Crystal Structures of Xanthomonas Small Heat Shock Protein Provide a Structural Basis for an Active Molecular Chaperone Oligomer

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

Isolation and in vitro hydrolysis of lentil protein fractions by trypsin

The albumin and globulin fractions from lentil seeds were isolated and characterised by gel filtration. The latter was shown to be homogeneous and the former heterogeneous on PAGE. The aminoacid analysis revealed high values of amidic amino acids for both fractions with great differences in the sulphur-containing amino acids. Native albumin, globulin and salt-soluble proteins were markedly resistant to trypsin hydrolysis compared to casein. The SDS-PAGE of native salt-soluble proteins indicated that the globulin fragments (20 to 30 kD) were slowly digested in the presence of albumin. The heating increased the hydrolysis of the proteins in the order: salt-soluble, albumin and globulin. The facilitated hydrolysis of the heated salt-soluble fraction seemed to be due to protein-protein interactions induced by heat.

Whydration effects on DNA double helix stability modulates ligand binding to natural DNA in response to changes in water activity

In this work we present evidence that water molecules are actively involved on the control of binding affinity and binding site discrimination of a drug to natural DNA. In a previous study, the effect of water activity (a(w)) on the energetic parameters of actinomycin-D intercalation to natural DNA was determined using the osmotic stress method (39). This earlier study has shown evidence that water molecules act as an allosteric regulator of ligand binding to DNA via the effect of water activity on the long-range stability of the DNA secondary structure. In this work we have carried out DNA circularization experiments using the plasmid pUC18 in the absence of drugs and in the presence of different neutral solutes to evaluate the contribution of water activity to the energetics of DNA helix unwinding. The contribution of water to these independent reactions were made explicit by the description of how the changes in the free energy of ligand binding to DNA and in the free energy associated with DNA helix torsional deformation are linked to a(w) via changes in structural hydration. Taken together, the results of these studies reveal an extensive linkage between ligand binding affinity and site binding discrimination, and long range helix conformational changes and DNA hydration, This is strong evidence that water molecules work as a classical allosteric regulator of ligand binding to the DNA via its contribution to the stability of the double helix secondary structure, suggesting a possible mechanism by which the biochemical machinery of DNA processing takes advantage of the low activity of water into the cellular milieu.

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

Disulfide Biochemistry in 2-Cys Peroxiredoxin: Requirement of Glu50 and Arg146 for the Reduction of Yeast Tsa1 by Thioredoxin

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

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.

Protein deficiency and nutritional recovery modulate insulin secretion and the early steps of insulin action in rats

Maternal malnutrition was shown to affect early growth and leads to permanent alterations in insulin secretion and sensitivity of offspring. In addition, epidemiological studies showed an association between low birth weight and glucose intolerance in adult life. To understand these interactions better, we investigated the insulin secretion by isolated islets and the early events related to insulin action in the hind-limb muscle of adult rats fed a diet of 17% protein (control) or 6% protein [low (LP) protein] during fetal life, suckling and after weaning, and in rats receiving 6% protein during fetal life and suckling followed by a 17% protein diet after weaning (recovered). The basal and maximal insulin secretion by islets from rats fed LP diet and the basal release by islets from recovered rats were significantly lower than that of control rats. The dose-response curves to glucose of islets from LP and recovered groups were shifted to the right compared to control islets, with the half-maximal response (EC 50) occurring at 16.9 ± 1.3, 12.4 ± 0.5 and 8.4 ± 0.1 mmol/L, respectively. The levels of insulin receptor, as well as insulin receptor substrate-1 and phosphorylation and the association between insulin receptor substrate-1 and phosphatidylinositol 3-kinase were greater in rats fed a LP diet than in control rats. In recovered rats, these variables were not significantly different from those of the other two groups. These results suggest that glucose homeostasis is maintained in LP and recovered rats by an increased sensitivity to insulin as a result of alterations in the early steps of the insulin signal transduction pathway.

Targeting Plasmodium ligands on mosquito salivary glands and midgut with a phage display peptide library

Despite vast efforts and expenditures in the past few decades, malaria continues to kill millions of persons every year, and new approaches for disease control are urgently needed. To complete its life cycle in the mosquito, Plasmodium, the causative agent of malaria, has to traverse the epithelia of the midgut and salivary glands. Although strong circumstantial evidence indicates that parasite interactions with the two organs are specific, hardly any information is available about the interacting molecules. By use of a phage display library, we identified a 12-aa peptide-salivary gland and midgut peptide 1 (SM1)-that binds to the distal lobes of the salivary gland and to the luminal side of the midgut epithelium, but not to the midgut surface facing the hemolymph or to ovaries. The coincidence of the tissues with which parasites and the SM1 peptide interact suggested that the parasite and peptide recognize the same surface ligand. In support of this hypothesis, the SM1 peptide strongly inhibited Plasmodium invasion of salivary gland and midgut epithelia. These experiments suggest a new strategy for the genetic manipulation of mosquito vectorial capacity.

A novel biological activity for galectin-1: Inhibition of leukocyte-endothelial cell interactions in experimental inflammation

Galectin-1 (Gal-1), the prototype of a family of β -galactoside-binding proteins, has been shown to attenuate experimental acute and chronic inflammation. In view of the fact that endothelial cells (ECs), but not human polymorphonuclear leukocytes (PMNs), expressed Gal-1 we tested here the hypothesis that the protein could modulate leukocyte-EC interaction in inflammatory settings. In vitro, human recombinant (hr) Gal-1 inhibited PMN chemotaxis and trans-endothelial migration. These actions were specific as they were absent if Gal-1 was boiled or blocked by neutralizing antiserum. In vivo, hrGal-1 (optimum effect at 0.3 μg equivalent to 20 pmol) inhibited interleukin-1β-induced PMN recruitment into the mouse peritoneal cavity. Intravital microscopy analysis showed that leukocyte flux, but not their rolling velocity, was decreased by an anti-inflammatory dose of hrGal-1. Binding of biotinylated Gal-1 to resting and post-adherent human PMNs occurred at concentrations inhibitory in the chemotaxis and transmigration assays. In addition, the pattern of Gal-1 binding was differentially modulated by PMN or EC activation. In conclusion, these data suggest the existence of a previously unrecognized function of Gal-1, that is inhibition of leukocyte rolling and extravasation in experimental inflammation. It is possible that endogenous Gal-1 may be part of a novel anti-inflammatory loop in which the endothelium is the source of the protein and the migrating PMNs the target for its anti-inflammatory action.

Mapping eIF5A binding sites for Dys1 and Lia1: In vivo evidence for regulation of eIF5A hypusination

The evolutionarily conserved factor eIF5A is the only protein known to undergo hypusination, a unique posttranslational modification triggered by deoxyhypusine synthase (Dys1). Although eIF5A is essential for cell viability, the function of this putative translation initiation factor is still obscure. To identify eIF5A-binding proteins that could clarify its function, we screened a two-hybrid library and identified two eIF-5A partners in S. cerevisiae: Dys1 and the protein encoded by the gene YJR070C, named Lia1 (Ligand of eIF5A). The interactions were confirmed by GST pulldown. Mapping binding sites for these proteins revealed that both eIF5A domains can bind to Dys1, whereas the C-terminal domain is sufficient to bind Lia1. We demonstrate for the first time in vivo that the N-terminal α-helix of Dys1 can modulate enzyme activity by inhibiting eIF5A interaction. We suggest that this inhibition be abrogated in the cell when hypusinated and functional eIF5A is required. © 2003 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Probing the binding of the coumarin drugs using peptide fragments of DNA gyrase B protein

Bacterial DNA gyrase, has been identified as the target of several antibacterial agents, including the coumarin drugs. The coumarins inhibit the gyrase action by competitive binding to the ATP-binding site of DNA gyrase B (GyrB) protein. The high in vitro inhibitory potency of coumarins against DNA gyrase reactions has raised interest in studies on coumarin-gyrase interactions. In this context, a series of low-molecular weight peptides, including the coumarin resistance-determining region of subunit B of Escherichia coli gyrase, has been designed and synthesized. The first peptide model was built using the natural fragment 131-146 of GyrB and was able to bind to novobiocin (K a = 1.8 ± 0.2 × 105/M) and ATP (Ka = 1.9 ± 0.4 × 103/M). To build the other sequences, changes in the Arg136 residue were introduced so that the binding to the drug was progressively reduced with the hydrophobicity of this residue (Ka = 1.3 ± 0.1 × 105/M and 1.0 ± 0.2 × 105/M for Ser and His, respectively). No binding was observed for the change Arg136 to Leu. In contrast, the binding to ATP was not altered, independently of the changes promoted. On the contrary, for peptide-coumarin and peptide-ATP complexes, Mg2+ appears to modulate the binding process. Our results demonstrate the crucial role of Arg 136 residue for the stability of coumarin-gyrase complex as well as suggest a different binding site for ATP and in both cases the interactions are mediated by magnesium ions. Copyright Blackwell Munksgaard, 2005.

Aspectos estruturais da membrana eritrocitária

This article describes the structures and functions of the erythrocyte membrane and its importance in transfusional medicine. The erythrocyte membrane is one of the best known membranes in terms of structure, function and genetic disorders. As any other plasma membrane, it mediates transport functions. It also provides the erythrocytes with their resilience and deformability. According to the International Society of Blood Transfusion (ISBT), more than 500 antigens are expressed in the erythrocyte membrane, and around 270 are involved in transfusion reaction cases and hemolytic diseases of the fetus and newborn. In the ISBT classification, the high frequency series is represented by antigens in more than 99% of population (high prevalence antigen). In transfusion, the absence of these antigens determines severe problems as for example, one woman without the P antigen suffered 6 repetitive miscarriages due to placental insufficiency, which was caused by an antibody formed against the absent P antigen. Some important erythrocyte membrane proteins are described here including Band 3, Glycophorins and spectrin. The most abundant integral membrane protein is Band 3 and its main function is to mediate exchange of chloride and bicarbonate anions across the plasma membrane. The second most abundant integral membrane protein in the human erythrocyte is sialoglycoprotein glycophorin A (GPA). With its high sialic acid content, GPA is the main contributor to the net negative cell-surface charge and is thus critical for minimizing cell-cell interactions and preventing red cell aggregation. Glycophorin C (GPC) is the receptor for PfEBP-2 (baebl, EBA-140), the newly identified erythrocyte binding ligand of Plasmodium falciparum. The ternary complex of spectrin, actin and 4.1R defines the nodes of the erythrocyte membrane skeletal network, and is inseparable from membrane stability when under mechanical stress. This erythrocyte membrane review is important for a better understanding of transfusion reactions, where the antibody formation against high prevalence antigens makes compatible transfusions difficult. The study of antigen diversity and biochemical characterization of different proteins will contribute to healthcare, as well as diagnosis, development of technology such as monoclonal antibody production and the therapeutic conduct of many diseases.

UMP kinase from Mycobacterium tuberculosis: Mode of action and allosteric interactions, and their likely role in pyrimidine metabolism regulation

The pyrH-encoded uridine 5′-monophosphate kinase (UMPK) is involved in both de novo and salvage synthesis of DNA and RNA precursors. Here we describe Mycobacterium tuberculosis UMPK (MtUMPK) cloning and expression in Escherichia coli. N-terminal amino acid sequencing and electrospray ionization mass spectrometry analyses confirmed the identity of homogeneous MtUMPK. MtUMPK catalyzed the phosphorylation of UMP to UDP, using ATP-Mg 2+ as phosphate donor. Size exclusion chromatography showed that the protein is a homotetramer. Kinetic studies revealed that MtUMPK exhibits cooperative kinetics towards ATP and undergoes allosteric regulation. GTP and UTP are, respectively, positive and negative effectors, maintaining the balance of purine versus pyrimidine synthesis. Initial velocity studies and substrate(s) binding measured by isothermal titration calorimetry suggested that catalysis proceeds by a sequential ordered mechanism, in which ATP binds first followed by UMP binding, and release of products is random. As MtUMPK does not resemble its eukaryotic counterparts, specific inhibitors could be designed to be tested as antitubercular agents. © 2010 Elsevier Inc. All rights reserved.

Insect/Bt-cotton interactions: Are immunological variables and hemocyte ultrastructure in Podisus nigrispinus (Hemiptera: Pentatomidae) affected?

Recent studies have shown that ingestion by the army worm Spodoptera frugiperda of Cry1Ac toxin from Bt cotton promotes histochemical and ultrastructural changes in the digestive cells of the predatory pentatomid bug Podisus nigrispinus. Therefore, mindful of the changes in the midgut of the predator, which represents the first line of defence in this insect, our aim was to test the hypothesis that the consumption of Bt cotton-fed S. frugiperda by P. nigrispinus might lead to alterations in components of the immune system of P. nigrispinus. The Cry1Ac toxin level in the leaves of Bt cotton, nitric oxide, phenoloxidase activity, and total proteins were quantified by ELISA. Total and differential hemocyte counts were evaluated, and hemocyte ultrastructure analysis was undertaken. We found that ingestion of the prey fed daily with approximately 23 ± 0.70 ng g-1 Cry1Ac by wet weight of leaves, and expressed by the Bt cotton, induces small ultrastructural changes in the predator's granulocytes and plasmatocytes. However, these changes did not affect the total number and differential and humoral variables analyzed for the bug's hemocytes. © 2013 Copyright Taylor and Francis Group, LLC.

Fetal-Maternal Interactions in the Synepitheliochorial Placenta Using the eGFP Cloned Cattle Model

Background: To investigate mechanisms of fetal-maternal cell interactions in the bovine placenta, we developed a model of transgenic enhanced Green Fluorescent Protein (t-eGFP) expressing bovine embryos produced by nuclear transfer (NT) to assess the distribution of fetal-derived products in the bovine placenta. In addition, we searched for male specific DNA in the blood of females carrying in vitro produced male embryos. Our hypothesis is that the bovine placenta is more permeable to fetal-derived products than described elsewhere. Methodology/Principal Findings: Samples of placentomes, chorion, endometrium, maternal peripheral blood leukocytes and blood plasma were collected during early gestation and processed for nested-PCR for eGFP and testis-specific Y-encoded protein (TSPY), western blotting and immunohistochemistry for eGFP detection, as well as transmission electron microscopy to verify the level of interaction between maternal and fetal cells. TSPY and eGFP DNA were present in the blood of cows carrying male pregnancies at day 60 of pregnancy. Protein and mRNA of eGFP were observed in the trophoblast and uterine tissues. In the placentomes, the protein expression was weak in the syncytial regions, but intense in neighboring cells on both sides of the fetal-maternal interface. Ultrastructurally, our samples from t-eGFP expressing NT pregnancies showed to be normal, such as the presence of interdigitating structures between fetal and maternal cells. In addition, channels-like structures were present in the trophoblast cells. Conclusions/Significance: Data suggested that there is a delivery of fetal contents to the maternal system on both systemic and local levels that involved nuclear acids and proteins. It not clear the mechanisms involved in the transfer of fetal-derived molecules to the maternal system. This delivery may occur through nonclassical protein secretion; throughout transtrophoblastic-like channels and/or by apoptotic processes previously described. In conclusion, the bovine synepitheliochorial placenta displays an intimate fetal-maternal interaction, similar to other placental types for instance human and mouse. © 2013 Pereira et al.