RESUMO
Hippocampus is the brain area where aluminum (Al) accumulates in abundance and is widely associated with learning and memory. In the present study, we evaluate behavioral, tissue, and proteomic changes in the hippocampus of Wistar rats caused by exposure to doses that mimic human consumption of aluminum chloride (AlCl3) in urban areas. For this, male Wistar rats were divided into two groups: Control (distilled water) and AlCl3 (8.3 mg/kg/day), both groups were exposed orally for 60 days. After the Al exposure protocol, cognitive functions were assessed by the Water maze test, followed by a collection for analysis of the global proteomic profile of the hippocampus by mass spectrometry. Aside from proteomic analysis, we performed a histological analysis of the hippocampus, to the determination of cell body density by cresyl violet staining in Cornu Ammonis fields (CA) 1 and 3, and hilus regions. Our results indicated that exposure to low doses of aluminum chloride triggered a decreased cognitive performance in learning and memory, being associated with the deregulation of proteins expression, mainly those related to the regulation of the cytoskeleton, cellular metabolism, mitochondrial activity, redox regulation, nervous system regulation, and synaptic signaling, reduced cell body density in CA1, CA3, and hilus.
Assuntos
Alumínio , Proteômica , Humanos , Ratos , Masculino , Animais , Alumínio/toxicidade , Alumínio/metabolismo , Cloreto de Alumínio/toxicidade , Ratos Wistar , Hipocampo/metabolismo , Compostos de Alumínio/toxicidadeRESUMO
During tuberculosis, Mycobacterium uses host macrophage cholesterol as a carbon and energy source. To mimic these conditions, Mycobacterium smegmatis can be cultured in minimal medium (MM) to induce cholesterol consumption in vitro. During cultivation, M. smegmatis consumes MM cholesterol and changes the accumulation of cell wall compounds, such as PIMs, LM, and LAM, which plays an important role in its pathogenicity. These changes lead to cell surface hydrophobicity modifications and H2O2 susceptibility. Furthermore, when M. smegmatis infects J774A.1 macrophages, it induces granuloma-like structure formation. The present study aims to assess macrophage molecular disturbances caused by M. smegmatis after cholesterol consumption, using proteomics analyses. Proteins that showed changes in expression levels were analyzed in silico using OmicsBox and String analysis to investigate the canonical pathways and functional networks involved in infection. Our results demonstrate that, after cholesterol consumption, M. smegmatis can induce deregulation of protein expression in macrophages. Many of these proteins are related to cytoskeleton remodeling, immune response, the ubiquitination pathway, mRNA processing, and immunometabolism. The identification of these proteins sheds light on the biochemical pathways involved in the mechanisms of action of mycobacteria infection, and may suggest novel protein targets for the development of new and improved treatments.
RESUMO
Spider venoms, despite their toxicity, represent rich sources of pharmacologically active compounds with biotechnological potential. However, in view of the large diversity of the spider species, the full potential of their venom molecules is still far from being known. In this work, we report the purification and structural and functional characterization of GiTx1 (ß/κ-TRTX-Gi1a), the first toxin purified from the venom of the Brazilian tarantula spider Grammostola iheringi. GiTx1 was purified by chromatography, completely sequenced through automated Edman degradation and tandem mass spectrometry and its structure was predicted by molecular modeling. GiTx1 has a MW of 3.585 Da, with the following amino acid sequence: SCQKWMWTCDQKRPCCEDMVCKLWCKIIK. Pharmacological activity of GiTx1 was characterized by electrophysiology using whole-cell patch clamp on dorsal root ganglia neurons (DRG) and two-electrode voltage-clamp on voltage-gated sodium and potassium channels subtypes expressed in Xenopus laevis oocytes. GiTx1, at 2 µM, caused a partial block of inward (â¼40%) and outward (â¼20%) currents in DRG cells, blocked rNav1.2, rNav1.4 and mNav1.6 and had a significant effect on VdNav, an arachnid sodium channel isoform. IC50 values of 156.39 ± 14.90 nM for Nav1.6 and 124.05 ± 12.99 nM for VdNav, were obtained. In addition, this toxin was active on rKv4.3 and hERG potassium channels, but not Shaker IR or rKv2.1 potassium channels. In summary, GiTx1 is a promiscuous toxin with multiple effects on different types of ion channels.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Venenos de Aranha , Aranhas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Moscas Domésticas , Humanos , Camundongos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Domínios Proteicos , Ratos , Ratos Wistar , Venenos de Aranha/química , Venenos de Aranha/isolamento & purificação , Venenos de Aranha/toxicidade , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/isolamento & purificação , Bloqueadores do Canal de Sódio Disparado por Voltagem/toxicidade , Canais de Sódio Disparados por Voltagem/químicaRESUMO
Exposure to bisphenol A (BPA) and bisphenol S (BPS) has been associated with the development of metabolic disorders, such as obesity, dyslipidemias, and nonalcoholic fatty liver disease. Nonetheless, the associated mechanisms are still not fully understood. BPS is being used with no restrictions to replace BPA, which increases the concern regarding its safety and claims for further investigation on its potential mechanisms of toxicity. The present study aims to access liver molecular disturbances which could be associated with systemic metabolic disorders following exposure to BPA or BPS. Therefore, body weight gain and serum biochemical parameters were measured in male Wistar rats chronically exposed to 50 or 500 µg/kg/day of BPA or BPS, while an extensive evaluation of liver protein expression changes was conducted after exposure to 50 µg/kg/day of both compounds. Exposure to the lowest dose of BPA led to the development of hyperglycemia and hypercholesterolemia, while the BPS lowest dose led to the development of hypertriglyceridemia. Besides, exposure to 500 µg/kg/day of BPS significantly increased body weight gain and LDL-cholesterol levels. Hepatic proteins differentially expressed in BPA and BPS-exposed groups compared to the control group were mostly related to lipid metabolism and synthesis, with upregulation of glucokinase activity-related sequence 1 (1.8-fold in BPA and 2.4-fold in BPS), which is involved in glycerol triglycerides synthesis, and hydroxymethylglutaryl-CoA synthase cytoplasmic (2-fold in BPS), an enzyme involved in mevalonate biosynthesis. Essential mitochondrial proteins of the electron transport chain were upregulated after exposure to both contaminants. Also, BPA and BPS dysregulated expression of liver antioxidant enzymes, which are involved in cellular reactive oxygen species detoxification. Altogether, the results of the present study contribute to expand the scientific understanding of how BPA and BPS lead to the development of metabolic disorders and reinforce the risks associated with exposure to these contaminants.
Assuntos
Compostos Benzidrílicos , Fenóis , Proteômica , Sulfonas , Animais , Compostos Benzidrílicos/toxicidade , Fígado , Masculino , Fenóis/toxicidade , Ratos , Ratos Wistar , Sulfonas/toxicidadeRESUMO
To date, several families of peptide toxins specifically interacting with ion channels in scorpion venom have been described. One of these families comprise peptide toxins (called KTxs), known to modulate potassium channels. Thus far, 202 KTxs have been reported, belonging to several subfamilies of KTxs (called α, ß, γ, κ, δ, and λ-KTxs). Here we report on a previously described orphan toxin from Tityus serrulatus venom, named Ts11. We carried out an in-depth structure-function analysis combining 3D structure elucidation of Ts11 and electrophysiological characterization of the toxin. The Ts11 structure is highlighted by an Inhibitor Cystine Knot (ICK) type scaffold, completely devoid of the classical secondary structure elements (α-helix and/or ß-strand). This has, to the best of our knowledge, never been described before for scorpion toxins and therefore represents a novel, 6th type of structural fold for these scorpion peptides. On the basis of their preferred interaction with voltage-gated K channels, as compared to all the other targets tested, it can be postulated that Ts11 is the first member of a new subfamily, designated as ε-KTx.
Assuntos
Peptídeos/química , Venenos de Escorpião/química , Toxinas Biológicas/química , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes , Oócitos/metabolismo , Oócitos/fisiologia , Peptídeos/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Toxinas Biológicas/fisiologia , Canais de Sódio Disparados por Voltagem/metabolismo , Canais de Sódio Disparados por Voltagem/fisiologia , Xenopus laevisRESUMO
PURPOSE: We designed a peptide, PnPP-19, comprising the potential active core of the Phoneutria nigriventer native toxin PnTx2-6. We investigated its role on erectile function, and its toxicity and immunogenicity. MATERIALS AND METHODS: Erectile function was evaluated by the intracavernous pressure-to-mean arterial pressure ratio during electrical field stimulation on rat pelvic ganglia. Cavernous strips were contracted with phenylephrine and relaxation was induced by electrical field stimulation with or without PnPP-19 (10(-8) M). Activity on sodium channels was evaluated by electrophysiological screening of transfected channels on Xenopus oocytes and dorsal root ganglion cells. Antibodies were detected by indirect enzyme-linked immunosorbent assay in mice previously treated with the peptide. Histopathological studies were performed with mouse organs treated with different doses of PnPP-19. RESULTS: PnPP-19 was able to potentiate erection at 4 and 8 Hz in vivo and ex vivo. It showed no toxicity and low immunogenicity in mice, and did not affect sodium channels or rat hearts. PnPP-19 increased cyclic guanosine monophosphate levels at 8 Hz. This effect was inhibited by L-NAME (10(-4) M). Erectile function was partially inhibited by 7-nitroindazole (10(-5) M), a selective inhibitor of neuronal nitric oxide synthase. CONCLUSIONS: PnPP-19 potentiates erection in vivo and ex vivo via the nitric oxide/cyclic guanosine monophosphate pathway. It does not affect sodium channels or rat hearts and shows no toxicity and low immunogenicity. These findings make it a promising candidate as a novel drug in the therapy of erectile dysfunction.
Assuntos
GMP Cíclico/metabolismo , Disfunção Erétil/tratamento farmacológico , Neuropeptídeos/farmacologia , Óxido Nítrico Sintase Tipo I/metabolismo , Ereção Peniana/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Disfunção Erétil/fisiopatologia , Masculino , Camundongos , Neurotoxinas , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-DawleyRESUMO
Schizophrenia is a severe psychiatric disorder with multi-factorial characteristics. A number of findings have shown disrupted synaptic connectivity in schizophrenia patients and emerging evidence suggests that this results from dysfunctional oligodendrocytes, the cells responsible for myelinating axons in white matter to promote neuronal conduction. The exact cause of this is not known, although recent imaging and molecular profiling studies of schizophrenia patients have identified changes in white matter tracts connecting multiple brain regions with effects on protein signaling networks involved in the myelination process. Further understanding of oligodendrocyte dysfunction in schizophrenia could lead to identification of novel drug targets for this devastating disease.
RESUMO
Sea anemones represent one of the emerging groups of interest concerning venomous animals in toxinology and the goal of the present work was the prospection, and the structural and functional characterization of the compounds present in the secretion of the sea anemone Stichodactyla duerdeni from Brazilian coast. We used a combination of offline RPC-MALDI-TOF and online nano-RPC-ESI-LTQ-Orbitrap proteomic techniques as well as functional bioassays. The mucus was milked by electric stimulation and fractionated by gel filtration on Sephadex G-50 yielding 5 main fractions. The low molecular weight fractions were further submitted to RP-HPLC resulting in 35 new subfractions that were subsequently analyzed by offline MALDI-TOF mass spectrometry. MALDI peptide mass fingerprinting yielded up to 134 different molecular masses, ranging from m/z 901 to 10,833. Among these subfractions, a new peptide of 3431Da, named U-SHTX-Sdd1, was purified and completely sequenced by automated Edman's degradation and tandem mass spectrometry. An analysis of U-SHTX-Sdd1 revealed a modified O-HexNAc-Threonine at position 1, which, at the best of our knowledge, constitutes the first sea anemone toxin reported with such post-translational modification. Because of its sequence similarity with other sea anemone toxins, the pharmacological activity of U-SHTX-Sdd1 was assessed by electrophysiological measurements using the two electrode voltage-clamp technique on cloned voltage-gated potassium channel subtypes, expressed in Xenopus laevis oocytes. However, U-SHTX-Sdd1 did not show activity on these channels. A large-scale proteomic approach was also employed to shed lights on the sea anemone compounds, and a total 67 proteins and peptides were identified. BIOLOGICAL SIGNIFICANCE: In this manuscript, we report an extensive characterization of S. duerdeni secretion by means of peptide mass fingerprinting and high-throughput proteome analyses. Also, we report the structure of a new glycopeptide by a combination of biochemical techniques. Despite the previous studies that described proteinaceous compounds present in sea anemone secretions, the number of reported primary sequences is still low. Thus, to access the scenery of protein components from S. duerdeni mucus, including their biological functions, a robust proteomic approach was used together with bioinformatic tools. The demonstrated strategy of analysis is perfectly suitable to other sea anemone secretions and animal venoms. Moreover, new peptide structures can arise contributing to the knowledge of the diversity of these animal peptides.
Assuntos
Glicopeptídeos , Ativação do Canal Iônico/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Proteômica , Anêmonas-do-Mar , Animais , Glicopeptídeos/química , Glicopeptídeos/genética , Glicopeptídeos/metabolismo , Glicopeptídeos/farmacologia , Ativação do Canal Iônico/genética , Toxinas Marinhas/química , Toxinas Marinhas/genética , Toxinas Marinhas/metabolismo , Toxinas Marinhas/farmacologia , Oócitos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/biossíntese , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anêmonas-do-Mar/química , Anêmonas-do-Mar/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Xenopus laevisRESUMO
Sea anemones are known to contain a wide diversity of biologically active peptides, mostly unexplored according to recent peptidomic and transcriptomic studies. In the present work, the neurotoxic fractions from the exudates of Stichodactyla helianthus and Bunodosoma granulifera were analyzed by reversed-phase chromatography and mass spectrometry. The first peptide fingerprints of these sea anemones were assessed, revealing the largest number of peptide components (156) so far found in sea anemone species, as well as the richer peptide diversity of B. granulifera in relation to S. helianthus. The transcriptomic analysis of B. granulifera, performed by massive cDNA sequencing with 454 pyrosequencing approach allowed the discovery of five new APETx-like peptides (U-AITX-Bg1a-e - including the full sequences of their precursors for four of them), which together with type 1 sea anemone sodium channel toxins constitute a very distinguishable feature of studied sea anemone species belonging to genus Bunodosoma. The molecular modeling of these new APETx-like peptides showed a distribution of positively charged and aromatic residues in putative contact surfaces as observed in other animal toxins. On the other hand, they also showed variable electrostatic potentials, thus suggesting a docking onto their targeted channels in different spatial orientations. Moreover several crab paralyzing toxins (other than U-AITX-Bg1a-e), which induce a variety of symptoms in crabs, were isolated. Some of them presumably belong to new classes of crab-paralyzing peptide toxins, especially those with molecular masses below 2kDa, which represent the smallest peptide toxins found in sea anemones.