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1.
Pharmaceuticals (Basel) ; 17(2)2024 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-38399400

RESUMO

Monomeric ubiquitin (Ub) is a 76-amino-acid highly conserved protein found in eukaryotes. The biological activity of Ub first described in the 1970s was extracellular, but it quickly gained relevance due to its intracellular role, i.e., post-translational modification of intracellular proteins (ubiquitination) that regulate numerous eukaryotic cellular processes. In the following years, the extracellular role of Ub was relegated to the background, until a correlation between higher survival rate and increased serum Ub concentrations in patients with sepsis and burns was observed. Although the mechanism of action (MoA) of extracellular ubiquitin (eUb) is not yet well understood, further studies have shown that it may ameliorate the inflammatory response in tissue injury and multiple sclerosis diseases. These observations, compounded with the high stability and low immunogenicity of eUb due to its high conservation in eukaryotes, have made this small protein a relevant candidate for biotherapeutic development. Here, we review the in vitro and in vivo effects of eUb on immunologic, cardiovascular, and nervous systems, and discuss the potential MoAs of eUb as an anti-inflammatory, antimicrobial, and cardio- and brain-protective agent.

2.
Int J Mol Sci ; 24(15)2023 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-37569569

RESUMO

Testicular cancer is the most prevalent tumor among males aged 15 to 35, resulting in a significant number of newly diagnosed cases and fatalities annually. Non-coding RNAs (ncRNAs) have emerged as key regulators in various cellular processes and pathologies, including testicular cancer. Their involvement in gene regulation, coding, decoding, and overall gene expression control suggests their potential as targets for alternative treatment approaches for this type of cancer. Furthermore, epigenetic modifications, such as histone modifications, DNA methylation, and the regulation by microRNA (miRNA), have been implicated in testicular tumor progression and treatment response. Epigenetics may also offer critical insights for prognostic evaluation and targeted therapies in patients with testicular germ cell tumors (TGCT). This comprehensive review aims to present the latest discoveries regarding the involvement of some proteins and ncRNAs, mainly miRNAs and lncRNA, in the epigenetic aspect of testicular cancer, emphasizing their relevance in pathogenesis and their potential, given the fact that their specific expression holds promise for prognostic evaluation and targeted therapies.


Assuntos
MicroRNAs , Neoplasias Embrionárias de Células Germinativas , Neoplasias Testiculares , Masculino , Humanos , Neoplasias Testiculares/genética , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Epigênese Genética , Neoplasias Embrionárias de Células Germinativas/genética
3.
Pathogens ; 12(1)2023 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-36678426

RESUMO

To understand whether protein Tv-PSP1 from Trichomonas vaginalis recognizes mRNA parasite stem-loop structures, we conducted REMSA and intrinsic fluorescence assays. We found the recombinant Tv-PSP1 structure, determined with X-ray crystallography, showed unusual thermal stability of the quaternary structure, associated with a disulfide bridge CYS76-CYS104. To gain deeper insight into the Tv-PSP1 interaction with mRNA stem-loops (mRNAsl) and its relationship with thermal stability, we also used an integrated computational protocol that combined molecular dynamics simulations, docking assays, and binding energy calculations. Docking models allowed us to determine a putative contact surface interaction region between Tv-PSP1 and mRNAsl. We determined the contributions of these complexes to the binding free energy (ΔGb) in the electrostatic (ΔGelec) and nonelectrostatic (ΔGnon-elec) components using the Adaptive Poisson-Boltzmann Solver (APBS) program. We are the first, to the best of our knowledge, to show the interaction between Tv-PSP1 and the stem-loop structures of mRNA.

4.
Artigo em Inglês | MEDLINE | ID: mdl-35636129

RESUMO

The goal of this work is to compile and discuss molecules of marine origin reported in the scientific literature with anti-parasitic activity against Trichomonas, Giardia, and Entamoeba, parasites responsible for diseases that are major global health problems, and Microsporidial parasites as an emerging problem. The presented data correspond to metabolites with anti-parasitic activity in human beings that have been isolated by chromatographic techniques from marine sources and structurally elucidated by spectroscopic and spectrometric procedures. We also highlight some semi-synthetic derivatives that have been successful in enhancing the activity of original compounds. The biological oceanic reservoir offers the possibility to discover new biologically active molecules as lead compounds to develop new drug candidates. The molecular variety is extensive and must be correctly explored and managed. Also, it will be necessary to take some actions to preserve the source species from extinction or overharvest (e.g., by cryopreservation of coral spermatozoa, oocytes, embryos, and larvae) and coordinate appropriate exploitation to increase the chemical knowledge of the natural products generated in the oceans. Additional initiatives such as the total synthesis of complex natural products and their derivatives can help to prevent overharvest of the marine ecosystems and at the same time contribute to the discovery of new molecules.


Assuntos
Antiprotozoários , Produtos Biológicos , Parasitos , Animais , Antiprotozoários/química , Produtos Biológicos/farmacologia , Ecossistema , Giardia , Humanos
5.
Mediators Inflamm ; 2019: 9130868, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31827384

RESUMO

Major depressive disorder (MDD) has a prevalence of 5% in adolescents. Several studies have described the association between the inflammatory response and MDD, but little is known about the relationship between MDD and growth factors, such as IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF. It must be appointed that there are scarce reports on growth factors in adolescents with MDD and even fewer with a clinical follow-up. In this work, we evaluated the levels of growth factors (IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF) in MDD adolescents and the clinical follow-up during eight weeks of treatment with fluoxetine. Methods. All patients were diagnosed according to the DSM-IV-TR, and the severity of the symptoms was evaluated using the Hamilton Depression Rating Scale (HDRS). Growth factors IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF were quantified by cytometric bead array using serum samples from 22 adolescents with MDD and 18 healthy volunteers. Results. All patients showed clinical improvement since the fourth week of pharmacological treatment according to the HDRS. Considerably higher levels of IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF were detected in MDD adolescents as compared to healthy volunteers. A significant but temporal decrease was detected in basic FGF, G-CSF, and GM-CSF at week four of fluoxetine administration. Conclusions. To the best of our knowledge, this is the first report to show alterations in the levels of growth factors, such as IL-7, IL-9, IL-17A, VEGF, basic FGF, G-CSF, and GM-CSF in MDD adolescents during eight weeks of clinical follow-up. These disturbances might be involved in the physiopathology of MDD since such growth factors have been proven to participate in the neural development and correct functioning of the CNS; therefore, subtle alterations in it may contribute to MDD.


Assuntos
Transtorno Depressivo Maior/tratamento farmacológico , Fluoxetina/uso terapêutico , Adolescente , Adulto , Transtorno Depressivo Maior/sangue , Feminino , Fator 2 de Crescimento de Fibroblastos/sangue , Fator Estimulador de Colônias de Granulócitos/sangue , Fator Estimulador de Colônias de Granulócitos e Macrófagos/sangue , Humanos , Interleucina-17/sangue , Interleucina-7/sangue , Interleucina-9/sangue , Estudos Longitudinais , Masculino , Fator A de Crescimento do Endotélio Vascular/sangue , Adulto Jovem
6.
Biometals ; 32(6): 887-899, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31586273

RESUMO

Metallothioneins (MTs) have been identified in a wide variety of organisms from bacteria to humans. The biological functions of these MTs have a key role in metalloregulatory metabolism and its expression is induced in response to different stimuli, particularly by divalent metal cations. Also, the action of MTs have been implicated in the survival of pathogens in presence of microbicidal concentration of divalent cations, which allows the establishment of the infection. Trichomonas vaginalis is a protozoan parasite that adapts to the microenvironment of the male urogenital tract, where cations such as zinc (Zn2+) and cadmium (Cd2+) are present. Nevertheless, the molecular mechanisms of metal tolerance and homeostasis is not yet dilucidated in this parasite. In this study, we have identified 4 potential MT-like sequences (tvmt´s) in T. vaginalis genome. Because tvmt-2, -3, and -4 corresponds to truncated partial genes, we characterized the trichomonad tvmt-1 gene. The bioinformatic analyses and the predicted protein (TvMT-1) show similar properties to the reported in other MTs. The expression patterns of tvmt-1 in the presence of several divalent cations (Fe2+, Mn2+, Zn2+ and Cd2+) were analyzed and we demonstrated that Cd2+ induce significantly their expression. By indirect immunofluorescence assays, we corroborated this positive regulation of TvMT-1 in the cytoplasm of parasites grown in the presence of Cd2+. The tvmt-1 promoter contains putative metal responsive elements, which are probably the responsible for the Cd2+-dependent expression of this gene. Our results suggest that tvmt-1 gene encode a metallothionein that may be responsible for the homeostatis and detoxification of Cd+2 in T. vaginalis.


Assuntos
Cádmio/farmacologia , Metalotioneína/genética , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/genética , Homeostase/efeitos dos fármacos , Metalotioneína/metabolismo , Trichomonas vaginalis/metabolismo
7.
Front Oncol ; 9: 1370, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31921634

RESUMO

During angiogenesis, new vessels emerge from existing endothelial lined vessels to promote the degradation of the vascular basement membrane and remodel the extracellular matrix (ECM), followed by endothelial cell migration, and proliferation and the new generation of matrix components. Matrix metalloproteinases (MMPs) participate in the disruption, tumor neovascularization, and subsequent metastasis while tissue inhibitors of metalloproteinases (TIMPs) downregulate the activity of these MMPs. Then, the angiogenic response can be directly or indirectly mediated by MMPs through the modulation of the balance between pro- and anti-angiogenic factors. This review analyzes recent knowledge on MMPs and their participation in angiogenesis.

8.
Mol Biotechnol ; 60(8): 563-575, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29936696

RESUMO

Previously, metalloproteinase was isolated and identified from Trichomonas vaginalis, belonging to the aminopeptidase P-like metalloproteinase subfamily A/B, family M24 of clan MG, named TvMP50. The native and recombinant TvMP50 showed proteolytic activity, determined by gelatin zymogram, and a 50 kDa band, suggesting that TvMP50 is a monomeric active enzyme. This was an unexpected finding since other Xaa-Pro aminopeptidases/prolidases are active as a biological unit formed by dimers/tetramers. In this study, the evolutionary history of TvMP50 and the preliminary crystal structure of the recombinant enzyme determined at 3.4 Å resolution is reported. TvMP50 was shown to be a type of putative, eukaryotic, monomeric aminopeptidase P, and the crystallographic coordinates showed a monomer on a "pseudo-homodimer" array on the asymmetric unit that resembles the quaternary structure of the M24B dimeric family and suggests a homodimeric aminopeptidase P-like enzyme as a likely ancestor. Interestingly, TvMP50 had a modified N-terminal region compared with other Xaa-Pro aminopeptidases/prolidases with three-dimensional structures; however, the formation of the standard dimer is structurally unstable in aqueous solution, and a comparably reduced number of hydrogen bridges and lack of saline bridges were found between subunits A/B, which could explain why TvMP50 portrays monomeric functionality. Additionally, we found that the Parabasalia group contains two protein lineages with a "pita bread" fold; the ancestral monomeric group 1 was probably derived from an ancestral dimeric aminopeptidase P-type enzyme, and group 2 has a probable dimeric kind of ancestral eukaryotic prolidase lineage. The implications of such hypotheses are also presented.


Assuntos
Aminopeptidases/metabolismo , Metaloproteases/metabolismo , Proteínas de Protozoários/metabolismo , Trichomonas vaginalis/enzimologia , Sequência de Aminoácidos , Aminopeptidases/química , Aminopeptidases/genética , Cristalografia por Raios X , Dipeptidases/química , Dipeptidases/genética , Dipeptidases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Metaloproteases/química , Metaloproteases/genética , Peso Molecular , Filogenia , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Trichomonas vaginalis/classificação , Trichomonas vaginalis/genética
9.
Sci Rep ; 8(1): 9063, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899533

RESUMO

Murine leprosy, caused by Mycobacterium lepraemurium (MLM), is a chronic disease that closely resembles human leprosy. Even though this disease does not directly involve the nervous system, we investigated a possible effect on working memory during this chronic infection in Balb/c mice. We evaluated alterations in the dorsal region of the hippocampus and measured peripheral levels of cytokines at 40, 80, and 120 days post-infection. To evaluate working memory, we used the T-maze while a morphometric analysis was conducted in the hippocampus regions CA1, CA2, CA3, and dentate gyrus (DG) to measure morphological changes. In addition, a neurochemical analysis was performed by HPLC. Our results show that, at 40 days post-infection, there was an increase in the bacillary load in the liver and spleen associated to increased levels of IL-4, working memory deterioration, and changes in hippocampal morphology, including degeneration in the four subregions analyzed. Also, we found a decrease in neurotransmitter levels at the same time of infection. Although MLM does not directly infect the nervous system, these findings suggest a possible functional link between the immune system and the central nervous system.


Assuntos
Hipocampo/fisiopatologia , Transtornos da Memória/fisiopatologia , Infecções por Mycobacterium/fisiopatologia , Animais , Doença Crônica , Giro Denteado/microbiologia , Giro Denteado/patologia , Giro Denteado/fisiopatologia , Hipocampo/microbiologia , Hipocampo/patologia , Interações Hospedeiro-Patógeno , Interleucina-4/metabolismo , Masculino , Aprendizagem em Labirinto , Transtornos da Memória/metabolismo , Transtornos da Memória/microbiologia , Memória de Curto Prazo , Camundongos Endogâmicos BALB C , Infecções por Mycobacterium/metabolismo , Infecções por Mycobacterium/microbiologia , Mycobacterium lepraemurium/fisiologia , Neurotransmissores/metabolismo , Fatores de Tempo
10.
Mediators Inflamm ; 2018: 4074051, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30662368

RESUMO

Changes in cytokine levels in major depression and during treatment have been reported in adults. However, few studies have examined cytokine levels in an adolescent sample despite this being a common age of onset. Methods. We measured proinflammatory (IL-2, IFN-γ, IL-1ß, TNF-α, IL-6, IL-12, and IL-15) and anti-inflammatory (IL-4, IL-5, IL-13, IL-1Ra, and IL-10) cytokine serum levels in 22 adolescents with major depression and 18 healthy volunteers. Cytokines were measured by multiplex bead-based immunoassays at baseline, and 4 and 8 weeks after commencement of fluoxetine administration in the clinical group. Results. Compared to healthy volunteers, adolescents with major depression at baseline showed significant increases in all pro- and anti-inflammatory cytokines, except IL-1Ra and IL-10. Significant changes were observed in fluoxetine treatment compared to baseline: proinflammatory cytokines IFN-γ, IL-1ß, TNF-α, IL-6, IL-12, and IL-15 were decreased only at week 4 whereas IL-2 was increased only at week 8; anti-inflammatory cytokines IL-4 and IL-5 were increased at week 8 while IL-1Ra was reduced only at week 4. There were no significant correlations between cytokine levels and symptomatic improvement in HDRS. Discussion. The results suggest a significant interplay between cytokine levels, the depressive state, and the stage of treatment with an SSRI. To the best of our knowledge, this is the first report in depressed adolescents with elevated IL-12, IL-13, and IL-15 levels. Further studies are necessary to clarify the role and mechanisms of altered cytokine levels in the pathogenesis and physiopathology of major depressive disorder.


Assuntos
Antidepressivos de Segunda Geração/uso terapêutico , Transtorno Depressivo Maior/tratamento farmacológico , Fluoxetina/uso terapêutico , Adolescente , Adulto , Citocinas/sangue , Depressão/sangue , Depressão/tratamento farmacológico , Depressão/imunologia , Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/imunologia , Feminino , Humanos , Interleucina-10/sangue , Interleucina-1beta/sangue , Masculino , Fator de Necrose Tumoral alfa/sangue , Adulto Jovem
11.
Biometals ; 30(6): 861-872, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28993928

RESUMO

The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental stimulus by biometals such as Zn2+. Many of these proteins have the classical C2H2 zinc finger motifs (C2H2-ZNFm) of approximately 30 amino acids, where a Zn2+ ion is coordinated by two cysteine and two histidine residues. Trichomonas vaginalis is a protozoan parasite than responds to environmental changes including Zn2+. Until now has not been described any ZNF that could be involved in the regulation of genic expression of T. vaginalis. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from T. vaginalis and isolated the gene, tvznf1 encoding it. TvZNF1 have eight C2H2-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn2+ upregulation expression of tvznf1 gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in T. vaginalis.


Assuntos
Dedos de Zinco CYS2-HIS2 , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Trichomonas vaginalis/genética , Sítios de Ligação , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Estrutura Secundária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Elementos de Resposta , Fatores de Transcrição/genética , Trichomonas vaginalis/química , Trichomonas vaginalis/metabolismo , Zinco/metabolismo
12.
J Immunol Res ; 2016: 3160486, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27795960

RESUMO

Dopamine (DA), a neurotransmitter in the central nervous system (CNS), has modulatory functions at the systemic level. The peripheral and central nervous systems have independent dopaminergic system (DAS) that share mechanisms and molecular machinery. In the past century, experimental evidence has accumulated on the proteins knowledge that is involved in the synthesis, reuptake, and transportation of DA in leukocytes and the differential expression of the D1-like (D1R and D5R) and D2-like receptors (D2R, D3R, and D4R). The expression of these components depends on the state of cellular activation and the concentration and time of exposure to DA. Receptors that are expressed in leukocytes are linked to signaling pathways that are mediated by changes in cAMP concentration, which in turn triggers changes in phenotype and cellular function. According to the leukocyte lineage, the effects of DA are associated with such processes as respiratory burst, cytokine and antibody secretion, chemotaxis, apoptosis, and cytotoxicity. In clinical conditions such as schizophrenia, Parkinson disease, Tourette syndrome, and multiple sclerosis (MS), there are evident alterations during immune responses in leukocytes, in which changes in DA receptor density have been observed. Several groups have proposed that these findings are useful in establishing clinical status and clinical markers.


Assuntos
Dopamina/metabolismo , Imunomodulação , Animais , Doenças Autoimunes/genética , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Sistema Nervoso Central/citologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/metabolismo , Proteínas da Membrana Plasmática de Transporte de Dopamina/genética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Regulação da Expressão Gênica , Humanos , Leucócitos/imunologia , Leucócitos/metabolismo , Transtornos Mentais/genética , Transtornos Mentais/imunologia , Transtornos Mentais/metabolismo , Redes e Vias Metabólicas , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/imunologia , Doenças Neurodegenerativas/metabolismo , Especificidade de Órgãos , Sistema Nervoso Periférico/imunologia , Sistema Nervoso Periférico/metabolismo , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Transdução de Sinais
13.
J Immunol Res ; 2015: 354957, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25961058

RESUMO

Serotonin (5-HT) induces concentration-dependent metabolic effects in diverse cell types, including neurons, entherochromaffin cells, adipocytes, pancreatic beta-cells, fibroblasts, smooth muscle cells, epithelial cells, and leukocytes. Three classes of genes regulating 5-HT function are constitutively expressed or induced in these cells: (a) membrane proteins that regulate the response to 5-HT, such as SERT, 5HTR-GPCR, and the 5HT3-ion channels; (b) downstream signaling transduction proteins; and (c) enzymes controlling 5-HT metabolism, such as IDO and MAO, which can generate biologically active catabolites, including melatonin, kynurenines, and kynurenamines. This review covers the clinical and experimental mechanisms involved in 5-HT-induced immunomodulation. These mechanisms are cell-specific and depend on the expression of serotonergic components in immune cells. Consequently, 5-HT can modulate several immunological events, such as chemotaxis, leukocyte activation, proliferation, cytokine secretion, anergy, and apoptosis. The effects of 5-HT on immune cells may be relevant in the clinical outcome of pathologies with an inflammatory component. Major depression, fibromyalgia, Alzheimer disease, psoriasis, arthritis, allergies, and asthma are all associated with changes in the serotonergic system associated with leukocytes. Thus, pharmacological regulation of the serotonergic system may modulate immune function and provide therapeutic alternatives for these diseases.


Assuntos
Imunomodulação/imunologia , Leucócitos/imunologia , Receptores de Serotonina/imunologia , Serotonina/imunologia , Animais , Artrite/imunologia , Asma/imunologia , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Quimiotaxia de Leucócito/imunologia , Humanos , Camundongos , Neoplasias/imunologia , Transporte Proteico/genética , Receptores de Serotonina/metabolismo , Serotonina/metabolismo , Transdução de Sinais/imunologia
14.
J Immunol Res ; 2015: 401630, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25961060

RESUMO

The benefits of garlic to health have been proclaimed for centuries; however, only recently have Allium sativum and its derivatives been proposed as promising candidates for maintaining the homeostasis of the immune system. The complex biochemistry of garlic makes it possible for variations in processing to yield different preparations with differences in final composition and compound proportion. In this review, we assess the most recent experimental results, which indicate that garlic appears to enhance the functioning of the immune system by stimulating certain cell types, such as macrophages, lymphocytes, natural killer (NK) cells, dendritic cells, and eosinophils, by mechanisms including modulation of cytokine secretion, immunoglobulin production, phagocytosis, and macrophage activation. Finally, because immune dysfunction plays an important role in the development and progress of several diseases, we critically examined immunoregulation by garlic extracts and compounds isolated, which can contribute to the treatment and prevention of pathologies such as obesity, metabolic syndrome, cardiovascular disorders, gastric ulcer, and even cancer. We concluded that A. sativum modulates cytokine secretion and that such modulation may provide a mechanism of action for many of their therapeutic effects.


Assuntos
Alho/metabolismo , Imunomodulação/efeitos dos fármacos , Inflamação/tratamento farmacológico , Extratos Vegetais/farmacologia , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/imunologia , Humanos , Inflamação/imunologia , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Ativação de Macrófagos/imunologia , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Fagocitose/efeitos dos fármacos , Fagocitose/imunologia
15.
Insect Biochem Mol Biol ; 41(6): 400-9, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21396445

RESUMO

Triosephosphate isomerase (TIM) is an enzyme with a role in glycolysis and gluconeogenesis by catalyzing the interconversion between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. This enzyme has been used as a target in endoparasite drug development. In this work we cloned, expressed, purified and studied kinetic and structural characteristics of TIM from tick embryos, Rhipicephalus (Boophilus) microplus (BmTIM). The Km and Vmax of the recombinant BmTIM with glyceraldehyde 3-phosphate as substrate, were 0.47 mM and 6031 µmol min⁻¹ mg protein⁻¹, respectively. The resolution of the diffracted crystal was estimated to be 2.4 Å and the overall data showed that BmTIM is similar to other reported dimeric TIMs. However, we found that, in comparison to other TIMs, BmTIM has the highest content of cysteine residues (nine cysteine residues per monomer). Only two cysteines could make disulfide bonds in monomers of BmTIM. Furthermore, BmTIM was highly sensitive to the action of the thiol reagents dithionitrobenzoic acid and methyl methane thiosulfonate, suggesting that there are five cysteines exposed in each dimer and that these residues could be employed in the development of species-specific inhibitors.


Assuntos
Embrião não Mamífero/enzimologia , Proteínas Recombinantes/metabolismo , Rhipicephalus/enzimologia , Triose-Fosfato Isomerase/metabolismo , Zigoto/enzimologia , Sequência de Aminoácidos , Animais , Catálise , Clonagem Molecular , Cristalografia por Raios X , Cisteína/química , Cisteína/metabolismo , Fosfato de Di-Hidroxiacetona/metabolismo , Dimerização , Escherichia coli , Gliceraldeído 3-Fosfato/metabolismo , Humanos , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica/efeitos dos fármacos , Proteínas Recombinantes/genética , Rhipicephalus/embriologia , Alinhamento de Sequência , Reagentes de Sulfidrila/farmacologia , Triose-Fosfato Isomerase/antagonistas & inibidores , Triose-Fosfato Isomerase/genética , Triose-Fosfato Isomerase/isolamento & purificação
16.
Biochem Biophys Res Commun ; 382(3): 626-30, 2009 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-19303397

RESUMO

It is assumed that amino acid sequence differences in highly homologous enzymes would be found at the peripheral level, subtle changes that would not necessarily affect catalysis. Here, we demonstrate that, using the same set of mutations at the level of the interface loop 3, the activity of a triosephosphate isomerase monomeric enzyme is ten times higher than that of a homologous enzyme with 74% identity and 86% similarity, whereas the activity of the native, dimeric enzymes is essentially the same. This is an example of how the dimeric biological unit evolved to compensate for the intrinsic differences found at the monomeric species level. Biophysical techniques of size exclusion chromatography, dynamic light scattering, X-ray crystallography, fluorescence and circular dichroism, as well as denaturation/renaturation assays with guanidinium hydrochloride and ANS binding, allowed us to fully characterize the properties of the new monomer.


Assuntos
Triose-Fosfato Isomerase/química , Sequência de Aminoácidos/genética , Animais , Catálise , Cristalografia por Raios X , Estabilidade Enzimática/genética , Guanidina/química , Luz , Mutação , Conformação Proteica , Desnaturação Proteica , Espalhamento de Radiação , Triose-Fosfato Isomerase/genética , Trypanosoma cruzi/enzimologia
17.
J Bioenerg Biomembr ; 40(4): 359-69, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18770013

RESUMO

We studied for the first time the ATP-synthase complex from shrimp as a model to understand the basis of crustacean bioenergetics since they are exposed to endogenous processes as molting that demand high amount of energy. We analyzed the cDNA sequence of two subunits of the Fo sector from mitochondrial ATP-synthase in the white shrimp Litopenaeus vannamei. The nucleus encoded atp9 subunit presents a 773 bp sequence, containing a signal peptide sequence only observed in crustaceans, and the mitochondrial encoded atp6 subunit presents a sequence of 675 bp, and exhibits high identity with homologous sequences from invertebrate species. ATP9 and ATP6 protein structural models interaction suggest specific functional characteristics from both proteins in the mitochondrial enzyme. Differences in the steady-state mRNA levels of atp9 and atp6 from five different tissues correlate with tissue function. Moreover, significant changes in the mRNA levels of both subunits at different molt stages were detected. We discussed some insights about the enzyme structure and the regulation mechanisms from both ATP-synthase subunits related to the energy requirements of shrimp.


Assuntos
Núcleo Celular/enzimologia , Mitocôndrias/enzimologia , Modelos Químicos , Modelos Moleculares , Penaeidae/enzimologia , ATPases Translocadoras de Prótons , Sequência de Aminoácidos , Animais , Sequência de Bases , Simulação por Computador , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Conformação Proteica , Subunidades Proteicas , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/ultraestrutura , RNA Mensageiro/genética
18.
Artigo em Inglês | MEDLINE | ID: mdl-18678934

RESUMO

The PyrR transcriptional regulator is widely distributed in bacteria. This RNA-binding protein is involved in the control of genes involved in pyrimidine biosynthesis, in which uridyl and guanyl nucleotides function as effectors. Here, the crystallization and preliminary X-ray diffraction analysis of two crystal forms of Bacillus halodurans PyrR are reported. One of the forms belongs to the monoclinic space group P2(1) with unit-cell parameters a = 59.7, b = 87.4, c = 72.1 A, beta = 104.4 degrees , while the other form belongs to the orthorhombic space group P22(1)2(1) with unit-cell parameters a = 72.7, b = 95.9, c = 177.1 A. Preliminary X-ray diffraction data analysis and molecular-replacement solution revealed the presence of four and six monomers per asymmetric unit; a crystallographic tetramer is formed in both forms.


Assuntos
Bacillus/química , Proteínas de Bactérias/química , Pentosiltransferases/química , Proteínas Repressoras/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Sequência de Bases , Cromatografia em Gel , Clonagem Molecular , Cristalografia por Raios X , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Modelos Moleculares , Pentosiltransferases/genética , Pentosiltransferases/isolamento & purificação , Conformação Proteica , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação
19.
Int J Biochem Cell Biol ; 40(10): 2206-17, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18403248

RESUMO

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH(2)THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.


Assuntos
Substituição de Aminoácidos , Escherichia coli/enzimologia , Ácido Fólico/metabolismo , Lisina/metabolismo , Proteínas Mutantes/química , Timidilato Sintase/química , Timidilato Sintase/metabolismo , Sítios de Ligação , Calorimetria , Catálise , Dicroísmo Circular , Cristalografia por Raios X , Antagonistas do Ácido Fólico/química , Ligantes , Mutação , Nucleotídeos/metabolismo , Estrutura Secundária de Proteína , Relação Estrutura-Atividade , Tetra-Hidrofolatos/química , Termodinâmica , Triptofano/metabolismo
20.
FEBS Lett ; 551(1-3): 63-70, 2003 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-12965206

RESUMO

Glucosamine-6-phosphate deaminase (EC 3.5.99.6) is an allosteric enzyme that catalyzes the reversible conversion of D-glucosamine-6-phosphate into D-fructose-6-phosphate and ammonium. Here we describe the existence of two mammalian glucosamine-6-phosphate deaminase enzymes. We present the crystallographic structure of one of them, the long human glucosamine-6-phosphate deaminase, at 1.75 A resolution. Crystals belong to the space group P2(1)2(1)2(1) and present a whole hexamer in the asymmetric unit. The active-site lid (residues 162-182) presented significant structural differences among monomers. Interestingly the region with the largest differences, when compared with the Escherichia coli homologue, was found to be close to the active site. These structural differences can be related to the kinetic and allosteric properties of both mammalian enzymes.


Assuntos
Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Sítio Alostérico , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bovinos , Cricetinae , Cristalografia por Raios X , Escherichia coli/enzimologia , Humanos , Isoenzimas/química , Isoenzimas/genética , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos
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