28 resultados para Thioester-containing protein (TEP)


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Nedd4 and Nedd4-2 are ubiquitin-protein ligases known to regulate a number of membrane proteins including receptors and ion transporters. Regulation of the epithelial Na+ channel by Nedd4 and Nedd4-2 is mediated via interactions between the PY motifs of the epithelial sodium channel subunits and the Nedd4/Nedd4-2 WW domains. This example serves as a model for the regulation of other PY motif-containing ion channels by Nedd4 and Nedd4-2. We found that the carboxyl termini of the six voltage-gated Na+ (Na-v) channels contain typical PY motifs (PPXY), and a further Na-v contains a PY motif variant (LPXY). Not only did we demonstrate by Far-Western analysis that Nedd4 and Nedd4-2 interact with the PY motif-containing Na-v channels, but we also showed that these channels have conserved WW domain binding specificity. We further showed that the carboxyl termini fusion proteins of one central nervous system and one peripheral nervous system-derived Na+ channel (Na(v)1.2 and Na(v)1.7, respectively) are readily ubiquitinated by Nedd4-2. In Xenopus oocytes, Nedd4-2 strongly inhibited the activities of all three Na(v)s (Na(v)1.2, Na(v)1.7, and Na(v)1.8) tested. Interestingly, Nedd4 suppressed the activity of Na(v)1.2 and Na(v)1.7 but was a poor inhibitor of Na(v)1.8. Our results provide evidence that Nedd4 and Nedd4-2 are likely to be key regulators of specific neuronal Na-v channels in vivo.

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Bacterial DNA activates mouse macrophages, B cells, and dendritic cells in a TLR9-dependent manner. Although short ssCpG-containing phosphodiester oligonucleotides (PO-ODN) can mimic the action of bacterial DNA on macrophages, they are much less immunostimulatory than Escherichia coli DNA. In this study we have assessed the structural differences between E. coli DNA and PO-ODN, which may explain the high activity of bacterial DNA on macrophages. DNA length was found to be the most important variable. Double-strandedness was not responsible for the increased activity of long DNA. DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) methylation of E. coli DNA did not enhance macrophage NO production. The presence of two CpG motifs on one molecule only marginally improved activity at low concentration, suggesting that ligand-mediated TLR9 cross-linking was not involved. The major contribution was from DNA length. Synthetic ODN > 44 nt attained the same levels of activity as bacterial DNA. The response of macrophages to CpG DNA requires endocytic uptake. The length dependence of the CpG ODN response was found to correlate with the presence in macrophages of a length-dependent uptake process for DNA. This transport system was absent from B cells and fibroblasts.

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Oxysterol binding protein (OSBP) and its homologs have been shown to regulate lipid metabolism and vesicular transport. However, the exact molecular function of individual OSBP homologs remains uncharacterized. Here we demonstrate that the yeast OSBP homolog, Osh6p, bound phosphatidic acid and phosphoinositides via its N-terminal half containing the conserved OSBP-related domain (ORD). Using a green fluorescent protein fusion chimera, Osh6p was found to localize to the cytosol and patch-like or punctate structures in the vicinity of the plasma membrane. Further examination by domain mapping demonstrated that the N-terminal half was associated with FM4-64 positive membrane compartments; however, the C-terminal half containing a putative coiled-coil was localized to the nucleoplasm. Functional analysis showed that the deletion of OSH6 led to a significant increase in total cellular ergosterols, whereas OSH6 overexpression caused both a significant decrease in ergosterol levels and resistance to nystatin. Oleate incorporation into sterol esters was affected in OSH6 overexpressing cells. However, Lucifer yellow internalization, and FM4-64 uptake and transport were unaffected in both OSH6 deletion and overexpressing cells. Furthermore, osh6 Delta exhibited no defect in carboxypeptidase Y transport and maturation. Lastly, we demonstrated that both the conserved ORD and the putative coiled-coil motif were indispensable for the in vivo function of Osh6p. These data suggest that Osh6p plays a role primarily in regulating cellular sterol metabolism, possibly stero transport.

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The function of the prion protein gene (PRNP) and its normal product PrPC is elusive. We used comparative genomics as a strategy to understand the normal function of PRNP. As the reliability of comparisons increases with the number of species and increased evolutionary distance, we isolated and sequenced a 66.5 kb BAC containing the PRNP gene from a distantly related mammal, the model Australian marsupial Macropus eugenii (tammar wallaby). Marsupials are separated from eutherians such as human and mouse by roughly 180 million years of independent evolution. We found that tammar PRNP, like human PRNP, has two exons. Prion proteins encoded by the tammar wallaby and a distantly related marsupial, Monodelphis domestica (Brazilian opossum) PRNP contain proximal PrP repeats with a distinct, marsupial-specific composition and a variable number. Comparisons of tammar wallaby PRNP with PRNPs from human, mouse, bovine and ovine allowed us to identify non-coding gene regions conserved across the marsupial-eutherian evolutionary distance, which are candidates for regulatory regions. In the PRNP 3' UTR we found a conserved signal for nuclear-specific polyadenylation and the putative cytoplasmic polyadenylation element (CPE), indicating that post-transcriptional control of PRNP mRNA activity is important. Phylogenetic footprinting revealed conserved potential binding sites for the MZF-1 transcription factor in both upstream promoter and intron/intron 1, and for the MEF2, MyTI, Oct-1 and NFAT transcription factors in the intron(s). The presence of a conserved NFAT-binding site and CPE indicates involvement of PrPC in signal transduction and synaptic plasticity. (c) 2004 Elsevier B.V. All rights reserved.

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Objective: To investigate the effects of recombinant human activated protein C (rhAPC) on pulmonary function in acute lung injury (ALI) resulting from smoke inhalation in association with a bacterial challenge. Design: Prospective, randomized, controlled, experimental animal study with repeated measurements. Setting: Investigational intensive care unit at a university hospital. Subjects: Eighteen sheep (37.2 +/- 1.0 kg) were operatively prepared and randomly allocated to either the sham, control, or rhAPC group (n = 6 each). After a tracheotomy had been performed, ALI was produced in the control and rhAPC group by insufflation of 4 sets of 12 breaths of cotton smoke. Then, a 30 mL suspension of live Pseudomonas aeruginosa bacteria (containing 2-5 x 10(11) colony forming units) was instilled into the lungs according to an established protocol. The sham group received only the vehicle, i.e., 4 sets of 12 breaths of room air and instillation of 30 mL normal saline. The sheep were studied in the awake state for 24 hrs and were ventilated with 100% oxygen. RhAPC (24 mu g/kg/hr) was intravenously administered. The infusion was initiated 1 hr post-injury and lasted until the end of the experiment. The animals were resuscitated with Ringer's lactate solution to maintain constant pulmonary artery occlusion pressure. Measurements and Main Results., In comparison with nontreatment in controls, the infusion of rhAPC significantly attenuated the fall in PaO2/FiO(2) ratio (control group values were 521 +/- 22 at baseline [BL], 72 +/- 5 at 12 hrs, and 74 +/- 7 at 24 hrs, vs. rhAPC group values of 541 +/- 12 at BL, 151 +/- 29 at 12 hours [p < .05 vs. control], and 118 +/- 20 at 24 hrs), and significantly reduced the increase in pulmonary microvascular shunt fraction (Qs/Qt; control group at BL, 0.14 +/- 0.02, and at 24 hrs, 0.65 +/- 0.08; rhAPC group at BL, 0.24 +/- 0.04, and at 24 hrs, 0.45 +/- 0.02 [p < .05 vs. control]) and the increase in peak airway pressure (mbar; control group at BL, 20 +/- 1, and at 24 hrs, 36 +/- 4; rhAPC group at BL, 21 +/- 1, and at 24 hrs, 28 +/- 2 [p < .05 vs. control]). In addition, rhAPC limited the increase in lung 3-nitrotyrosine (after 24 hrs [%]: sham, 7 +/- 2; control, 17 +/- 1; rhAPC, 12 +/- 1 [p < .05 vs. control]), a reliable indicator of tissue injury. However, rhAPC failed to prevent lung edema formation. RhAPC-treated sheep showed no difference in activated clotting time or platelet count but exhibited less fibrin degradation products (1/6 animals) than did controls (4/6 animals). Conclusions. Recombinant human activated protein C attenuated ALI after smoke inhalation and bacterial challenge in sheep, without bleeding complications.

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In the process of internalization of molecules from the extracellular milieu, a cell uses multiple endocytic pathways, consequently generating different endocytic vesicles. These primary endocytic vesicles are targeted to specific destinations inside the cell. Here, we show that GPI-anchored proteins are internalized by an Arf6-independent mechanism into GPI-anchored protein-enriched early endosomal compartments (GEECs). Internalized GPI-anchored proteins and the fluid phase are first visualized in GEECs that are acidic, primary endocytic structures, negative for early endosomal markers, Rab4, Rab5, and early endosome antigen (EEA)1. They subsequently acquire Rab5 and EEA1 before homotypic fusion with other GEECs, and heterotypic fusion with endosomes containing cargo from the clathrin-dependent endocytic pathway. Although, the formation of GEECs is unaffected by inhibition of Rab5 GTPase and phosphatidylinositol-3'-kinase (PI3K) activity, their fusion with sorting endosomes is dependent on both activities. Overexpression of Rab5 reverts PI3K inhibition of fusion, providing evidence that Rab5 effectors play important roles in heterotypic fusion between the dynamin-independent GEECs and clathrin- and dynamin-dependent sorting endosomes.

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Purpose. The purpose of this study was to investigate the immunogenicity of liposomes containing mannosylated lipid core peptide (manLCP) constructs, both in vitro and in vivo, with or without the addition of the immune stimulating adjuvant Quil A. Methods. Mouse bone marrow dendritic cells (BMDC) were cultured with liposome formulations for 48 h, and the resulting level of BMDC activation was determined by flow cytometry. BMDC pulsed with liposome formulations were incubated with 5,6-carboxyfluoroscein diacetate succinimidyl ester-labeled T cells for 72 h and the resulting T cell proliferation was determined by flow cytometry. To investigate the immunogenicity of formulations in vivo, groups of C57Bl/6J mice were immunized by subcutaneous injection, and the resulting antigen-specific cytotoxic and protective immune responses toward tumor challenge evaluated. Results. Despite being unable to demonstrate the activation of BMDC, BMDC pulsed with liposomes containing manLCP constructs were able to stimulate the proliferation of naive T cells in vitro. However, in vivo only liposomes containing both manLCP and Quil A were able to stimulate a strong antigen-specific cytotoxic immune response. Liposomes containing manLCP and Quil A within the same particle were able to protect against the growth of tumor cells to a similar level as if the antigen was administered in alum with CD4 help. Conclusion. ManLCPs administered in liposomes are able to stimulate strong cytotoxic and protective immune responses if Quil A is also incorporated as an adjuvant.

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A common feature associated with the replication of most RNA viruses is the formation of a unique membrane environment encapsulating the viral replication complex. For their part, flaviviruses are no exception, whereupon infection causes a dramatic rearrangement and induction of unique membrane structures within the cytoplasm of infected cells. These virus-induced membranes, termed paracrystalline arrays, convoluted membranes, and vesicle packets, all appear to have specific functions during replication and are derived from different organelles within the host cell. The aim of this study was to identify which protein(s) specified by the Australian strain of West Nile virus, Kunjin virus (KUNV), are responsible for the dramatic membrane alterations observed during infection. Thus, we have shown using immunolabeling of ultrathin cryosections of transfected cells that expression of the KUNV polyprotein intermediates NS4A-4B and NS213-34A, as well as that of individual NS4A proteins with and without the C-terminal transmembrane domain 2K, resulted in different degrees of rearrangement of cytoplasmic membranes. The formation of the membrane structures characteristic for virus infection required coexpression of an NS4A-NS4B cassette with the viral protease NS2B-3pro which was shown to be essential for the release of the individual NS4A and NS4B proteins. Individual expression of NS4A protein retaining the C-terminal transmembrane domain 2K resulted in the induction of membrane rearrangements most resembling virus-induced structures, while removal of the 2K domain led to a less profound membrane rearrangement but resulted in the redistribution of the NS4A protein to the Golgi apparatus. The results show that cleavage of the KUNV polyprotein NS4A-4B by the viral protease is the key initiation event in the induction of membrane rearrangement and that the NS4A protein intermediate containing the uncleaved C-terminal transmembrane domain plays an essential role in these membrane rearrangements.

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Pyrin domain (PYD)-containing proteins are key components of pathways that regulate inflammation, apoptosis, and cytokine processing. Their importance is further evidenced by the consequences of mutations in these proteins that give rise to autoimmune and hyperinflammatory syndromes. PYDs, like other members of the death domain ( DD) superfamily, are postulated to mediate homotypic interactions that assemble and regulate the activity of signaling complexes. However, PYDs are presently the least well characterized of all four DD subfamilies. Here we report the three-dimensional structure and dynamic properties of ASC2, a PYD-only protein that functions as a modulator of multidomain PYD-containing proteins involved in NF-KB and caspase-1 activation. ASC2 adopts a six-helix bundle structure with a prominent loop, comprising 13 amino acid residues, between helices two and three. This loop represents a divergent feature of PYDs from other domains with the DD fold. Detailed analysis of backbone N-15 NMR relaxation data using both the Lipari-Szabo model-free and reduced spectral density function formalisms revealed no evidence of contiguous stretches of polypeptide chain with dramatically increased internal motion, except at the extreme N and C termini. Some mobility in the fast, picosecond to nanosecond timescale, was seen in helix 3 and the preceding alpha 2-alpha 3 loop, in stark contrast to the complete disorder seen in the corresponding region of the NALP1 PYD. Our results suggest that extensive conformational flexibility in helix 3 and the alpha 2-alpha 3 loop is not a general feature of pyrin domains. Further, a transition from complete disorder to order of the alpha 2-alpha 3 loop upon binding, as suggested for NALP1, is unlikely to be a common attribute of pyrin domain interactions.

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Two experiments were conducted to measure urea recycling and rumen flow dynamics in young rusa deer fed low (LP) or high (HP) protein diets. Pool size and flux rate of labelled urea. into and out of the blood pool were measured using single intravenous (i.v.) injection solutions containing [C-14] - and [N-15]-urea. A curve peeling technique was used to fit the enrichment of N-15 or specific radioactivity (SRA) of C-14 to exponential equations. Body urea-N pool size was significantly greater (P < 0.05) when a HP, compared to a LP diet, was fed. Urea space, expressed as a percent of live weight, total flux rate of urea through the blood pool and the irreversible loss of urea was similar for both diets. The mean (+/- S.E.M.) concentration of plasma urea-N was greater when animals were fed the HP diet compared to the LP diet (2 1.1 +/- 0.3 versus 14.4 +/- 1.4 mg/100 ml, respectively). Voluntary feed intake and digestibility of dietary components were also measured. Daily dry matter intakes were not affected by the crude protein (CP) content of the diet, although apparent DM digestibility was significantly greater for HP diet fed in both experiments. An intraruminal infusion of CrEDTA was used to determine rumen flow dynamics. Ruminal mean retention time, relative net outflow rate of water and passage rate constant (k(w)) were significantly greater (P < 0.05) when the HP diet was fed compared to the LP diet. The extent of urea metabolism and flux rates of urea between the blood and secondary pools appear similar to those previously reported for other ruminants fed diets contrasting in CP content. (c) 2005 Elsevier B.V. All rights reserved.

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The virulence of Pseudomonas aeruginosa and other surface pathogens involves the coordinate expression of a wide range of virulence determinants, including type IV pili. These surface filaments are important for the colonization of host epithelial tissues and mediate bacterial attachment to, and translocation across, surfaces by a process known as twitching motility. This process is controlled in part by a complex signal transduction system whose central component, ChpA, possesses nine potential sites of phosphorylation, including six histidine-containing phosphotransfer (HPt) domains, one serine-containing phosphotransfer domain, one threonine-containing phosphotransfer domain, and one CheY-like receiver domain. Here, using site-directed mutagenesis, we show that normal twitching motility is entirely dependent on the CheY-like receiver domain and partially dependent on two of the HPt domains. Moreover, under different assay conditions, point mutations in several of the phosphotransfer domains of ChpA give rise to unusual "swarming" phenotypes, possibly reflecting more subtle perturbations in the control of P. aeruginosa motility that are not evident from the conventional twitching stab assay. Together, these results suggest that ChpA plays a central role in the complex regulation of type IV pilus-mediated motility in P. aeruginosa

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To determine if low dietary protein concentration in the first two trimesters of pregnancy alters placental development, genetically similar heifers from closed herd were fed diets containing different levels of protein in the first and second trimesters of gestation. There were four animals per treatment group, the groups being: L/L = fed a diet containing 7% crude protein (CP) (low protein) in the first and second trimesters; H/H = fed a diet containing 14% (P thigh protein) in the first and second trimesters; L/H = fed low protein in the first trimester and high in the second trimester and vice versa for the H/L group. Low protein diets in the first trimester increased dry cotyledon weight at term. Trophectoderm volume density increased in the H/L and L/H group compared to the L/L and H/H groups. Blood vessel volume and volume density in foetal villi decreased in the H/L and L/H groups compared with the H/H and L/L groups. There was no effect of diet treatment on cotyledon number, diameter or wet weight and no effect on the volume density of connective tissue or fibroblasts in the foetal villi. These results show that a low dietary protein concentration in the first trimester of pregnancy followed by increased protein in the second trimester enhanced placental development. Further, trophectoderm volume was highly correlated with birth weight. Early protein restriction in the pregnant cow may enhance foetal growth in part by stimulating placental growth and function. (C) 1999 Published by Elsevier Science B.V. All rights reserved.

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Plant resistance proteins (R proteins) recognize corresponding pathogen avirulence (Avr) proteins either indirectly through detection of changes in their host protein targets or through direct R-Avr protein interaction. Although indirect recognition imposes selection against Avr effector function, pathogen effector molecules recognized through direct interaction may overcome resistance through sequence diversification rather than loss of function. Here we show that the flax rust fungus AvrLS67 genes, whose products are recognized by the L5, L6, and L7 R proteins of flax, are highly diverse, with 12 sequence variants identified from six rust strains. Seven AvrL567 variants derived from Avr alleles induce necrotic responses when expressed in flax plants containing corresponding resistance genes (R genes), whereas five variants from avr alleles do not. Differences in recognition specificity between AvA567 variants and evidence for diversifying selection acting on these genes suggest they have been involved in a gene-specific arms race with the corresponding flax R genes. Yeast two-hybrid assays indicate that recognition is based on direct R-Avr protein interaction and recapitulate the interaction specificity observed in planta. Biochemical analysis of Escherichia coli-produced AvrL567 proteins shows that variants that escape recognition nevertheless maintain a conserved structure and stability, suggesting that the amino acid sequence differences directly affect the R-Avr protein interaction. We suggest that direct recognition associated with high genetic diversity at corresponding R and Avr gene loci represents an alternative outcome of plant-pathogen coevolution to indirect recognition associated with simple balanced polymorphisms for functional and nonfunctional R and Avr genes.