An antiviral response directed by PKR phosphorylation of the RNA helicase A

The double-stranded RNA-activated protein kinase R (PKR) is a key regulator of the innate immune response. Activation of PKR during viral infection culminates in phosphorylation of the α subunit of the eukaryotic translation initiation factor 2 (eIF2α) to inhibit protein translation. A broad range of regulatory functions has also been attributed to PKR. However, as few additional PKR substrates have been identified, the mechanisms remain unclear. Here, PKR is shown to interact with an essential RNA helicase, RHA. Moreover, RHA is identified as a substrate for PKR, with phosphorylation perturbing the association of the helicase with double-stranded RNA (dsRNA). Through this mechanism, PKR can modulate transcription, as revealed by its ability to prevent the capacity of RHA to catalyze transactivating response (TAR)–mediated type 1 human immunodeficiency virus (HIV-1) gene regulation. Consequently, HIV-1 virions packaged in cells also expressing the decoy RHA peptides subsequently had enhanced infectivity. The data demonstrate interplay between key components of dsRNA metabolism, both connecting RHA to an important component of innate immunity and delineating an unanticipated role for PKR in RNA metabolism.

Cotranslational assembly of the yeast SET1C histone methyltransferase complex

 While probing the role of RNA for the function of SET1C/COMPASS histone methyltransferase, we identified SET1RC (SET1 mRNA-associated complex), a complex that contains SET1 mRNA and Set1, Swd1, Spp1 and Shg1, four of the eight polypeptides that constitute SET1C. Characterization of SET1RC showed that SET1 mRNA binding did not require associated Swd1, Spp1 and Shg1 proteins or RNA recognition motifs present in Set1. RNA binding was not observed when Set1 protein and SET1 mRNA were derived from independent genes or when SET1 transcripts were restricted to the nucleus. Importantly, the protein-RNA interaction was sensitive to EDTA, to the translation elongation inhibitor puromycin and to the inhibition of translation initiation in prt1-1 mutants. Taken together, our results support the idea that SET1 mRNA binding was dependent on translation and that SET1RC assembled on nascent Set1 in a cotranslational manner. Moreover, we show that cellular accumulation of Set1 is limited by the availability of certain SET1C components, such as Swd1 and Swd3, and suggest that cotranslational protein interactions may exert an effect in the protection of nascent Set1 from degradation.

Anti-metastatic and differential effects on protein expression of epigallocatechin-3-gallate in HCCLM6 hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide and the third highest cause of cancer-related mortality in humans. Epigallocatechin-3-gallate (EGCG) has been shown to inhibit the metastatic activity of certain cancer cells. The aim of this study was to determine the effects and molecular mechanism(s) of action of EGCG in human HCC cells. A migration and invasion assay for the metastatic behavior of HCCLM6 cells was performed. The anti-metastatic effects of EGCG were investigated by RT-PCR and gelatin zymography. A total cellular protein profile was obtained using 2-dimensional gel electrophoresis (2-DE), followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) analyses of proteins with significant differences in expression following treatment with EGCG. The results revealed that EGCG induced apoptosis and inhibited the metastasis of HCCLM6 cells. The anti-metastatic effects of EGCG were associated with the inhibition of matrix metalloproteinase (MMP)-2 and MMP-9 activity. The expression levels of far upstream element (FUSE) binding protein 1 (FUBP1), heat shock protein beta 1 (HSPB1), heat shock 60 kDa protein 1 (chaperonin) (CH60) and nucleophosmin (NPM) proteins, which are associated with metastasis, were significantly altered in the EGCG-treated HCCLM6 cells. The data from the present study suggest that EGCG has potential as a therapeutic agent for the treatment of HCC.

Binding to DNA Protects Neisseria meningitidis Fumarate and Nitrate Reductase Regulator (FNR) from Oxygen

Here, we report the overexpression, purification, and characterization of the transcriptional activator fumarate and nitrate reductase regulator from the pathogenic bacterium Neisseria meningitidis (NmFNR). Like its homologue from Escherichia coli (EcFNR), NmFNR binds a 4Fe-4S cluster, which breaks down in the presence of oxygen to a 2Fe-2S cluster and subsequently to apo-FNR. The kinetics of NmFNR cluster disassembly in the presence of oxygen are 2–3× slower than those previously reported for wild-type EcFNR, but similar to constitutively active EcFNR* mutants, consistent with earlier work in which we reported that the activity of FNR-dependent promoters in N. meningitidis is only weakly inhibited by the presence of oxygen (Rock, J. D., Thomson, M. J., Read, R. C., and Moir, J. W. (2007) J. Bacteriol. 189, 1138–1144). NmFNR binds to DNA containing a consensus FNR box sequence, and this binding stabilizes the iron-sulfur cluster in the presence of oxygen. Partial degradation of the 4Fe-4S cluster to a 3Fe-4S occurs, and this form remains bound to the DNA. The 3Fe-4S cluster is converted spontaneously back to a 4Fe-4S cluster under subsequent anaerobic reducing conditions in the presence of ferrous iron. The finding that binding to DNA stabilizes FNR in the presence of oxygen such that it has a half-life of ∼30 min on the DNA has implications for our appreciation of how oxygen switches off FNR activatable genes in vivo.

Cytokine expression and secretion by skeletal muscle cells: regulatory mechanisms and exercise effects

Cytokines are important mediators of various aspects of health and disease, including appetite, glucose and lipid metabolism, insulin sensitivity, skeletal muscle hypertrophy and atrophy. Over the past decade or so, considerable attention has focused on the potential for regular exercise to counteract a range of disease states by modulating cytokine production. Exercise stimulates moderate to large increases in the circulating concentrations of interleukin (IL)-6, IL-8, IL- 10, IL-1 receptor antagonist, granulocyte-colony stimulating factor, and smaller increases in tumor necrosis factor-α, monocyte chemotactic protein-1, IL-1β, brain-derived neurotrophic factor, IL-12p35/p40 and IL-15. Although many of these cytokines are also expressed in skeletal muscle, not all are released from skeletal muscle into the circulation during exercise. Conversely, some cytokines that are present in the circulation are not expressed in skeletal muscle after exercise. The reasons for these discrepant cytokine responses to exercise are unclear. In this review, we address these uncertainties by summarizing the capacity of skeletal muscle cells to produce cytokines, analyzing other potential cellular sources of circulating cytokines during exercise, and discussing the soluble factors and intracellular signaling pathways that regulate cytokine synthesis (e.g., RNA-binding proteins, microRNAs, suppressor of cytokine signaling proteins, soluble receptors).

The bacterial chromosome segregation protein Spo0J spreads along DNA from parS nucleation sites

Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.

The Saccharomyces cerevisiae COQ10 gene encodes a START domain protein required for function of coenzyme Q in respiration

Deletion of the Saccharomyces cerevisiae gene YOL008W, here referred to as COQ10, elicits a respiratory defect as a result of the inability of the mutant to oxidize NADH and succinate. Both activities are restored by exogenous coenzyme Q(2). Respiration is also partially rescued by COQ2, COQ7, or COQ8/ABC1, when these genes are present in high copy. Unlike other coq mutants, all of which lack Q(6), the coq10 mutant has near normal amounts of Q(6) in mitochondria. Coq10p is widely distributed in bacteria and eukaryotes and is homologous to proteins of the aromatic-rich protein family Pfam03654 and to members of the START domain superfamily that have a hydrophobic tunnel implicated in binding lipophilic molecules such as cholesterol and polyketides. Analysis of coenzyme Q in polyhistidine-tagged Coq10p purified from mitochondria indicates the presence 0.032-0.034 mol of Q(6)/mol of protein. We propose that Coq10p is a Q(6)-binding protein and that in the coq10 mutant Q(6) it is not able to act as an electron carrier, possibly because of improper localization.

Leishmania replication protein A-1 binds in vivo single-stranded telomeric DNA

Replication protein A (RPA) is a highly conserved heterotrimeric single-stranded DNA-binding protein involved in different events of DNA metabolism. In yeast, subunits 1 (RPA-1) and 2 (RPA-2) work also as telomerase recruiters and, in humans, the complex unfolds G-quartet structures formed by the 3' G-rich telomeric strand. In most eukaryotes, RPA-1 and RPA-2 bind DNA using multiple OB fold domains. In trypanosomatids, including Leishmania, RPA-1 has a canonical OB fold and a truncated RFA-1 structural domain. In Leishmania amazonensis, RPA-1 alone can form a complex in vitro with the telomeric G-rich strand. In this work, we show that LaRPA-1 is a nuclear protein that associates in vivo with Leishmania telomeres. We mapped the boundaries of the OB fold DNA-binding domain using deletion mutants. Since Leishmania and other trypanosomatids lack homologues of known telomere end binding proteins, our results raise questions about the function of RPA-1 in parasite telomeres. (C) 2007 Elsevier B.V. All rights reserved.

DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Protein-energy status and 15N-glycine kinetic study of child a cirrhotic patients fed low- to high-protein energy diets

In five male cirrhotic patients (Child A) and in four age- and sex-matched healthy control subjects, whole-body protein turnover was measured using a single oral dose of N-15-glycine as a tracer and urinary ammonia as end product. Subjects were studied in the fasting and feeding state, with different levels of protein and energy intake. The patients were underweight and presented lower plasma transthyretin and retinol-binding protein levels. When compared with controls, the kinetic studies showed patients to be hypometabolic in the fasting (Do) state and with the control diet [D-1 = (0.85 g of protein/154 kJ). kg(-1). day(-1)]. However, when corrected by body weight, the kinetic differences between groups disappeared, whereas the N-retention in the feeding state showed better results for the patients due mainly to their efficient breakdown decrease. When fed high-level protein or energy diets [D-2 = (0.9 g protein/195 kJ) and D-3 = (1.56 g protein/158 kJ). kg(-1). day(-1)], the patients showed D-0 = D-1 = D-2 < D-3 for N-flux and (D-0 = D-1) < D-3 (D-2 is intermediary) for protein synthesis. Thus, the present data suggest that the remaining mass of the undernourished mild cirrhotic patients has fairly good protein synthesis activity and also that protein, rather than energy intake, would be the limiting factor for increasing their whole-body protein synthesis.

Laminin-binding epitope on gp43 from Paracoccidioides brasiliensis is recognized by a monoclonal antibody raised against Staphylococcus aureus laminin receptor

Adhesion is regarded as an important step in the pathogenesis of several microorganisms. Thus, the ability to recognize extracellular matrix proteins, such as laminin or fibronectin, has been correlated with invasiveness. Studying the already characterized laminin-binding protein of Paracoccidioides brasiliensis, the 43 kDa glycoprotein (gp43), we evaluated whether MAb 1.H12, raised against the laminin-binding protein from Staphylococcus aureus, cross-reacts with that fungal protein. By immunoblot analysis we show that MAb 1.H12 recognizes gp43. This interaction is able to inhibit the laminin-mediated adhesion to epithelial cells as well as the P. brasiliensis infection in vivo. Moreover, through immunoenzymatic assays, we show that MAb 1.H12 recognizes gp43 in solid phase and that this interaction is partially inhibited by the addition of anti-gp43 MAbs. These results show that MAb 1.H12 recognizes the gp43, suggesting the presence of an epitope similar to those found in the other laminin-binding proteins from phylogenetically very distant cells. These findings reinforce the possibility of evolutionary conservation of such epitopes.

Preparation of N2, N2,7-trimethylguanosine affinity columns

2,2,7-trimethylguanosine (TMG) binding proteins from human cells were purified through TMG-affinity columns. TMG synthesis was improved and the TMG obtained was shown to be similar to the TMG in the 5' cap of the UsnRNAs. The eluates obtained with TMG-affinity chromatographies were very different from those isolated with m7G-affinity columns, thus suggesting that specific TMG- binding proteins were obtained. The fraction may be enriched with factors associated with import and/or hypermethylation of UsnRNPs.

Conserved sequences in the β subunit of archaeal and eukaryal translation initiation factor 2 (elF2), absent from elF5, mediate interaction with elF2γ

The eukaryotic translation initiation factor 2 (eIF2) binds the methionyl-initiator tRNA in a GTP-dependent mode. This complex associates with the 40 S ribosomal particle, which then, with the aid of other factors, binds to the 5' end of the mRNA and migrates to the first AUG codon, where eIF5 promotes GTP hydrolysis, followed by the formation of the 80 S ribosome. Here we provide a comparative sequence analysis of the β subunit of eIF2 and its archaeal counterpart (aIF2β). aIF2β differs from eIF2β in not possessing an N-terminal extension implicated in binding RNA, eIF5 and eIF2B. The remaining sequences are highly conserved, and are shared with eIF5. Previously isolated mutations in the yeast eIF2β, which allow initiation of translation at UUG codons due to the uncovering of an intrinsic GTPase activity in eIF2, involve residues that are conserved in aIF2β, but not in eIF5. We show that the sequence of eIF2B homologous to aIF2β is sufficient for binding eIF2γ, the only subunit with which it interacts, and comprises, at the most, 78 residues, eIF5 does not interact with eIF2γ, despite its similarity with eIF2β, probably because of a gap in homology in this region. These observations have implications for the evolution of the mechanism of translation initiation.

The plant energy-dissipating mitochondrial systems: Depicting the genomic structure and the expression profiles of the gene families of uncoupling protein and alternative oxidase in monocots and dicots

The simultaneous existence of alternative oxidases and uncoupling proteins in plants has raised the question as to why plants need two energy-dissipating systems with apparently similar physiological functions. A probably complete plant uncoupling protein gene family is described and the expression profiles of this family compared with the multigene family of alternative oxidases in Arabidopsis thaliana and sugarcane (Saccharum sp.) employed as dicot and monocot models, respectively. In total, six uncoupling protein genes, AtPUMP1-6, were recognized within the Arabidopsis genome and five (SsPUMP1-5) in a sugarcane EST database. The recombinant AtPUMP5 protein displayed similar biochemical properties as AtPUMP1. Sugarcane possessed four Arabidopsis AOx1-type orthologues (SsAOx1a-1d); no sugarcane orthologue corresponding to Arabidopsis AOx2-type genes was identified. Phylogenetic and expression analyses suggested that AtAOx1d does not belong to the AOx1-type family but forms a new (AOx3-type) family. Tissue-enriched expression profiling revealed that uncoupling protein genes were expressed more ubiquitously than the alternative oxidase genes. Distinct expression patterns among gene family members were observed between monocots and dicots and during chilling stress. These findings suggest that the members of each energy-dissipating system are subject to different cell or tissue/organ transcriptional regulation. As a result, plants may respond more flexibly to adverse biotic and abiotic conditions, in which oxidative stress is involved. © The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.

Inhibition of eukaryotic translation initiation factor 5A (eIF5A) hypusination impairs melanoma growth

The eukaryotic translation initiation factor 5A (eIF5A) undergoes a specific post-translational modification called hypusination. This modification is required for the functionality of this protein. The compound N1-guanyl-1,7-diaminoheptane (GC7) is a potent and selective inhibitor of deoxyhypusine synthase, which catalyses the first step of eIF5A hypusination process. In the present study, the effects of GC7 on cell death were investigated using two cell lines: melan-a murine melanocytes and Tm5 marine melanoma. In vitro treatment with GC7 increased by 3-fold the number of cells presenting DNA fragmentation in Tm5 cells. Exposure to GC7 also decreased viability to both cell lines. This study also describes, for the first time, the in vivo antitumour effect of GC7, as indicated by impaired melanoma growth in C57BL/6 mice. Copyright © 2006 John Wiley & Sons, Ltd.