Institute Bioquimica Medica

Rio de Janeiro, Brazil

Institute Bioquimica Medica

Rio de Janeiro, Brazil
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Puntel R.L.,Federal University of Pampa | Roos D.H.,Federal University of Santa Maria | Folmer V.,Federal University of Pampa | Nogueira C.W.,Federal University of Santa Maria | And 3 more authors.
Toxicological Sciences | Year: 2010

Ebselen (Ebs) and diphenyl diselenide [(PhSe)2] readily oxidize thiol groups. Here we studied mitochondrial swelling changes in mitochondrial potential (ΔΨm), NAD(P)H oxidation, reactive oxygen species production, protein aggregate formation, and oxygen consumption as ending points of their in vitro toxicity. Specifically, we tested the hypothesis that organochalchogens toxicity could be associated with mitochondrial dysfunction via oxidation of vicinal thiol groups that are known to be involved in the regulation of mitochondrial permeability (Petronilli et al. J. Biol. Chem., 269; 16638; 1994). Furthermore, we investigated the possible mechanism(s) by which these organochalchogens could disrupt liver mitochondrial function. Ebs and (PhSe)2 caused mitochondrial depolarization and swelling in a concentrationdependent manner. Furthermore, both organochalchogens caused rapid oxidation of the mitochondrial pyridine nucleotides (NAD(P)H) pool, likely reflecting the consequence and not the cause of increased mitochondrial permeability (Costantini, P., Chernyak, B. V., Petronilli, V., and Bernardi, P. (1996). Modulation of the mitochondrial permeability transition pore (PTP) by pyridine nucleotides and dithiol oxidation at two separate sites. J. Biol. Chem. 271, 6746-6751). The organochalchogens-induced mitochondrial dysfunction was prevented by the reducing agent dithiothreitol (DTT). Ebsand (PhSe)2-induced mitochondrial depolarization and swelling were unchanged by ruthenium red (4μM), butylated hydroxytoluene (2.5μM), or cyclosporine A(1μM). N-ethylmaleimide enhanced the organochalchogensinduced mitochondrial depolarization, without affecting the magnitude of the swelling response. In contrast, iodoacetic acid did not modify the effects of Ebs or (PhSe)2 on the mitochondria. Additionally, Ebs and (PhSe)2 decreased the basal 2' 7' dichlorofluorescin diacetate (H2-DCFDA) oxidation and oxygen consumption rate in state 3 and increased it during the state 4 of oxidative phosphorylation and induced the formation of protein aggregates, which were prevented by DTT. However, DTT failed to reverse the formation of protein aggregates, when it was added after a preincubation of liver mitochondria with Ebs or (PhSe)2.Similarly, DTT did not reverse the Ebsor (PhSe)2-induced ΔΨm collapse or swelling, when it was added after a preincubation period of mitochondria with chalcogenides. These results show that Ebs and (PhSe)2 can effectively induce mitochondrial dysfunction and suggest that effects of these compounds are associated with mitochondrial thiol groups oxidation. The inability of cyclosporine A to reverse the Ebs-and (PhSe)2-induced mitochondrial effects suggests that the redox-regulated mitochondrial permeability transition (MPT) pore was mechanistically regulated in a manner that is distinct from the classical MPT pore. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved.

De Paula V.S.,Institute Bioquimica Medica | Razzera G.,Institute Bioquimica Medica | Barreto-Bergter E.,Federal University of Rio de Janeiro | Almeida F.C.L.,Institute Bioquimica Medica | Valente A.P.,Institute Bioquimica Medica
Structure | Year: 2011

Defensins are essentially ancient natural antibiotics with potent activity extending from lower organisms to humans. Sd5 is a recently described antifungal defensin that appears to be the result of a recent gain of function. We reported here the solution NMR structure of Sd5 and characterized the backbone dynamics in the free state and in the presence of membrane models. 15N relaxation dispersion measurements indicate intrinsic conformational exchange processes, showing two clear distinct kex, 490 and 1800 s-1. These multiple motions may be related to transient twisting or breathing of the α helix and β sheet. The stages of membrane recognition and disruption by Sd5 over a large timescale range were mapped and demonstrated that Sd5 in solution sampled an ensemble of different conformations, of which a subset is selected upon membrane binding. Defensins share similar structures, but we demonstrated here that their dynamics can be extremely diverse. © 2011 Elsevier Ltd All rights reserved.

Silva J.L.,Institute Bioquimica Medica | Vieira T.C.R.G.,Institute Bioquimica Medica | Gomes M.P.B.,Institute Bioquimica Medica | Ano Bom A.P.,Institute Bioquimica Medica | And 5 more authors.
Accounts of Chemical Research | Year: 2010

(Figure Presented) Protein misfolding has been implicated in a large number of diseases termed protein- folding disorders (PFDs), which include Alzheimer's disease, Parkinson's disease, transmissible spongiform encephalopathies, familial amyloid polyneuropathy, Huntington's disease, and type II diabetes. In these diseases, large quantities of incorrectly folded proteins undergo aggregation, destroying brain cells and other tissues. The interplay between ligand binding and hydration is an important component of the formation of misfolded protein species. Hydration drives various biological processes, including protein folding, ligand binding, macromolecular assembly, enzyme kinetics, and signal transduction. The changes in hydration and packing, both when proteins fold correctly or when folding goes wrong, leading to PFDs, are examined through several biochemical, biophysical, and structural approaches. Although in many cases the binding of a ligand such as a nucleic acid helps to prevent misfolding and aggregation, there are several examples in which ligands induce misfolding and assembly into amyloids. This occurs simply because the formation of structured aggregates (such as protofibrillar and fibrillar amyloids) involves decreases in hydration, formation of a hydrogen-bond network in the secondary structure, and burying of nonpolar amino acid residues, processes that also occur in the normal folding landscape. In this Account, we describe the present knowledge of the folding and misfolding of different proteins, with a detailed emphasis on mammalian prion protein (PrP) and tumoral suppressor protein p53; we also explore how ligand binding and hydration together influence the fate of the proteins. Anfinsen's paradigm that the structure of a protein is determined by its amino add sequence is to some extent contradicted by the observation that there are two isoforms of the prion protein with the same sequence: the cellular and the misfolded isoform. The cellular isoform of PrP has a disordered N-terminal domain and a highly flexible, not-well-packed C-terminal domain, which might account for its significant hydration. When PrP binds to biological molecules, such as glycosaminoglycans and nucleic adds, the disordered segments appear to fold and become less hydrated. Formation of the PrP-nucleic acid complex seems to accelerate the conversion of the cellular form of the protein into the disease-causing isoform. For p53, binding to some ligands, including nucleic acids, would prevent misfolding of the protein. Recently, several groups have begun to analyze the folding-misfolding of the individual domains of p53, but several questions remain unanswered. We discuss the implications of these findings for understanding the productive and incorrect folding pathways of these proteins in normal physiological states and in human disease, such as prion disorders and cancer. These studies are shown to lay the groundwork for the development of new drugs. © 2010 American Chemical Society.

Silva J.L.,Institute Bioquimica Medica | Vieira T.C.R.G.,Institute Bioquimica Medica | Gomes M.P.B.,Institute Bioquimica Medica | Rangel L.P.,Institute Bioquimica Medica | And 2 more authors.
Methods | Year: 2011

The concept that transmissible spongiform encephalopathies (TSEs) are caused only by proteins has changed the traditional paradigm that disease transmission is due solely to an agent that carries genetic information. The central hypothesis for prion diseases proposes that the conversion of a cellular prion protein (PrPC) into a misfolded, β-sheet-rich isoform (PrPSc) accounts for the development of (TSE). There is substantial evidence that the infectious material consists chiefly of a protein, PrPSc, with no genomic coding material, unlike a virus particle, which has both. However, prions seem to have other partners that chaperone their activities in converting the PrPC into the disease-causing isoform. Nucleic acids (NAs) and glycosaminoglycans (GAGs) are the most probable accomplices of prion conversion. Here, we review the recent experimental approaches that have been employed to characterize the interaction of prion proteins with nucleic acids and glycosaminoglycans. A PrP recognizes many nucleic acids and GAGs with high affinities, and this seems to be related to a pathophysiological role for this interaction. A PrP binds nucleic acids and GAGs with structural selectivity, and some PrP:NA complexes can become proteinase K-resistant, undergoing amyloid oligomerization and conversion to a β-sheet-rich structure. These results are consistent with the hypothesis that endogenous polyanions (such as NAs and GAGs) may accelerate the rate of prion disease progression by acting as scaffolds or lattices that mediate the interaction between PrPC and PrPSc molecules. In addition to a still-possible hypothesis that nucleic acids and GAGs, especially those from the host, may modulate the conversion, the recent structural characterization of the complexes has raised the possibility of developing new diagnostic and therapeutic strategies. © 2010 Elsevier Inc.

Vieira T.C.R.G.,Institute Bioquimica Medica | Reynaldo D.P.,Institute Bioquimica Medica | Gomes M.P.B.,Institute Bioquimica Medica | Almeida M.S.,Institute Bioquimica Medica | And 2 more authors.
Journal of the American Chemical Society | Year: 2011

The conversion of cellular prion protein (PrPC) into the pathological conformer PrPSc requires contact between both isoforms and probably also requires a cellular factor, such as a nucleic acid or a glycosaminoglycan (GAG). Little is known about the structural features implicit in the GAG-PrP interaction. In the present work, light scattering, fluorescence, circular dichroism, and nuclear magnetic resonance (NMR) spectroscopy were used to describe the chemical and physical properties of the murine recombinant PrP 23-231 interaction with low molecular weight heparin (LMWHep) at pH 7.4 and 5.5. LMWHep interacts with rPrP 23-231, thereby inducing transient aggregation. The interaction between murine rPrP and heparin at pH 5.5 had a stoichiometry of 2:1 (LMWHep:rPrP 23-231), in contrast to a 1:1 binding ratio at pH 7.4. At binding equilibrium, NMR spectra showed that rPrP complexed with LMWHep had the same general fold as that of the free protein, even though the binding can be indicated by significant changes in few residues of the C-terminal domain, especially at pH 5.5. Notably, the soluble LMWHep:rPrP complex prevented RNA-induced aggregation. We also investigated the interaction between LMWHep and the deletion mutants rPrP ?51-90 and ?32-121. Heparin did not bind these constructs at pH 7.4 but was able to interact at pH 5.5, indicating that this glycosaminoglycan binds the octapeptide repeat region at pH 7.4 but can also bind other regions of the protein at pH 5.5. The interaction at pH 5.5 was dependent on histidine residues of the murine rPrP 23-231. Depending on the cellular milieu, the PrP may expose different regions that can bind GAG. These results shed light on the role of GAGs in PrP conversion. The transient aggregation of PrP may explain why some GAGs have been reported to induce the conversion into the misfolded, scrapie conformation, whereas others are thought to protect against conversion. The acquired resistance of the complex against RNA-induced aggregation explains some of the unique properties of the PrP interaction with GAGs. © 2010 American Chemical Society.

Conceicao T.M.,Institute Bioquimica Medica | El-Bacha T.,Institute Bioquimica Medica | Villas-Boas C.S.A.,Institute Bioquimica Medica | Coello G.,National Autonomous University of Mexico | And 3 more authors.
Journal of Infection | Year: 2010

Objectives: Liver damage occurs during Dengue Virus infection and constitutes a characteristic of severe forms of the disease. The present study was focused on the modulation of gene expression in a human hepatic cell lineage, HepG2, in response to Dengue Virus infection. Methods: The global gene expression changes in HepG2 cells after 6, 24 and 48 h of infection with Dengue Virus were investigated using a new tool of microarray data analysis and real-time PCR. Results: HepG2 cells infected with Dengue Virus showed alterations in several signaling pathways involved in innate immune response. The analysis of pattern recognition pathways genes demonstrated that TLR3, TLR8, RIG-I and MDA5 mRNAs were up-regulated during Dengue Virus infection along with an increase in the expression of the type I interferon, IFN-β and pro-inflammatory cytokines IL-6, IL-8 and RANTES genes. Conclusions: Our results suggest that innate immune pathways are involved in the recognition of Dengue Virus by HepG2 cells. These observations may contribute to the understanding of the inflammatory responses induced by Dengue Virus-hepatocytes interaction during dengue diseases. © 2009 The British Infection Society.

Kozlowski E.O.,Institute Bioquimica Medica | Pavao M.S.G.,Institute Bioquimica Medica
Frontiers in Bioscience - Scholar | Year: 2011

Metastasis is the most devastating aspect of the tumor, being the main cause of morbidity and mortality in cancer patients. The events that lead to tumor invasion and metastasis depend fundamentally on the initial aquisition of a mesenchymal phenotype by the primary carcinoma, which provides the necessary machinary for invasion, intravasation, vascular transport, extravasation and tumor colonization. These events are orquestrated by different growth factors, proteoglycans and adhesion molecules, acting at the surface of cells. The anticoagulant heparin binds several of these molecules and can regulate the interactions that occur during tumor invasion and metastasis. For example, heparin modulates the binding of FGF-2 to its tyrosine kinase receptor during tumor proliferation, and the binding of growth factors involved in epithelial to mesenchymal transition during tumor invasion. It also binds P-selectin on activated platelets, preventing tumor cell-platelet interaction during hematogeneous metastasis. In this review, we discuss the role of sulfated glycosaminoglycans during tumor invasion and metastasis, and the possible therapeutic use of heparin analogs on cancer treatment.

Rojas C.A.,Institute Bioquimica Medica
GM crops | Year: 2010

Genetically modified crops (GMCs) have been developed to accelerate the creation of new varieties with improved characteristics such as disease resistance, stress tolerance and higher quality composition. However, agriculture, without minimizing its role in food, feed and fiber source, has become important for the energy matrix of many countries. GMCs are also attractive systems that could fulfill the requirements for these new necessities. An increase of crop yields in an environmental friendly system is a new goal for plant biology research in the twenty-first century. In particular, biomass yield improvement is needed to render the use of biofuels economically feasible. In this context, research directed toward increasing biomass production has attracted much attention and a considerable effort is being made to reach new goals. Nonetheless, in some cases differentiated strategies are needed, as biomass improvement requires approaches other than those employed with traditional crops. This review summarizes the various approaches applied so far to modulate plant growth applying molecular biology-based strategies and increase biomass production, and it highlights several outstanding issues about the developmental constraints that must be addressed. © 2010 Landes Bioscience

Da-Silva W.S.,University of Miami | Da-Silva W.S.,Institute Bioquimica Medica | Ribich S.,University of Miami | Drigo R.A.E.,University of Miami | And 3 more authors.
FEBS Letters | Year: 2011

Exposure of cell lines endogenously expressing the thyroid hormone activating enzyme type 2 deiodinase (D2) to the chemical chaperones tauroursodeoxycholic acid (TUDCA) or 4-phenylbutiric acid (4-PBA) increases D2 expression, activity and T3 production. In brown adipocytes, TUDCA or 4-PBA induced T3-dependent genes and oxygen consumption (∼2-fold), an effect partially lost in D2 knockout cells. In wild type, but not in D2 knockout mice, administration of TUDCA lowered the respiratory quotient, doubled brown adipose tissue D2 activity and normalized the glucose intolerance associated with high fat feeding. Thus, D2 plays a critical role in the metabolic effects of chemical chaperones. © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

p53 is a key protein that participates in cell-cycle control, and its malfunction can lead to cancer. This tumour suppressor protein has three main domains; the N-terminal transactivation domain, the CTD (C-terminal domain) and the core domain (p53C) that constitutes the sequence-specific DBD (DNA-binding region). Most p53 mutations related to cancer development are found in the DBD. Aggregation of p53 into amyloid oligomers and fibrils has been shown. Moreover, amyloid aggregates of both the mutant and WT (wild-type) forms of p53 were detected in tumour tissues. We propose that if p53 aggregation occurred, it would be a crucial aspect of cancer development, as p53 would lose its WT functions in an aggregated state. Mutant p53 can also exert a dominant-negative regulatory effect on WT p53. Herein, we discuss the dominant-negative effect in light of p53 aggregation and the fact that amyloid-like mutant p53 can convert WT p53 into more aggregated species, leading into gain of function in addition to the loss of tumour suppressor function. In summary, the results obtained in the last decade indicate that cancer may have characteristics in common with amyloidogenic and prion diseases.

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