An interaction between two RNA binding proteins, Nab2 and Pub1, links mRNA Processing/Export and mRNA stability

mRNA stability is modulated by elements in the mRNA transcript and their cognate RNA binding proteins. Poly(U) binding protein 1 (Pub1) is a cytoplasmic Saccharomyces cerevisiae mRNA binding protein that stabilizes transcripts containing AU-rich elements (AREs) or stabilizer elements (STEs). In a yeast two-hybrid screen, we identified nuclear poly(A) binding protein 2 (Nab2) as being a Pub1-interacting protein. Nab2 is an essential nucleocytoplasmic shuttling mRNA binding protein that regulates poly(A) tail length and mRNA export. The interaction between Pub1 and Nab2 was confirmed by copurification and in vitro binding assays. The interaction is mediated by the Nab2 zinc finger domain. Analysis of the functional link between these proteins reveals that Nab2, like Pub1, can modulate the stability of specific mRNA transcripts. The half-life of the RPS16B transcript, an ARE-like sequence-containing Pub1 target, is decreased in both nab2-1 and nab2-67 mutants. In contrast, GCN4, an STE-containing Pub1 target, is not affected. Similar results were obtained for other ARE- and STE-containing Pub1 target transcripts. Further analysis reveals that the ARE-like sequence is necessary for Nab2-mediated transcript stabilization. These results suggest that Nab2 functions together with Pub1 to modulate mRNA stability and strengthen a model where nuclear events are coupled to the control of mRNA turnover in the cytoplasm.

Heparin-binding proteins of canine seminal plasma

Heparin-binding proteins (HBP) from seminal plasma have been expected to participate in modulation of the acrosomal reaction, and have been correlated with fertility in some species. However, they have not been described in the dog. The aim of this study was to document the HBPs of canine seminal plasma. Six pooled samples of seminal plasma from three crossbred dogs were used. The HBPs were isolated by heparin affinity chromatography and the fractions recovered were pooled. One-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out on 12 and 18% vertical minigels. The stained gels were scanned and the molecular weight (kDa) values for each band within a lane were calculated by image analysis software. The electrophoresis analysis of the pooled eluded fractions identified 19 bands, with molecular weights varying from 61.5 to 5.2 kDa. Previous studies, using one-dimensional SDS-PAGE, identified two bands (67 and 58.6 kDa), which were positively correlated with some semen parameters (sperm motility, sperm vigor, percentage of morphologically normal sperm and plasma membrane integrity). The 61.5 kDa band detected in the present study apparently corresponded to the 58.6 kDa band identified previously. Canine seminal plasma contained HBP; since HBP modulate the acrosome reaction in other species, they may have the same function in the dog. Further studies are necessary to better characterize this protein and determine if it is associated with fertility in the dog. (c) 2006 Elsevier B.V. All rights reserved.

Heparin-binding proteins of seminal plasma in Nellore bulls

O objetivo deste estudo foi identificar proteínas ligadoras à heparina no plasma seminal de touros Nelore (Bos taurus indicus). Para tanto, foram selecionados quatro touros entre 30 e 36 meses de idade e peso aproximado de 500-550kg. Após centrifugação, amostras do plasma seminal foram misturadas e as proteínas ligadoras à heparina foram isoladas por meio da cromatografia por afinidade. As frações após a eluição foram agrupadas para caracterização das bandas protéicas (SDSPAGE, 12,5%). Foram identificadas oito bandas protéicas variando entre 15 e 63kDa. Duas proteínas com 22 e 25kDa foram similares às descritas em touros Bos taurus taurus. Outras proteínas identificadas com 39, 53, 58 e 63kDa ainda não foram descritas e possivelmente sejam específicas para Bos taurus indicus.

A systematic approach to identify STRE-binding proteins of the gsn glycogen synthase gene promoter in Neurospora crassa

The gene encoding glycogen synthase in Neurospora crassa (gsn) is transcriptionally down-regulated when mycelium is exposed to a heat shock from 30 to 45 degrees C. The gsn promoter has one stress response element (STRE) motif that is specifically bound by heat shock activated nuclear proteins. In this work, we used biochemical approaches together with mass spectrometric analysis to identify the proteins that bind to the STRE motif and could participate in the gsn transcription regulation during heat shock. Crude nuclear extract of heat-shocked mycelium was prepared and fractionated by affinity chromatography. The fractions exhibiting DNA-binding activity were identified by electrophoretic mobility shift assay (EMSA) using as probe a DNA fragment containing the STRE motif DNA-protein binding activity was confirmed by Southwestern analysis. The molecular mass (MM) of proteins was estimated by fractionating the crude nuclear extract by SDS-PAGE followed by EMSA analysis of the proteins corresponding to different MM intervals. Binding activity was detected at the 30-50 MM kDa interval. Fractionation of the crude nuclear proteins by IEF followed by EMSA analysis led to the identification of two active fractions belonging to the pIs intervals 3.54-4.08 and 6.77-7.31. The proteins comprising the MM and pI intervals previously identified were excised from a 2-DE gel, and subjected to mass spectrometric analysis (MALDI-TOF/TOF) after tryptic digestion. The proteins were identified by search against the MIPS and MIT N. crassa databases and five promising candidates were identified. Their structural characteristics and putative roles in the gsn transcription regulation are discussed.

Temporal changes in calcium-binding proteins in the medial geniculate nucleus of the monkey Sapajus apella

The subdivisions of the medial geniculate complex can be distinguished based on the immunostaining of calcium-binding proteins and by the properties of the neurons within each subdivision. The possibility of changes in neurochemistry in this and other central auditory areas are important aspects to understand the basis that contributing to functional variations determined by environmental cycles or the animal's cycles of activity and rest. This study investigated, for the first time, day/night differences in the amounts of parvalbumin-, calretinin- and calbindin-containing neurons in the thalamic auditory center of a non-human primate, Sapajus apella. The immunoreactivity of the PV-IR, CB-IR and CR-IR neurons demonstrated different distribution patterns among the subdivisions of the medial geniculate. Moreover, a high number of CB- and CR-IR neurons were found during day, whereas PV-IR was predominant at night. We conclude that in addition to the chemical heterogeneity of the medial geniculate nucleus with respect to the expression of calcium-binding proteins, expression also varied relative to periods of light and darkness, which may be important for a possible functional adaptation of central auditory areas to environmental changes and thus ensure the survival and development of several related functions.

Use of a synthetic lethal screen to identify genes related to TIF51A in Saccharomyces cerevisiae

The putative eukaryotic translation initiation factor 5A (eIF5A) is an essential protein for cell viability and the only cellular protein known to contain the unusual amino acid residue hypusine. eIF5A has been implicated in translation initiation, cell proliferation, nucleocytoplasmic transport, mRNA decay, and actin polarization, but the precise biological function of this protein is not clear. However, eIF5A was recently shown to be directly involved with the translational machinery. A screen for synthetic lethal mutations was carried out with one of the temperature-sensitive alleles of TIF51A (tif51A-3) to identify factors that functionally interact with eIF5A and revealed the essential gene YPT1. This gene encodes a small GTPase, a member of the rab family involved with secretion, acting in the vesicular trafficking between endoplasmatic reticulum and the Golgi. Thus, the synthetic lethality between TIF51A and YPT1 may reveal the connection between translation and the polarized distribution of membrane components, suggesting that these proteins work together in the cell to guarantee proper protein synthesis and secretion necessary for correct bud formation during G1/ S transition. Future studies will investigate the functional interaction between eIF5A and Ypt1 in order to clarify this involvement of eIF5A with vesicular trafficking. ©FUNPEC-RP.

Positive selection results in frequent reversible amino acid replacements in the G protein gene of human respiratory syncytial virus

Human respiratory syncytial virus (HRSV) is the major cause of lower respiratory tract infections in children under 5 years of age and the elderly, causing annual disease outbreaks during the fall and winter. Multiple lineages of the HRSVA and HRSVB serotypes co-circulate within a single outbreak and display a strongly temporal pattern of genetic variation, with a replacement of dominant genotypes occurring during consecutive years. In the present study we utilized phylogenetic methods to detect and map sites subject to adaptive evolution in the G protein of HRSVA and HRSVB. A total of 29 and 23 amino acid sites were found to be putatively positively selected in HRSVA and HRSVB, respectively. Several of these sites defined genotypes and lineages within genotypes in both groups, and correlated well with epitopes previously described in group A. Remarkably, 18 of these positively selected tended to revert in time to a previous codon state, producing a flipflop phylogenetic pattern. Such frequent evolutionary reversals in HRSV are indicative of a combination of frequent positive selection, reflecting the changing immune status of the human population, and a limited repertoire of functionally viable amino acids at specific amino acid sites.

Splice variants of the human ZC3H14 gene generate multiple isoforms of a zinc finger polyadenosine RNA binding protein

The human ZC3H14 gene encodes an evolutionarily conserved Cys(3)His zinc finger protein that binds specifically to polyadenosine RNA and is thus postulated to modulate post-transcriptional gene expression. Expressed sequence tag (EST) data predicts multiple splice variants of both human and mouse ZC3H14. Analysis of ZC3H14 expression in both human cell lines and mouse tissues confirms the presence of multiple alternatively spliced transcripts. Although all of these transcripts encode protein isoforms that contain the conserved C-terminal zinc finger domain, suggesting that they could all bind to polyadenosine RNA, they differ in other functionally important domains. Most of the alternative transcripts encode closely related proteins (termed isoforms 1, 2. 3, and 3short) that differ primarily in the inclusion of three small exons, 9, 10, and 11, resulting in predicted protein isoforms ranging from 82 to 64 kDa. Each of these closely related isoforms contains predicted classical nuclear localization signals (cNLS) within exons 7 and 11. Consistent with the presence of these putative nuclear targeting signals, these ZC3H14 isoforms are all localized to the nucleus. In contrast, an additional transcript encodes a smaller protein (34 kDa) with an alternative first exon (isoform, 4). Consistent with the absence of the predicted cNLS motifs located in exons 7 and 11, ZC3H14 isoform 4 is localized to the cytoplasm. Both EST data and experimental data suggest that this variant is enriched in testes and brain. Using an antibody that detects endogenous ZC3H14 isoforms 1-3 reveals localization of these isoforms to nuclear speckles. These speckles co-localize with the splicing factor, SC35, suggesting a role for nuclear ZC3H14 in mRNA processing. Taken together, these results demonstrate that multiple transcripts encoding several ZC3H14 isoforms exist in vivo. Both nuclear and cytoplasmic ZC3H14 isoforms could have distinct effects on gene expression mediated by the common Cys(3)His zinc finger polyadenosine RNA binding domain. (C) 2009 Elsevier B.V. All rights reserved.

LaTBP1: A Leishmania amazonensis DNA-binding protein that associates in vivo with telomeres and GT-rich DNA using a Myb-like domain

Different species of Leishmania can cause a variety of medically important diseases, whose control and treatment are still health problems. Telomere binding proteins (TBPs) have potential as targets for anti-parasitic chemotherapy because of their importance for genome stability and cell viability. Here, we describe LaTBP1 a protein that has a Myb-like DNA-binding domain, a feature shared by most double-stranded telomeric proteins. Binding assays using full-length and truncated LaTBP1 combined with spectroscopy analysis were used to map the boundaries of the Myb-like domain near to the protein only tryptophan residue. The Myb-like domain of LaTBP1 contains a conserved hydrophobic cavity implicated in DNA-binding activity. A hypothetical model helped to visualize that it shares structural homology with domains of other Myb-containing proteins. Competition assays and chromatin immunoprecipitation confirmed the specificity of LaTBP1 for telomeric and GT-rich DNAs, suggesting that LaTBP1 is a new TBP. (C) 2007 Elsevier B.V. All rights reserved.

Proteomic profiling of the molecular targets of interactions of the mastoparan peptide Protopolybia MP-III at the level of endosomal membranes from rat mast cells

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

Macrophage suppression following phagocytosis of apoptotic neutrophils is mediated by the S100A9 calcium-binding protein

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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.

Plasma levels of transthyretin and retinol-binding protein in child-a cirrhotic patients in relation to protein-calorie status and plasma amino acids, zinc, vitamin a and plasma thyroid hormones

Transthyretin and retinal-binding protein are sensitive markers of acute protein-calorie malnutrition both for early diagnosis and dietary evaluation. A preliminary study showed that retinal-binding protein is the most sensitive marker of protein-calorie malnutrition in cirrhotic patients, even those with the mild form of the disease (Child A). However, in addition to being affected by protein-calorie malnutrition, the levels of these short half-life-liver-produced proteins are also influenced by other factors of a nutritional (zinc, tryptophan, vitamin A, etc) and non-nutritional (sex, aging, hormones, renal and liver functions and inflammatory activity) nature. These interactions were investigated in 11 adult male patients (49.9 ± 9.2 years of age) with alcoholic cirrhosis (Child-Pugh grade A) and with normal renal function. Both transthyretin and retinol binding protein were reduced below normal levels in 55% of the patients, in close agreement with their plasma levels of retinal. In 67% of the patients (4/6), the reduced levels of transthyretin and retinal-binding protein were caused by altered liver function and in 50% (3/6) they were caused by protein-calorie malnutrition. Thus, the present data, taken as a whole, indicate that reduced transthyretin and retinal-binding protein levels in mild cirrhosis of the liver are mainly due to liver failure and/or vitamin A status rather than representing an isolated protein-calorie malnutrition indicator.

Arginine methylation and binding of Hrp1p to the efficiency element for mRNA 3'-end formation

Hrp1p is a heterogeneous ribonucleoprotein (hnRNP) from the yeast Saccharomyces cerevisiae that is involved in the cleavage and polyadenylation of the 3'-end of mRNAs and mRNA export. In addition, Hrp1p is one of several RNA-binding proteins that are posttranslationally modified by methylation at arginine residues. By using-functional recombinant Hrp1p, we have identified RNA sequences with specific high affinity binding sites. These sites correspond to the efficiency element for mRNA 3'-end formation, UAUAUA. To examine the effect of methylation on specific RNA binding, purified recombinant arginine methyltransferase (Hmt1p) was used to methylate Hrp1p. Methylated Hrp1p binds with the same affinity to UAUAUA-containing RNAs as unmethylated Hrp1p indicating that methylation does not affect specific RNA binding. However, RNA itself inhibits the methylation of Hrp1p and this inhibition is enhanced by RNAs that specifically bind Hrp1p. Taken together, these data support a model in which protein methylation occurs prior to protein-RNA binding in the nucleus.

Genetic interactions of yeast eukaryotic translation initiation factor 5a (eIF5A) reveal connections to poly(A)-binding protein and protein kinase C signaling

The highly conserved eukaryotic translation initiation factor eIF5A has been proposed to have various roles in the cell, from translation to mRNA decay to nuclear protein export. To further our understanding of this essential protein, three temperature-sensitive alleles of the yeast TIF51A gene have been characterized. Two mutant eIF5A proteins contain mutations in a proline residue at the junction between the two eIFSA domains and the third, strongest allele encodes a protein with a single mutation in each domain, both of which are required for the growth defect. The stronger tif51A alleles cause defects in degradation of short-lived mRNAs, supporting a role for this protein in mRNA decay. A multicopy suppressor screen revealed six genes, the overexpression of which allows growth of a tif51A-1 strain at high temperature; these genes include PAB1, PKC1, and PKC1 regulators WSC1, WSC2, and WSC3. Further results suggest that eIFSA may also be involved in ribosomal synthesis and the WSC/PKC1 signaling pathway for cell wall integrity or related processes.