Analysis of agonist and antagonist effects on thyroid hormone receptor conformation by hydrogen/deuterium exchange

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T 3) or antagonist (NH3). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/ deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, Cterminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T 3, but not NH3, increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T 3 but not NH3.Wepresent data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes. (Molecular Endocrinology 25: 15-31, 2011). Copyright © 2011 by The Endocrine Society.

Agelaia MP-I: A peptide isolated from the venom of the social wasp, Agelaia pallipes pallipes, enhances insulin secretion in mice pancreatic islets

Peptides isolated from animal venoms have shown the ability to regulate pancreatic beta cell function. Characterization of wasp venoms is important, since some components of these venoms present large molecular variability, and potential interactions with different signal transduction pathways. For example, the well studied mastoparan peptides interact with a diversity of cell types and cellular components and stimulate insulin secretion via the inhibition of ATP dependent K + (K ATP) channels, increasing intracellular Ca 2+ concentration. In this study, the insulin secretion of isolated pancreatic islets from adult Swiss mice was evaluated in the presence of synthetic Agelaia MP-I (AMP-I) peptide, and some mechanisms of action of this peptide on endocrine pancreatic function were characterized. AMP-I was manually synthesized using the Fmoc strategy, purified by RP-HPLC and analyzed using ESI-IT-TOF mass spectrometry. Isolated islets were incubated at increasing glucose concentrations (2.8, 11.1 and 22.2 mM) without (Control group: CTL) or with 10 μM AMP-I (AMP-I group). AMP-I increased insulin release at all tested glucose concentrations, when compared with CTL (P < 0.05). Since molecular analysis showed a potential role of the peptide interaction with ionic channels, insulin secretion was also analyzed in the presence of 250 μM diazoxide, a K ATP channel opener and 10 μM nifedipine, a Ca 2+ channel blocker. These drugs abolished insulin secretion in the CTL group in the presence of 2.8 and 11.1 mM glucose, whereas AMP-I also enhanced insulin secretory capacity, under these glucose conditions, when incubated with diazoxide and nifedipine. In conclusion, AMP-I increased beta cell secretion without interfering in K ATP and L-type Ca 2+ channel function, suggesting a different mechanism for this peptide, possibly by G protein interaction, due to the structural similarity of this peptide with Mastoparan-X, as obtained by modeling. © 2012 Elsevier Ltd.

Fibronectin induces MMP2 expression in human prostate cancer cells

High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the tumor cell lines commonly employed for prostate cancer research express only small amounts of MMPs when cultivated as monolayer cultures, in common culture media. The present study was conducted to ascertain whether culture conditions that include fibronectin can alter MMP2 and MMP9 expression by the human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2×104cells/cm2, cultivated until they reached 80% confluence, and then exposed for 4h to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 cells express detectable amounts of MMP9 when cultivated in common medium, whereas the addition of fibronectin induced high expression levels of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumor prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies of transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions. © 2012 Elsevier Inc..

EIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer

The protein eukaryotic initiation factor 5A (eIF5A) is highly conserved among archaea and eukaryotes, but not in bacteria. Bacteria have the elongation factor P (EF-P), which is structurally and functionally related to eIF5A. eIF5A is essential for cell viability and the only protein known to contain the amino acid residue hypusine, formed by post-translational modification of a specific lysine residue. Although eIF5A was initially identified as a translation initiation factor, recent studies strongly support a function for eIF5A in the elongation step of translation. However, the mode of action of eIF5A is still unknown. Here, we analyzed the oligomeric state of yeast eIF5A. First, by using size-exclusion chromatography, we showed that this protein exists as a dimer in vitro, independent of the hypusine residue or electrostatic interactions. Protein-protein interaction assays demonstrated that eIF5A can form oligomers in vitro and in vivo, in an RNA-dependent manner, but independent of the hypusine residue or the ribosome. Finally, small-angle X-ray scattering (SAXS) experiments confirmed that eIF5A behaves as a stable dimer in solution. Moreover, the molecular envelope determined from the SAXS data shows that the eIF5A dimer is L-shaped and superimposable on the tRNAPhe tertiary structure, analogously to the EF-P monomer. © 2012 Springer-Verlag.

Cooperative RNA Polymerase Molecules Behavior on a Stochastic Sequence-Dependent Model for Transcription Elongation

The transcription process is crucial to life and the enzyme RNA polymerase (RNAP) is the major component of the transcription machinery. The development of single-molecule techniques, such as magnetic and optical tweezers, atomic-force microscopy and single-molecule fluorescence, increased our understanding of the transcription process and complements traditional biochemical studies. Based on these studies, theoretical models have been proposed to explain and predict the kinetics of the RNAP during the polymerization, highlighting the results achieved by models based on the thermodynamic stability of the transcription elongation complex. However, experiments showed that if more than one RNAP initiates from the same promoter, the transcription behavior slightly changes and new phenomenona are observed. We proposed and implemented a theoretical model that considers collisions between RNAPs and predicts their cooperative behavior during multi-round transcription generalizing the Bai et al. stochastic sequence-dependent model. In our approach, collisions between elongating enzymes modify their transcription rate values. We performed the simulations in Mathematica® and compared the results of the single and the multiple-molecule transcription with experimental results and other theoretical models. Our multi-round approach can recover several expected behaviors, showing that the transcription process for the studied sequences can be accelerated up to 48% when collisions are allowed: the dwell times on pause sites are reduced as well as the distance that the RNAPs backtracked from backtracking sites. © 2013 Costa et al.

The Deoxyhypusine Synthase Mutant dys1-1 Reveals the Association of eIF5A and Asc1 with Cell Wall Integrity

The putative eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein among archaea and eukaryotes that has recently been implicated in the elongation step of translation. eIF5A undergoes an essential and conserved posttranslational modification at a specific lysine to generate the residue hypusine. The enzymes deoxyhypusine synthase (Dys1) and deoxyhypusine hydroxylase (Lia1) catalyze this two-step modification process. Although several Saccharomyces cerevisiae eIF5A mutants have importantly contributed to the study of eIF5A function, no conditional mutant of Dys1 has been described so far. In this study, we generated and characterized the dys1-1 mutant, which showed a strong depletion of mutated Dys1 protein, resulting in more than 2-fold decrease in hypusine levels relative to the wild type. The dys1-1 mutant demonstrated a defect in total protein synthesis, a defect in polysome profile indicative of a translation elongation defect and a reduced association of eIF5A with polysomes. The growth phenotype of dys1-1 mutant is severe, growing only in the presence of 1 M sorbitol, an osmotic stabilizer. Although this phenotype is characteristic of Pkc1 cell wall integrity mutants, the sorbitol requirement from dys1-1 is not associated with cell lysis. We observed that the dys1-1 genetically interacts with the sole yeast protein kinase C (Pkc1) and Asc1, a component of the 40S ribosomal subunit. The dys1-1 mutant was synthetically lethal in combination with asc1Δ and overexpression of TIF51A (eIF5A) or DYS1 is toxic for an asc1Δ strain. Moreover, eIF5A is more associated with translating ribosomes in the absence of Asc1 in the cell. Finally, analysis of the sensitivity to cell wall-perturbing compounds revealed a more similar behavior of the dys1-1 and asc1Δ mutants in comparison with the pkc1Δ mutant. These data suggest a correlated role for eIF5A and Asc1 in coordinating the translational control of a subset of mRNAs associated with cell integrity. © 2013 Galvão et al.

IL-4 and IL-13 inhibit IL-1β and TNF-α induced kinin B 1 and B2 receptors through a STAT6-dependent mechanism

Background and Purpose Bone resorption induced by interleukin-1β (IL-1β) and tumour necrosis factor (TNF-α) is synergistically potentiated by kinins, partially due to enhanced kinin receptor expression. Inflammation-induced bone resorption can be impaired by IL-4 and IL-13. The aim was to investigate if expression of B1 and B2 kinin receptors can be affected by IL-4 and IL-13. Experimental Approach We examined effects in a human osteoblastic cell line (MG-63), primary human gingival fibroblasts and mouse bones by IL-4 and IL-13 on mRNA and protein expression of the B1 and B2 kinin receptors. We also examined the role of STAT6 by RNA interference and using Stat6-/- mice. Key Results IL-4 and IL-13 decreased the mRNA expression of B1 and B2 kinin receptors induced by either IL-1β or TNF-α in MG-63 cells, intact mouse calvarial bones or primary human gingival fibroblasts. The burst of intracellular calcium induced by either bradykinin (B2 agonist) or des-Arg10-Lys-bradykinin (B1 agonist) in gingival fibroblasts pretreated with IL-1β was impaired by IL-4. Similarly, the increased binding of B1 and B2 ligands induced by IL-1β was decreased by IL-4. In calvarial bones from Stat6-deficient mice, and in fibroblasts in which STAT6 was knocked down by siRNA, the effect of IL-4 was decreased. Conclusions and Implications These data show, for the first time, that IL-4 and IL-13 decrease kinin receptors in a STAT6-dependent mechanism, which can be one important mechanism by which these cytokines exert their anti-inflammatory effects and impair bone resorption. © 2013 The Authors. British Journal of Pharmacology © 2013 The British Pharmacological Society.

Intermolecular interactions of the malate synthase of Paracoccidioides spp

Background: The fungus Paracoccidioides spp is the agent of paracoccidioidomycosis (PCM), a pulmonary mycosis acquired by the inhalation of fungal propagules. Paracoccidioides malate synthase (PbMLS) is important in the infectious process of Paracoccidioides spp because the transcript is up-regulated during the transition from mycelium to yeast and in yeast cells during phagocytosis by murine macrophages. In addition, PbMLS acts as an adhesin in Paracoccidioides spp. The evidence for the multifunctionality of PbMLS indicates that it could interact with other proteins from the fungus and host. The objective of this study was to identify and analyze proteins that possibly bind to PbMLS (PbMLS-interacting proteins) because protein interactions are intrinsic to cell processes, and it might be possible to infer the function of a protein through the identification of its ligands. Results: The search for interactions was performed using an in vivo assay with a two-hybrid library constructed in S. cerevisiae; the transcripts were sequenced and identified. In addition, an in vitro assay using pull-down GST methodology with different protein extracts (yeast, mycelium, yeast-secreted proteins and macrophage) was performed, and the resulting interactions were identified by mass spectrometry (MS). Some of the protein interactions were confirmed by Far-Western blotting using specific antibodies, and the interaction of PbMLS with macrophages was validated by indirect immunofluorescence and confocal microscopy. In silico analysis using molecular modeling, dynamics and docking identified the amino acids that were involved in the interactions between PbMLS and PbMLS-interacting proteins. Finally, the interactions were visualized graphically using Osprey software. Conclusion: These observations indicate that PbMLS interacts with proteins that are in different functional categories, such as cellular transport, protein biosynthesis, modification and degradation of proteins and signal transduction. These data suggest that PbMLS could play different roles in the fungal cell. © 2013 de Oliveira et al.; licensee BioMed Central Ltd.

RBPI21 interacts with negative membranes endothermically promoting the formation of rigid multilamellar structures

rBPI21 belongs to the antimicrobial peptide and protein (AMP) family. It has high affinity for lipopolysaccharide (LPS), acting mainly against Gram-negative bacteria. This work intends to elucidate the mechanism of action of rBPI21 at the membrane level. Using isothermal titration calorimetry, we observed that rBPI21 interaction occurs only with negatively charged membranes (mimicking bacterial membranes) and is entropically driven. Differential scanning calorimetry shows that membrane interaction with rBPI21 is followed by an increase of rigidity on negatively charged membrane, which is corroborated by small angle X-ray scattering (SAXS). Additionally, SAXS data reveal that rBPI21 promotes the multilamellarization of negatively charged membranes. The results support the proposed model for rBPI21 action: first it may interact with LPS at the bacterial surface. This entropic interaction could cause the release of ions that maintain the packed structure of LPS, ensuring peptide penetration. Then, rBPI21 may interact with the negatively charged leaflets of the outer and inner membranes, promoting the interaction between the two bacterial membranes, ultimately leading to cell death. © 2013 Elsevier B.V.

Sodium dodecyl sulfate (SDS) effect on the thermal stability of oxy-HbGp: Dynamic light scattering (DLS) and small angle X-ray scattering (SAXS) studies

Glossoscolex paulistus (HbGp) hemoglobin is an oligomeric protein, presenting a quaternary structure constituted by 144 globin and 36 non-globin chains (named linkers) with a total molecular mass of 3.6MDa. SDS effects on the oxy-HbGp thermal stability were studied, by DLS and SAXS, at pH 5.0, 7.0 and 9.0. DLS and SAXS data show that the SDS-oxy-HbGp interactions induce a significant decrease of the protein thermal stability, with the formation of larger aggregates, at pH 5.0. At pH 7.0, oxy-HbGp undergoes complete oligomeric dissociation, with increase of temperature, in the presence of SDS. Besides, oxy-HbGp 3.0mg/mL, pH 7.0, in the presence of SDS, has the oligomeric dissociation process reduced as compared to 0.5mg/mL of protein. At pH 9.0, oxy-HbGp starts to dissociate at 20°C, and the protein is totally dissociated at 50°C. The thermal dissociation kinetic data show that oxy-HbGp oligomeric dissociation at pH 7.0, in the presence of SDS, is strongly dependent on the protein concentration. At 0.5mg/mL of protein, the oligomeric dissociation is complete and fast at 40 and 42°C, with kinetic constants of (2.1±0.2)×10-4 and (5.5±0.4)×10-4s-1, respectively, at 0.6mmol/L SDS. However, at 3.0mg/mL, the oligomeric dissociation process starts at 46°C, and only partial dissociation, accompanied by aggregates formation is observed. Moreover, our data show, for the first time, that, for 3.0mg/mL of protein, the oligomeric dissociation, denaturation and aggregation phenomena occur simultaneously, in the presence of SDS. Our present results on the surfactant-HbGp interactions and the protein thermal unfolding process correspond to a step forward in the understanding of SDS effects. © 2013 Elsevier B.V.

Analisando a determinação sexual de vertebrados com base em redes de interação entre proteínas

Pós-graduação em Ciências Biológicas (Genética) - IBB

Estudos de pré-formulação e formulação de Vismia guianensis (Aubl.) Choisy

A obtenção e a avaliação da formulação fitoterápica foi traçada através de parâmetros de controle de qualidade realizando-se a caracterização físico-química da matéria-prima vegetal desidratada e triturada, do seu derivado (extrato liofilizado da tintura hidroalcóolica) e da formulação semi-sólida fitoterápica antimicrobiana contendo a tintura das folhas de Vismia guianensis (Aubl) Choisy. Para tais caracterizações utilizaram-se os parâmetros específicos para as drogas vegetais contidos na Farmacopéia Brasileira 4. Ed., a análise térmica: termogravimetria, análise térmica diferencial e calorimetria exploratória diferencial e a espectroscopia na região do infravermelho e do ultravioleta. A avaliação da atividade antimicrobiana pelo método de difusão em disco em meio sólido identificou a sensibilidade de S. aureus (ATCC 25923) ao extrato seco da tintura dissolvido em DMSO, nas concentrações de 500, 250, 125 e 62,5 mg/mL. A CLAE traçou o perfil de composição das sub-frações A e B provenientes da fração acetato de etila da tintura e evidenciou o alcance máximo de absorção em 290 nm para a fração acetato de etila semelhante ao alcance máximo da emodia, sendo essa então utilizada como padrão de referência e marcador externo. A validação do método foi realizada através da espectrofotometria no ultravioleta, demonstrando ser um método seletivo, linear, repetitivo e robusto. A análise térmica evidenciou possíveis incompatibilidades existentes entre a mistura binária do extrato liofilizado da tintura com a hidroxietilcelulose e o propilenoglicol; obtendo-se então um gel com características não homogêneas devido à precipitação de proteínas, ocorrida pela interação polifenol-protéina. A avaliação da estabilidade preliminar da formulação permaneceu dentro dos parâmetros, apresentando resultados dentro dos limites aceitáveis para os testes de centrifugação, estresse térmico e características organolépticas.

Caracterização dos genes codificadores da hemaglutinina e PB2 do vírus Influenza A (H1N1) pandêmico isolado na mesorregião metropolitana de Belém

A recente pandemia de gripe de 2009/2010 causada pelo vírus A (H1N1) pandêmico mostrou um perfil de gravidade diferente da gripe sazonal, pois um percentual considerável de casos graves e fatais ocorreu em indivíduos adultos jovens, sem comorbidade. A virulência dos vírus Influenza A (H1N1) pandêmico resulta de interações protéicas complexas e depende essencialmente de alguns genes virais. O objetivo deste estudo foi caracterizar os genes codificadores da hemaglutinina (H1) e polimerase básica 2 (PB2) do vírus Influenza A (H1N1) pandêmico mediante a obtenção de cepas provenientes de pacientes com gripe procedente da mesorregião metropolitana de Belém-PA. O tamanho amostral foi constituído de 87 amostras aleatórias de ambos os sexos de 0 a 96 anos, com síndrome respiratória aguda grave (SRAG) sem nenhuma comorbidade relatada, no período de maio de 2009 a agosto de 2010. As amostras foram isoladas em cultura de célula MDCK e analisadas por técnicas de biologia molecular que compreenderam três etapas principais: a) extração do RNA viral (RNAv) a partir do sobrenadante celular; b) amplificação do RNAv pela técnica de Reação em Cadeia mediada pela Polimerase precedida de Transcrição Reversa (RT-PCR); c) sequenciamento completo dos genes codificadores da H1 e PB2. Das 87 cepas amplificadas pelo RT-PCR, em 82 tornou-se possível a obtenção e análise de sequências para o gene HA, enquanto que de 81 amostras virais obteve-se sequências para o gene PB2. A análise comparativa das sequências obtidas com a sequência da cepa vacinal (A/California/07/2009(H1N1)) revelou substituições aminoacídicas na HA (P83S; D97N; S203T; D222G; Q293H e I321V) e na PB2 (K340N; K526R e M631L), no entanto sem associação a hospitalização. Ao nível de substituição na HA, a D97N isolada ou associada com a S203T, foi detectada com mais frequência na primeira onda. Já ao nível da PB2 a substituição K526R foi mais encontrada em cepas que circularam na primeira onda, enquanto que, a M631L foi mais evidenciada na segunda. A substituição D222G na HA só foi encontrada em casos de óbitos. Por fim, observou-se uma tendência de alterações nos sítios antigênicos da HA. Sendo assim, a contínua vigilância genética e antigênica do vírus Influenza A (H1N1) pdm em circulação, bem como o compartilhamento de informações é de extrema importância para a melhor recomendação possível para os vírus que entram na composição vacinal evitando assim maior risco de epidemias severas no futuro.

Construção, caracterização global e local de redes biológicas

A Biologia Sistêmica visa a compreensão da vida através de modelos integrativos que enfatizem as interações entre os diferentes agentes biológicos. O objetivo é buscar por leis universais, não nas partes componentes dos sistemas mas sim nos padrões de interação dos elementos constituintes. As redes complexas biológicas são uma poderosa abstração matemática que permite a representação de grandes volumes de dados e a posterior formulação de hipóteses biológicas. Nesta tese apresentamos as redes biológicas integradas que incluem interações oriundas do metabolismo, interação física de proteínas e regulação. Discutimos sua construção e ferramentas para sua análise global e local. Apresentamos também resultados do uso de ferramentas de aprendizado de máquina que nos permitem compreender a relação entre propriedades topológicas e a essencialidade gênica e a previsão de genes mórbidos e alvos para drogas em humanos

Estudo das interações entre a proteína TRF de Leishmania amazonensis (LaTRF) e suas possíveis parcerias

Pós-graduação em Ciências Biológicas (Genética) - IBB