainly by the distance (approximately 11 bp) from the stem-ssRNA junction. Purified DGCR8, but not Drosha, interacts with pri-miRNAs both directly and specifically, and the flanking ssRNA segments are vital for this binding to occur. Thus, DGCR8 may function as the molecular anchor that measures the distance from the dsRNA-ssRNA junction. Our current study thus facilitates the prediction of novel microRNAs and will assist in the rational design of small hairpin RNAs for RNA interference.

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