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Changes in the synaptic content of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)–type glutamate receptors lead to synaptic efficacy modifications, involved in synaptic plasticity mechanisms believed to underlie learning and memory formation. Early in development, GluR4 is highly expressed in the hippocampus, and GluR4-containing AMPA receptors are inserted into synapses. During synapse maturation, the number of AMPA receptors at the synapse is dynamically regulated, and both addition and removal of receptors from postsynaptic sites occur through regulated mechanisms. GluR4 delivery to synapses in rat hippocampal slices was shown to require protein kinase A (PKA)–mediated phosphorylation of GluR4 at serine 842 (Ser842). Protein kinase C (PKC) can also phosphorylate Ser842, and we have shown that PKCγ can associate with GluR4. Here we show that activation of PKC in retina neurons, or in human embryonic kidney 293 cells cotransfected with GluR4 and PKCγ, increases GluR4 surface expression and Ser842 phosphorylation. Moreover, mutation of amino acids R821A, K825A and R826A at the GluR4 C-terminal, within the interacting region of GluR4 with PKCγ, abolishes the interaction between PKCγ and GluR4 and prevents the stimulatory effect of PKCγ on GluR4 Ser842 phosphorylation and surface expression. These data argue for a role of anchored PKCγ in Ser842 phosphorylation and targeting to the plasma membrane. The triple GluR4 mutant is, however, phosphorylated by PKA, and it is targeted to the synapse in CA1 hippocampal neurons in organotypic rat hippocampal slices. The present findings show that the interaction between PKCγ and GluR4 is specifically required to assure PKC-driven phosphorylation and surface membrane expression of GluR4., We acknowledge support from FCT, Portugal, to A. R. G., S. S. C., C. B. D. and A. L. C. (POCI/SAU-NEU/58955/2004), and the NIH grant support MH070417 to J .A. E.

Biotechnology and Biological Sciences Research Council (BB/C005074/1) Enzyme systems that attack the plant cell wall contain noncatalytic carbohydrate-binding modules (CBMs) that mediate attachment to this composite structure and play a pivotal role in maximizing the hydrolytic process. Although xyloglucan, which includes a backbone of β-1,4-glucan decorated primarily with xylose residues, is a key component of the plant cell wall, CBMs that bind to this polymer have not been identified. Here we showed that the C-terminal domain of the modular Clostridium thermocellum enzyme CtCel9D-Cel44A (formerly known as CelJ) comprises a novel CBM (designated CBM44) that binds with equal affinity to cellulose and xyloglucan. We also showed that accommodation of xyloglucan side chains is a general feature of CBMs that bind to single cellulose chains. The crystal structures of CBM44 and the other CBM (CBM30) in CtCel9D-Cel44A display a β-sandwich fold. The concave face of both CBMs contains a hydrophobic platform comprising three tryptophan residues that can accommodate up to five glucose residues. The orientation of these aromatic residues is such that the bound ligand would adopt the twisted conformation displayed by cello-oligosaccharides in solution. Mutagenesis studies confirmed that the hydrophobic platform located on the concave face of both CBMs mediates ligand recognition. In contrast to other CBMs that bind to single polysaccharide chains, the polar residues in the binding cleft of CBM44 play only a minor role in ligand recognition. The mechanism by which these proteins are able to recognize linear and decorated β-1,4-glucans is discussed based on the structures of CBM44 and the other CBMs that bind single cellulose chains. publishersversion published

Nucleocytoplasmic shuttling activity of the African swine fever virus p37 protein, a major structural protein of this highly complex virus, has been recently reported. The systematic characterization of the nuclear export ability of this protein constituted the major purpose of the present study. We report that both the N- and C-terminal regions of p37 protein are actively exported from the nucleus to the cytoplasm of yeast and mammalian cells. Moreover, experiments using leptomycin B and small interfering RNAs targeting the CRM1 receptor have demonstrated that the export of p37 protein is mediated by both the CRM1-dependent and CRM1-independent nuclear export pathways. Two signals responsible for the CRM1-mediated nuclear export of p37 protein were identified at the N terminus of the protein, and an additional signal was identified at the C-terminal region, which mediates the CRM1-independent nuclear export. Interestingly, site-directed mutagenesis revealed that hydrophobic amino acids are critical to the function of these three nuclear export signals. Overall, our results demonstrate that two distinct pathways contribute to the strong nuclear export of full-length p37 protein, which is mediated by three independent nuclear export signals. The existence of overlapping nuclear export mechanisms, together with our observation that p37 protein is localized in the nucleus at early stages of infection and exclusively in the cytoplasm at later stages, suggests that the nuclear transport ability of this protein may be critical to the African swine fever virus replication cycle., This work was supported by a grant from the Portuguese Foundation for Science and Technology (POCTI/CVT/44854/2002). A. Eula´-lio and I. Nunes-Correia are recipients of fellowships from the Portuguese Foundation for Science and Technology. The work in the V. Citovsky laboratory was supported by grants from the National Institutes of Health, the National Science Foundation, the U.S. Department of Agriculture, the U.S.-Israel Binational Science Foundation (BSF), and the U.S.-Israel Binational Research and Development Fund (BARD). The work in the J. Salas and M. L. Salas laboratory was supported by a grant from Ministerio de Educacio´n y Ciencia (BFU2004-00298) and by an institutional grant from the Fundacio´n Ramo´n Areces.

J Biol Inorg Chem (2006) 11: 548–558 DOI 10.1007/s00775-006-0104-y Superoxide reductase (SOR) is a metalloprotein containing a non-heme iron centre, responsible for the scavenging of superoxide radicals in the cell. The crystal structure of Treponema pallidum (Tp) SOR was determined using soft X-rays and synchrotron radiation. Crystals of the oxidized form were obtained using poly(ethylene glycol) and MgCl2 and diffracted beyond 1.55 A resolution. The overall architecture is very similar to that of other known SORs but TpSOR contains an N-terminal domain in which the desulforedoxin-type Fe centre, found in other SORs, is absent. This domain conserves the beta-barrel topology with an overall arrangement very similar to that of other SOR proteins where the centre is present. The absence of the iron ion and its ligands, however, causes a decrease in the cohesion of the domain and some disorder is observed, particularly in the region where the metal would be harboured. The C-terminal domain exhibits the characteristic immunoglobulin-like fold and harbours the Fe(His)4(Cys) active site. The five ligands of the iron centre are well conserved despite some disorder observed for one of the four molecules in the asymmetric unit. The participation of a glutamate as the sixth ligand of some of the iron centres in Pyrococcus furiosus SOR was not observed in TpSOR. A possible explanation is that either X-ray photoreduction occurred or there was a mixture of redox states at the start of data collection. In agreement with earlier proposals, details in the TpSOR structure also suggest that Lys49 might be involved in attraction of superoxide to the active site.

Bovine acidic seminal fluid protein (aSFP) is a 1.29 kDa polypeptide of the spermadhesin family built by a single CUB domain architecture. The CUB domain is an extracellular module present in 16 functionally diverse proteins. To determine the three-dimensional structure of aSFP, the protein was crystallized at 21 degrees C by vapor diffusion in hanging drops, using ammonium sulfate, pH 4.7, and polyethyleneglycol 4,000 as precipitants, containing 10% dioxane to avoid the formation of clustered crystals. Elongated prismatic crystals with maximal size of 0.6 x 0.3 x 0.2 mm3 diffract to beyond 1.9 A resolution and belong to space group P2(1)2(1)2(1), with cell parameters a = 52.4 A, b = 41.5 A, c = 48.2 A. There is one aSFP molecule per asymmetric unit, which corresponds to a crystal volume per unit molecular mass of 2.04 A3/Da, and analytical ultracentrifugation analysis show that aSFP is a monomeric protein.

Superoxide reductase is a 14 kDa metalloprotein containing a catalytic non-haem iron centre [Fe(His)4Cys]. It is involved in defence mechanisms against oxygen toxicity, scavenging superoxide radicals from the cell. The oxidized form of Treponema pallidum superoxide reductase was crystallized in the presence of polyethylene glycol and magnesium chloride. Two crystal forms were obtained depending on the oxidizing agents used after purification: crystals grown in the presence of K3Fe(CN)6 belonged to space group P2(1) (unit-cell parameters a = 60.3, b = 59.9, c = 64.8 A, beta = 106.9 degrees) and diffracted beyond 1.60 A resolution, while crystals grown in the presence of Na2IrCl6 belonged to space group C2 (a = 119.4, b = 60.1, c = 65.6 A, beta = 104.9 degrees) and diffracted beyond 1.55 A. A highly redundant X-ray diffraction data set from the C2 crystal form collected on a copper rotating-anode generator (lambda = 1.542 A) clearly defined the positions of the four Fe atoms present in the asymmetric unit by SAD methods. A MAD experiment at the iron absorption edge confirmed the positions of the previously determined iron sites and provided better phases for model building and refinement. Molecular replacement using the P2(1) data set was successful using a preliminary trace as a search model. A similar arrangement of the four protein molecules could be observed.

Cells preferentially expressing GluR4-containing alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors are particularly sensitive to excitotoxicity mediated through non-N-methyl-D-aspartate receptors. However, the excitotoxic signalling pathways associated with GluR4-containing AMPA receptors are not known. In this work, we investigated the downstream signals coupled to excitotoxicity mediated by Ca2+-permeable GluR4-containing AMPA receptors, using a HEK 293 cell line constitutively expressing the GluR4flip subunit of AMPA receptors (HEK-GluR4). Glutamate stimulation of GluR4-containing AMPA receptors decreased cell viability, in a calcium-dependent manner, when the receptor desensitisation was prevented with cyclothiazide. The excitotoxic stimulation mediated through GluR4-containing AMPA receptors increased activator protein-1 (AP-1) DNA-binding activity. Inhibition of the AP-1 activity by overexpression of a c-Jun dominant-negative form protected HEK-GluR4 cells against excitotoxic damage. Taken together, the results indicate that overactivation of Ca2+-permeable GluR4-containing AMPA receptors is coupled to a death pathway mediated, at least in part, by the AP-1 transcription factor.

Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates. Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains. In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM. To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display beta-1,4- and beta-1,3-1,4-mixed linked endoglucanase activity, respectively. Here we show that CtCBM11 binds to both beta-1,4- and beta-1,3-1,4-mixed linked glucans, displaying K(a) values of 1.9 x 10(5), 4.4 x 10(4), and 2 x 10(3) m(-1) for Glc-beta1,4-Glc-beta1,4-Glc-beta1,3-Glc, Glc-beta1,4-Glc-beta1,4-Glc-beta1,4-Glc, and Glc-beta1,3-Glc-beta1,4-Glc-beta1,3-Glc, respectively, demonstrating that CBMs can display a preference for mixed linked glucans. To determine whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of the protein was solved to a resolution of 1.98 A. The protein displays a beta-sandwich with a concave side that forms a potential binding cleft. Site-directed mutagenesis revealed that Tyr(22), Tyr(53), and Tyr(129), located in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein. We propose, therefore, that CtCBM11 contains a single ligand-binding site that displays affinity for both beta-1,4- and beta-1,3-1,4-mixed linked glucans.

Ionotropic glutamate receptors mediate the majority of excitatory synaptic transmission in the brain and are thought to be involved in learning and memory formation. The activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors can be regulated by direct phosphorylation of their subunits, which affects the electrophysiological properties of the receptor, and the receptor association with numerous proteins that modulate membrane traffic and synaptic targeting of the receptor. In the present study we investigated the association of protein kinase C (PKC) gamma isoform with the GluR4 AMPA receptor subunit. PKC gamma was co-immunoprecipitated with GluR4 AMPA receptor subunit in rat cerebellum and in cultured chick retina cell extracts, and immunocytochemistry experiments showed co-localization of GluR4 and PKC gamma in cultured chick retinal neurons. Pull-down assays showed that native PKC gamma binds the GluR4 C-terminal membrane-proximal region, and recombinant PKC gamma was retained by GST-GluR4 C-terminal fusion protein, suggesting that the kinase binds directly to GluR4. Furthermore, GST-GluR4 C-terminal protein was phosphorylated on GluR4 Ser-482 by bound kinases, retained by the fusion protein, including PKC gamma. The GluR4 C-terminal segment that interacts with PKC gamma, which lacks the PKC phosphorylation sites, inhibited histone H1 phosphorylation by PKC, to the same extent as the PKC pseudosubstrate peptide 19-31, indicating that PKC gamma bound to GluR4 preferentially phosphorylates GluR4 to the detriment of other substrates. Additionally, PKC gamma expression in GluR4 transfected human embryonic kidney 293T cells increased the amount of plasma membrane-associated GluR4. Our results suggest that PKC gamma binds directly to GluR4, thereby modulating the function of GluR4-containing AMPA receptors.

Prostate-specific kallikrein, a member of the gene family of serine proteases, was initially discovered in semen and is the most useful serum marker for prostate cancer diagnosis and prognosis. We report the crystal structure at 1.42 A resolution of horse prostate kallikrein (HPK). This is the first structure of a serine protease purified from seminal plasma. HPK shares extensive sequence homology with human prostate-specific antigen (PSA), including a predicted chymotrypsin-like specificity, as suggested by the presence of a serine residue at position S1 of the specificity pocket. In contrast to other kallikreins, HPK shows a structurally distinct specificity pocket. Its entrance is blocked by the kallikrein loop, suggesting a possible protective or substrate-selective role for this loop. The HPK structure seems to be in an inactivated state and further processing might be required to allow the binding of substrate molecules. Crystal soaking experiments revealed a binding site for Zn2+ and Hg2+, two known PSA inhibitors.

We have previously reported that the activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors is potentiated by protein kinase C (PKC) in cultured chick retina amacrine neurons, and that constitutive PKC activity is necessary for basal AMPA receptor activity (Carvalho et al., 1998). In this study, we evaluated the phosphorylation of the GluR4 subunit, which is very abundant in cultured amacrine neurons, to correlate it with the effects of PKC on AMPA receptor activity in these cells. 32P-labelling of GluR4 increased upon AMPA receptor stimulation or cell treatment with phorbol 12-myristate 13-acetate (PMA) before stimulating with kainate. By contrast, phosphorylation of GluR4 was not changed when PKC was inhibited by treating the cells with the selective PKC inhibitor GF 109203X before stimulation with kainate. We conclude that GluR4 is phosphorylated upon PKC activation and/or stimulation of AMPA receptors in cultured amacrine cells. Additionally, AMPA receptor activation with kainate in cultured chick amacrine cells leads to translocation of conventional and novel PKC isoforms to the cell membrane, suggesting that PKC could be activated upon AMPA receptor stimulation in these cells.

Gold nanoparticles (AuNPs) exhibit unique properties that have made them a very attractive material for application in biological assays [1]. Given the potentially interesting interactions between AuNPs and biological macromolecules, we investigated AuNPs-induced protein crystal growth [2]. Differently functionalized AuNPs were tested as additives in co-crystallization studies with model proteins (hen egg white lysozyme (HEWL), ribonuclease A (RNase A) and proteinase K) as well as with case studies where there were problems in obtaining well-diffracting crystals. Trials were performed considering different crystallization drawbacks, from total absence of crystals to improvement of crystal morphology, size, twinning and number of crystals per drop. Improvement of some of these factors was observed in the cases of HEWL, RNase A, phenylalanine hydroxylase (PAH), myoglobin, native aldehyde oxidase (AOH) and human albumin. In these proteins, the presence of the AuNPs promoted an increase in the size and/or better crystal morphology. From the systematic trials and subsequent observations, it can be concluded that the introduction of AuNPs should definitely be considered in crystal optimization trials to improve previously determined crystallization conditions. Acknowledgments The work was supported by the Portuguese Science and Technology Foundation (FCT-MEC) through grants PTDC/CTM-NAN/112241/2009 to RF, PEst-C/EQB/LA0006/2011 and PEst-C/EQB/LA0006/2013 (to Associate Lab REQUIMTE).

The purification, crystallization and identification by X-ray diffraction analysis of a horse kallikrein is reported. The protein was purified from horse seminal plasma. Crystals belong to space group C2 and the structure was solved by the MIRAS method, with two heavy-atom derivatives of mercury and platinum. X-ray diffraction data to 1.42 A resolution were collected at the ESRF synchrotron-radiation source.

The aim of this work was to investigate whether excitotoxicity induced by overstimulation of different ionotropic glutamate receptors could trigger different intracellular signaling cascades. Cultured chick neuronal retina cells, essentially amacrine-like, were particularly sensitive to the toxicity induced by non-NMDA glutamate receptor agonists. One hour stimulation with 100 muM kainate induced a reduction of cell viability of about 44%, as assessed by the MTT test 24 hr after stimulation. Kainate-induced toxicity was mediated through AMPA receptors. Glutamate (100 muM, 1 hr) reduced cell viability by 26%, essentially acting through N-methyl-D-aspartate receptors. Five hours after stimulation, neuronal retina cells had an apoptotic-like nuclear morphology. In retinal neurons, the excitotoxic stimulation, with either glutamate or kainate, induced a calcium-dependent enhancement of the DNA-binding activity of the activating protein-1 (AP-1) transcription factor, which was maximal 2 hr after stimulation. Glutamate induced a greater increase in the AP-1 DNA-binding activity than did kainate. Supershift assays using antibodies directed against different members of the Fos and Jun protein families showed that the AP-1 complex in retinal neurons includes proteins of the Fos family, namely, Fra-2, c-Jun, and Jun D. The DNA-binding activity of the nuclear factor-kappaB transcription factor was not significantly changed upon excitotoxic stimulation with any agonist. Stimulation of glutamate receptors with 100 muM kainate or 100 muM glutamate for 2 min was sufficient to induce the activation of the extracellular signal-regulated kinase (ERK). Inhibition of the ERK activation with the MEK inhibitors U 0126 and PD 98059 increased the toxicity induced by kainate but was without effect on the toxicity induced by glutamate. These results indicate that, although stimulation with both glutamate receptor agonists increased ERK phosphorylation, only kainate-induced ERK activation correlates with the activation of a survival signaling pathway. Our results suggest that, in chick embryo retinal neurons, the signaling pathways that mediate excitotoxic cell death and neuroprotection are stimulus specific. J. Neurosci. Res. 66:643-655, 2001. © 2001 Wiley-Liss, Inc.

Desulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio gigas, is a small homodimeric (2 x 36 amino acids) protein. Each subunit contains a high-spin iron atom tetrahedrally bound to four cysteinyl sulfur atoms, a metal center similar to that found in rubredoxin (Rd) type proteins. The simplicity of the active center in Dx and the possibility of replacing the iron by other metals make this protein an attractive case for the crystallographic analysis of metal-substituted derivatives. This study extends the relevance of Dx to the bioinorganic chemistry field and is important to obtain model compounds that can mimic the four sulfur coordination of metals in biology. Metal replacement experiments were carried out by reconstituting the apoprotein with In3+, Ga3+, Cd2+, Hg2+, and Ni2+ salts. The In3+ and Ga3+ derivatives are isomorphous with the iron native protein; whereas Cd2+, Hg2+, and Ni2+ substituted Dx crystallized under different experimental conditions, yielding two additional crystal morphologies; their structures were determined by the molecular replacement method. A comparison of the three-dimensional structures for all metal derivatives shows that the overall secondary and tertiary structures are maintained, while some differences in metal coordination geometry occur, namely, bond lengths and angles of the metal with the sulfur ligands. These data are discussed in terms of the entatic state theory. publishersversion published

Recent studies have suggested that protein phosphorylation of glutamate receptors may play an important role in synaptic transmission. Specifically, the phosphorylation of AMPA receptors has been implicated in cellular models of synaptic plasticity. The phosphorylation of the glutamate receptor 1 (GluR1) subunit of AMPA receptors by protein kinase A (PKA), protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been characterized extensively. Phosphorylation of this subunit occurs exclusively on the intracellular C-terminal domain. However, the GluR1 subunit C terminus shows low homology to the other AMPA receptor subunits. In this paper we characterized the phosphorylation of AMPA receptor subunit GluR4, using site-specific mutagenesis and biochemical techniques. We found that GluR4 is phosphorylated on serine 842 within the C-terminal domainin vitroandin vivo. Serine 842 is phosphorylated by PKA, PKC, and CaMKIIin vitroand is phosphorylated in transfected cells by PKA. Two-dimensional phosphopeptide analysis indicates that serine 842 is the major phosphorylation site on GluR4. In addition, we identified threonine 830 as a potential PKC phosphorylation site. These results suggest that GluR4, which is the most rapidly desensitizing AMPA receptor subunit, may be modulated by phosphorylation.

The functional modulation of the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors by protein kinase C (PKC) was investigated in cultures enriched in retinal amacrine-like cells. The kainate-evoked [Ca2+]i increase is due to Ca2+ entry through open AMPA receptor channels, because it was blocked by the active isomer of a 2,3-benzodiazepine (LY 303070), an AMPA receptor antagonist. The AMPA receptor response to kainate was potentiated by phorbol 12-myristate 13-acetate, which specifically stimulates PKC, and it was decreased by bisindolylmaleimide I, a selective inhibitor of PKC, as well as by PKC down-regulation. The results indicate not only that the AMPA receptor activation has a PKC requirement, but also that PKC amplifies maximal receptor activation by 100 µM kainate. The effect of PKC activation or inhibition on voltage-gated Ca2+-channel activity was also investigated. Activation of PKC caused inhibition of Ca2+ channels, and the same effect was produced by inhibition of PKC, whereas the inactive analogue of the phorbol ester did not affect channel activity. Our results show an important role for PKC in regulating the function of both AMPA receptors and Ca2+ channels in cultured retina cells.

1. Glutamate is the neurotransmitter released by bipolar cells at their synapses with amacrine cells. The amacrine cells express ionotropic (NMDA, AMPA and kainate) and metabotropic (mGluR1, mGluR2, mGluR4 and mGluR7) glutamate receptors and may take up glutamate from the synaptic cleft. 2. Activation of the ionotropic glutamate receptors increases the intracellular free calcium concentration ([Ca2+]i), owing to Ca2+ entry through the receptor-associated channels as well as through voltage-gated Ca2+ channels. The [Ca2+]i response to glutamate may be amplified by Ca2+-induced Ca2+ release from intracellular sources. 3. Activation of NMDA and non-NMDA glutamate receptors stimulates the release of GABA and acetylcholine from amacrine cells. GABA is released by a Ca2+-dependent mechanism and by reversal of the neurotransmitter transporter. 4. Excessive activation of glutamate receptors during ischemia leads to amacrine cell death. An increase in [Ca2+]i due to Ca2+ influx through NMDA and AMPA/kainate receptor channels is related to cell death in studies in vitro. In other studies, it was shown that nitric oxide may also take part in the process of cell damage during ischemia.

Spermadhesins, 12,000-14,000 M(r) mammalian proteins, include lectins involved in sperm-egg binding and display a single CUB domain architecture. We report the crystal structures of porcine seminal plasma PSP-I/PSP-II, a heterodimer of two glycosylated spermadhesins, and bovine aSFP at 2.4 A and 1.9 A resolution respectively.

Glutamate is the major excitatory neurotransmitter in the brain, and glutamate receptors mediate rapid excitatory synaptic transmission in the CNS. Glutamate activates ionotropic receptors and receptors coupled to guanine nucleotide-binding proteins (Schoepp and Conn, 1993; Schoepfer et al., 1994). Based on ligand binding assays, and on molecular biological and electrophysiological properties, the mammalian ionotropic glutamate receptors were subdivided into three classes: the N-methyl-D-aspartate (NMDA), S-α-amino-3-hydroxy-5-methyl-4-isoxazole-propionie acid (AMPA) and kainate receptors. The activity of these receptors is modulated during neuronal plasticity, most likely by phosphorylation (Raymond et al., 1993a). In this chapter we will review the basic structure of the non-NMDA ionotropic glutamate receptors, and the modulation of the channel activity by protein kinase C-dependent mechanisms. Finally, the role of these receptors and PKC in long term depression in the cerebellum is also discussed.

Overexpression of the Thomsen-Friedenreich (TF) antigen in cell membrane proteins occurs in 90 % of adenocarcinomas. Additionally, the binding of the TF antigen to human galectin-3 (Gal-3), also frequently overexpressed in malignancy, promotes cancer progression and metastasis. In this context, structures that interfere with this specific interaction have the potential to prevent cancer metastasis. A multidisciplinary approach combining the optimized synthesis of a TF antigen mimetic with NMR, X-ray crystallography methods, and isothermal titration calorimetry assays was used to unravel the molecular structural details that govern the Gal-3/TF mimetic interaction. The TF mimetic has a binding affinity for Gal-3 similar to that of the TF natural antigen and retains the binding epitope and bioactive conformation observed for the native antigen. Furthermore, from a thermodynamic perspective, a decrease in the enthalpic contribution was observed for the Gal-3/TF mimetic complex; however, this behavior is compensated by a favorable gain in entropy. From a structural perspective, these results establish our TF mimetic as a scaffold to design multivalent solutions to potentially interfere with Gal-3 aberrant interactions and for likely use in hampering Gal-3-mediated cancer cell adhesion and metastasis.

Machado-Joseph disease (MJD/SCA3) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG tract in the coding portion of the ATXN3 gene. The presence of ubiquitin-positive aggregates of the defective protein in affected neurons is characteristic of this and most of the polyglutamine disorders. Recently, the accumulation of the neural precursor cell expressed developmentally downregulated 8 (NEDD8), a ubiquitin-like protein, in the inclusions of MJD brains was reported. Here, we report a new molecular interaction between wild-type ataxin-3 and NEDD8, using in vitro and in situ approaches. Furthermore, we show that this interaction is not dependent on the ubiquitin-interacting motifs in ataxin-3, since the presence of the Josephin domain is sufficient for the interaction to occur. The conservation of the interaction between the Caenorhabditis elegans ataxin-3 homologue (atx-3) and NEDD8 suggests its biological and functional relevance. Molecular docking studies of the NEDD8 molecule to the Josephin domain of ataxin-3 suggest that NEDD8 interacts with ataxin-3 in a substrate-like mode. In agreement, ataxin-3 displays deneddylase activity against a fluorogenic NEDD8 substrate., We thank Dr. H. Paulson, Dr. R. Hay, Dr. D. Bohmann, Dr. S.Elledge and PM laboratory members for the reagents and as-sistance provided. A.F. would like to address special thanks toCarlos Melo for the continuous support, Maria do Carmo Costaand Sandra Santos for all the assistance with the Y2H and cellculture assays. This work was funded by FCT (POCTI/MGI/47550/2002; SAU-MMO 60412/2004; POCI/SAU-MMO/60156/2004), Fundação Luso-Americana para o Desenvolvi-mento (Proc.3.L/A.II/I P.582/99) and the National Ataxia Foun-dation. A.F. (SFRH/BD/1288/2000), A.T.-C. (SFRH/BI/11844/2003) and A.-J.R. (SFRH/BD/17066/2004) are scholarship re-cipients from FCT.

Homeostatic synaptic scaling is a negative feedback response to fluctuations in synaptic strength induced by developmental or learning-related processes, which maintains neuronal activity stable. Although several components of the synaptic scaling apparatus have been characterized, the intrinsic regulatory mechanisms promoting scaling remain largely unknown. MicroRNAs may contribute to posttranscriptional control of mRNAs implicated in different stages of synaptic scaling, but their role in these mechanisms is still undervalued. Here, we report that chronic blockade of glutamate receptors of the AMPA and NMDA types in hippocampal neurons in culture induces changes in the neuronal mRNA and miRNA transcriptomes, leading to synaptic upscaling. Specifically, we show that synaptic activity blockade persistently down-regulates miR-186-5p. Moreover, we describe a conserved miR-186-5p-binding site within the 3′UTR of the mRNA encoding the AMPA receptor GluA2 subunit, and demonstrate that GluA2 is a direct target of miR-186-5p. Overexpression of miR-186 decreased GluA2 surface levels, increased synaptic expression of GluA2-lacking AMPA receptors, and blocked synaptic scaling, whereas inhibition of miR-186-5p increased GluA2 surface levels and the amplitude and frequency of AMPA receptor-mediated currents, and mimicked excitatory synaptic scaling induced by synaptic inactivity. Our findings elucidate an activity-dependent miRNA-mediated mechanism for regulation of AMPA receptor expression.

Mestrado em Engenharia do Ambiente As questões ambientais têm assumido um papel cada vez mais relevante na qualidade de vida da população e na componente económica de uma organização/região/país. Com efeito, tem-se verificado uma crescente consciencialização para a necessidade de garantir que todas as actividades se desenvolvem em respeito pelo ambiente, para que assim se alcance o tão desejável desenvolvimento sustentável. Contudo, continua a verificar-se que na maioria das obras de construção civil não há qualquer controlo/minimização dos impactes ambientais gerados e, mesmo nestas situações, as obras são licenciadas e construídas. Neste sentido, a cadeia produtiva da construção civil, provavelmente uma das maiores da economia, causadora de grandes impactes ambientais, consumo desmesurado de recursos naturais e produção de volumes cada vez maiores de resíduos gerados nas suas actividades, beneficiaria da implementação urgente de sistemas de gestão ambiental (SGA). Tem-se assistido mundialmente a uma crescente adesão à certificação ambiental. Em Portugal, estudos sobre esta matéria confirmam esta tendência, não obstante, para o caso concreto do sector da construção civil, não se conhecem trabalhos de investigação que fomentem as motivações, vantagens e dificuldades do sector na implementação e certificação de sistemas de gestão ambiental. O interesse em documentar o processo de adopção destes sistemas de gestão, no contexto das organizações portuguesas do sector da construção civil e obras públicas, justifica a elaboração do presente estudo. Perspectiva-se que o desenvolvimento de qualquer estratégia para potenciar a certificação ambiental no sector da construção, como ferramenta também importante no desenvolvimento da construção sustentável no nosso país, decorrerá, inevitavelmente, da caracterização inicial das organizações que a possuem. Concretamente o presente trabalho de investigação objectivou a identificação das motivações, vantagens e dificuldades da implementação/certificação de sistemas de gestão ambiental pelas empresas do sector da construção. Foi elaborado um inquérito enviado a todas as empresas construtoras certificadas pela ISO 14001 e decorrente dos 32 inquéritos válidos (43% de taxa de resposta), tornou-se ainda possível efectuar um conjunto de recomendações destinadas a orientar e facilitar o processo de adopção de sistemas de gestão ambiental neste sector. Conclui-se que a obtenção da certificação ambiental é potenciada pela experiência anterior que as organizações possam ter na implementação de outros sistemas de gestão e que o processo de implementação do SGA está intimamente relacionado com a disponibilidade de recursos das empresas. No entanto, e apesar da possibilidade de obtenção das vantagens esperadas, entre as quais se incluem, a melhoria de desempenho ambiental, a satisfação de clientes e outras partes interessadas, o cumprimento dos requisitos legais, a melhoria de imagem e o acesso a novos mercados, convém destacar que o processo poderá ser dificultado pela resistência interna à mudança das práticas existentes, pela relevância dos custos associados ao processo de implementação de SGA e pelas dificuldades inerentes ao próprio cumprimento da legislação ambiental. Para as empresas inquiridas a melhoria do seu desempenho ambiental dependerá, com certeza, da optimização dos sistemas de gestão ambiental implementados que, obrigatoriamente, terão de ser estendidos às empresas subcontratadas e à totalidade de obras em curso. Não obstante e indirectamente intrínseco às conclusões do presente estudo, identifica-se a grande problemática inerente às pequenas empresas do sector da construção. Na realidade, são estas as empresas que constituem a maioria do tecido sectorial da construção e para as quais não à há evidencias de preocupação ambiental nas obras executadas. Mais do que conseguir convergir as boas práticas ambientais com o fenómeno de outsourcing e dispersão dos estaleiros de obra das grandes empresas, a estratégia terá de ser governamental e atingir o pequeno empresário da construção: aquele que desconhece, que não tem recursos, que não tem visão, o que polui, o que procura desmesuradamente, consciente ou inconsciente, um desenvolvimento ainda que não sustentável. ABSTRACT: Environmental issues have assumed an increasingly important role in the quality of life and economic component of an organization / region / country. Indeed, it has been observed a growing awareness of the need to ensure that all activities are progressing in regard to the environment, to achieve the so desirable sustainable development. However, it continues to notice that most of the civil constructions have no control / mitigation of environmental impacts generated and, even in these situations the works are licensed and built. In this sense, with a supply chain of construction, probably one of the largest economies, causing large environmental impacts, disproportionate consumption of natural resources and production of increased volumes of waste generated in its activities, urges the interest and urgency of implementing and certification of environmental management systems in enterprises in the civil construction. There has been a growing worldwide membership of environmental certification. In Portugal, studies on this subject confirm this trend, however, in the case of the civil construction, there are no known researches that foster the motivations, advantages and difficulties of this sector in the implementation and certification of environmental management systems. The interest in documenting the process of adopting such management in the context of the Portuguese organizations of civil construction and public works, justifies the preparation of this study. It is expected that the development of any strategy to enhance the environmental certification in the construction sector, also as a tool in the development of sustainable construction in our country, place, inevitably, in the initial characterization of the organizations that have it. Specifically this research work objectifies the id of the motivation, advantages and difficulties of implementations/certifications for environmental management systems by enterprises in the sector of construction. It was elaborated an inquiry sent to all construction companies certified by ISO 14001 and caused the 32 valid surveys (43% response rate), it became even possible to perform a set for the recommended to guide and facilitate the process of adoption of environmental management systems in this sector. It is concluded that the achievement of environmental certification is enhanced by previous experience that organizations can have on the implementation of other management systems and that the process of EMS implementation is closely related to the availability of company’s resources. However, despite the possibility of obtaining the expected benefits, among which are included the improvement of environmental performance, customer satisfaction and other stakeholders, compliance with legal requirements, improved image and access to new markets, should be noted that the process may be hampered by internal resistance to change existing practices, the relevance of the costs associated with the process of EMS implementation and the difficulties inherent in environmental laws compliance. For the surveyed companies, the improving of their environmental performance will depend, of course, in the optimization of environmental management systems in place which, necessarily, must be extended to subcontractors and all works in progress. Despite this and indirectly intrinsic to the conclusions of this study, it is identified the major inherent problematic to small business of civil construction. In reality, these are the companies that make up the majority of the construction sector for which there is no evidence of the environmental concern in the works performed. More than getting good environmental practices converge with the phenomenon of outsourcing and dispersion of construction sites of large companies, the strategy must be government and to reach the small business of construction: the one who doesn’t know, who has no resources, has no vision, the one that pollutes, that looks disproportionately, conscious or unconscious, a development even not a sustainable one.

BACKGROUND: Machado-Joseph Disease (MJD), a form of dominantly inherited ataxia belonging to the group of polyQ expansion neurodegenerative disorders, occurs when a threshold value for the number of glutamines in Ataxin-3 (Atx3) polyglutamine region is exceeded. As a result of its modular multidomain architecture, Atx3 is known to engage in multiple macromolecular interactions, which might be unbalanced when the polyQ tract is expanded, culminating in the aggregation and formation of intracellular inclusions, a unifying fingerprint of this group of neurodegenerative disorders. Since aggregation is specific to certain brain regions, localization-dependent posttranslational modifications that differentially affect Atx3 might also contribute for MJD. METHODS: We combined in vitro and cellular approaches to address SUMOylation in the brain-predominant Atx3 isoform and assessed the impact of this posttranslational modification on Atx3 self-assembly and interaction with its native partner, p97. RESULTS: We demonstrate that Atx3 is SUMOylated at K356 both in vitro and in cells, which contributes for decreased formation of amyloid fibrils and for increased affinity towards p97. CONCLUSIONS AND GENERAL SIGNIFICANCE: These findings highlight the role of SUMOylation as a regulator of Atx3 function, with implications on Atx3 protein interaction network and self-assembly, with potential impact for further understanding the molecular mechanisms underlying MJD pathogenesis. This work was funded by the Fundação para a Ciência e a Tecnologia (FCT, Portugal) through grants PTDC/BIA-PRO/100059/2008 and PTDC/SAU-NMC/110602/2009 (EU-FEDER funding through COMPETEFCOMP-01-0124-FEDER-009031 and FCOMP-01-0124-FEDER-015860, respectively), and by QREN — Quadro de Referência Estratégica Nacional through Programa Operacional Regional do Norte ON. 2 (Neurodegenerative Disorders — NORTE-01-0124-FEDER-000001). C.M., S.F., I.A.A. and B.A. acknowledge the financial support from FCT through fellowships SFRH/BD/47160/2008, SFRH/BPD/77009/2011, Ciência 2008 Programme, and SFRH/BPD/70783/2010, respectively.

Mestrado Engenharia Química Atualmente é premente a existência de dados experimentais de coeficientes de difusão binária, em particular em dióxido de carbono supercrítico (SC–CO2). Os sistemas ternários não são exceção, dado o interesse de difusividades de solutos em misturas de solventes. Conhecido o papel determinante dos compostos bioativos na saúde humana, surge o principal objetivo desta dissertação, que tem por alicerce fundamental a medição de difusividades do ácido gálico tanto em etanol líquido puro como em SC–CO2 modificado com etanol (cossolvente). Realizaram-se ainda medições de difusividade de oxiacetilacetonato de titânio (TiO(acac)2) em etanol líquido puro. Recorreu-se ao método cromatográfico de abertura de pico (CPB) para todas as medições efetuadas. A determinação de difusividades em SC–CO2 com cossolvente foi feita pela primeira vez na Universidade de Aveiro com este trabalho. Preparou-se uma solução de ácido gálico em etanol (sistema binário), tendo-se medido a sua difusividade primeiro em etanol líquido puro no intervalo de temperaturas 303,15−333,15 K e de pressões 1−100 bar, encontrando-se os seus valores entre 4,85×10−6 a 9,05×10−6 cm2∙s−1, e, posteriormente, em SC–CO2 modificado com etanol, no intervalo 313,15−333,15 K e 150−250 bar, obtendo-se valores entre 3,26×10−5 a 4,98×10−5 cm2∙s−1. As medições do TiO(acac)2 em etanol líquido puro forneceram valores entre 1,03×10−5 a 2,00×10−5 cm2∙s−1 no intervalo de temperaturas 303,15−333,15 K e de pressões 1−100 bar. Procedeu-se ainda à pesquisa, programação e validação de equações para estimar a densidade e a viscosidade do etanol líquido puro e de SC–CO2 modificado com etanol. Utilizaram-se as equações de Tait (densidade) e de Mamedov (viscosidade) para etanol líquido puro e, para misturas SC–CO2 com etanol, a equação de estado Soave-Redlich-Kwong (densidade) e a equação de Kendall-Monroe (viscosidade). Finalmente estudou-se o comportamento da difusividade com a temperatura, a pressão, a densidade do solvente e em coordenadas Stokes-Einstein. Recorrendo-se à modelação, compararam-se os valores experimentais com os calculados por equações hidrodinâmicas, de volume livre e empíricas. Alcançaram-se erros globais médios pequenos pelos modelos de volume livre de Dymond-Hildebrand-Batchinsky (1,6%−5,7%), TLSMd (5,6%) e pelas correlações empíricas de Magalhães et al. (0,9%−3,7%). Provou-se que as equações empíricas de Magalhães et al. são particularmente adequadas para calcular a difusividade do ácido gálico nas condições indicadas, tanto em etanol líquido puro como em SC–CO2 modificado com etanol. Currently there is a demand for experimental data comprising diffusion coefficients, in particular for systems that contain supercritical carbon dioxide (SC−CO2) as a solvent. The ternary systems are no exception, due to the interest of such data in solvent mixtures. It is known the determining role of the bioactive compounds on human health, so the main purpose of this dissertation is fundamentally based on the experimental measurements of diffusivity of gallic acid both in pure liquid ethanol and SC−CO2 modified with ethanol (cosolvent). Measurements of tracer diffusion coefficients of Titanium (IV) oxyacetylacetonate (TiO(acac)2) in pure liquid ethanol, were also performed. Furthermore, the Taylor-Aris chromatographic peak broadening (CPB) method was used for the measurements of diffusivity. With this work, the determination of diffusivity in SC−CO2 with cosolvent was accomplished at the University of Aveiro for the first time. A solution of gallic acid was prepared in ethanol (binary system), and diffusivities were firstly measured in pure liquid ethanol for the temperature range of 303.15−333.15 K and pressure range of 1−100 bar. The obtained diffusivities were between 4.85×10−6 and 9.05×10−6 cm2∙s−1. Thereafter, for gallic acid in SC−CO2 containing ethanol as a modifier, the diffusivity was measured for the ranges of 313.15−333.15 K and 150−250 bar, yielding values between 3.26×10−5 and 4.98×10−5 cm2∙s−1. Thirdly, diffusivity measurements of TiO(acac)2 in pure liquid ethanol gave rise to values between 1.03×10−5 and 2.00×10−5 cm2∙s−1 for the temperature and pressure ranges of 303.15−333.15 K and 1−100 bar, respectively. Also in this work, programming and validation of equations to estimate the density and viscosity for pure liquid ethanol and SC−CO2 modified with ethanol, was performed. Accordingly, the Tait equation (density) and Mamedov equation (viscosity) for pure liquid ethanol were used, as well as the Soave-Redlich-Kwong equation of state (density) and the Kendall-Monroe equation (viscosity) for SC−CO2 mixtures with ethanol. Finally, the behaviour of diffusivity with temperature, pressure, solvent density and in Stokes-Einstein coordinates was also studied. A set of hydrodynamic, free-volume and empirical equations from the literature was used to compare the experimental diffusivity values with those calculated. The best results were achieved by the free-volume models of Dymond-Hildebrand-Batchinsky (1.6%−5.7%), TLSMd (5.6%) and through the empirical correlations of Magalhães et al. (0.9%−3.7%). It was proved that the empirical equations of Magalhães et al. were particularly suitable to calculate the diffusivity of gallic acid under the studied conditions, either of pure liquid ethanol or SC−CO2 modified with ethanol.