RESUMO
Phenotypic drug discovery (PDD) involves screening compounds for their effects on cells, tissues, or whole organisms without necessarily understanding the underlying molecular targets. PDD differs from target-based strategies as it does not require knowledge of a specific drug target or its role in the disease. This approach can lead to the discovery of drugs with unexpected therapeutic effects or applications and allows for the identification of drugs based on their functional effects, rather than through a predefined target-based approach. Ultimately, disease definitions are mostly symptom-based rather than mechanism-based, and the therapeutics should be likewise. In recent years, there has been a renewed interest in PDD due to its potential to address the complexity of human diseases, including the holistic picture of multiple metabolites engaging with multiple targets constituting the central hub of the metabolic host-microbe interactions. Although PDD presents challenges such as hit validation and target deconvolution, significant achievements have been reached in the era of big data. This article explores the experiences of researchers testing the effect of a thymic peptide hormone, thymosin alpha-1, in preclinical and clinical settings and discuss how its therapeutic utility in the precision medicine era can be accommodated within the PDD framework.
RESUMO
The study of mechanism of action of Thymosin alpha 1 (Tα1) and the basis of the pleiotropic effect in health and disease, is one of the main focus of our ongoing research. Tα1 is a thymic peptide that demonstrates a peculiar ability to restore homeostasis in different physiological and pathological conditions (i.e., infections, cancer, immunodeficiency, vaccination, and aging) acting as multitasking protein depending on the host state of inflammation or immune dysfunction. However, few are the information about mechanisms of action mediated by specific Tα1-target protein interaction that could explain its pleiotropic effect. We investigated the interaction of Tα1 with Galectin-1 (Gal-1), a protein belonging to an oligosaccharide binding protein family involved in a variety of biological and pathological processes, including immunoregulation, infections, cancer progression and aggressiveness. Using molecular and cellular methodological approaches, we demonstrated the interaction between these two proteins. Tα1 specifically inhibited the hemagglutination activity of Gal-1, the Gal-1 dependent in vitro formation of endothelial cell tubular structures, and the migration of cancer cells in wound healing assay. Physico-chemical methods revealed the details of the molecular interaction of Tα1 with Gal-1. Hence, the study allowed the identification of the not known until now specific interaction between Tα1 and Gal-1, and unraveled a novel mechanism of action of Tα1 that could support understanding of its pleiotropic activity.
Assuntos
Neoplasias , Timosina , Humanos , Timalfasina , Galectina 1RESUMO
BACKGROUND: Thymosin alpha 1 (Tα1) is a well-recognized immune response modulator in a wide range of disorders, particularly infections and cancer. The bioinformatic analysis of public databases allows drug repositioning, predicting a new potential area of clinical intervention. We aimed to decipher the cellular network induced by Tα1 treatment to confirm present use and identify new potential clinical applications. RESEARCH DESIGN AND METHODS: We used the transcriptional profile of human peripheral blood mononuclear cells treated in vitro with Tα1 to perform the enrichment network analysis by the Metascape online tools and the disease enrichment analysis by the DAVID online tool. RESULTS: Networked cellular responses reflected Tα1 regulated biological processes including immune and metabolic responses, response to compounds and oxidative stress, ion homeostasis, peroxisome biogenesis and drug metabolic process. Beyond cancer and infections, the analysis evidenced the association with disorders such as kidney chronic failure, diabetes, cardiovascular, chronic respiratory, neuropsychiatric, neurodegenerative and autoimmune diseases. CONCLUSIONS: In addition to the known ability to promote immune response pathways, the network enrichment analysis demonstrated that Tα1 regulates cellular metabolic processes and oxidative stress response. Notable, the analysis highlighted the association with several diseases, suggesting new translational implication of Tα1 treatment in pathological conditions unexpected until now.
Assuntos
Infecções/tratamento farmacológico , Leucócitos Mononucleares/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Timalfasina/uso terapêutico , Transcriptoma/efeitos dos fármacos , Doenças Autoimunes/tratamento farmacológico , Fenômenos Biológicos/efeitos dos fármacos , Fenômenos Biológicos/genética , Perfilação da Expressão Gênica , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Infecções/sangue , Infecções/genética , Leucócitos Mononucleares/metabolismo , Análise em Microsséries , Neoplasias/sangue , Neoplasias/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genéticaRESUMO
Thymosin α1 (Tα1), is a peptidic hormone, whose immune regulatory properties have been demonstrated both in vitro and in vivo and approved in different countries for treatment of several viral infections and cancers. Tα1 assumes a conformation in negative membranes upon insertion into the phosphatidylserine exposure as found in several pathologies and in apoptosis. These findings are in agreement with the pleiotropy of Tα1, which targets both normal and tumor cells, interacting with multiple cellular components, and have generated renewed interest in the topic. Hyaluronan (HA) occurs ubiquitously in the extracellular matrix and on cell surfaces and has been related to a variety of diseases, and developmental and physiological processes. Proteins binding HA, among them CD44 and the Receptor for HA-mediated motility (RHAMM) receptors, mediate its biological effects. NMR spectroscopy indicated preliminarily that an interaction of Tα1 with HA occurs specifically around lysine residues of the sequence LKEKK of Tα1 and is suggestive of a possible interference of Tα1 in the binding of HA with CD44 and RHAMM. Further studies are needed to deepen these observations because Tα1 is known to potentiate the T-cell immunity and anti-tumor effect. The binding inhibitory activity of Tα1 on HA-CD44 or HA-RHAMM interactions can suppress both T-cell reactivity and tumor progression.
Assuntos
Sequência de Aminoácidos , Ácido Hialurônico/química , Domínios e Motivos de Interação entre Proteínas , Eletricidade Estática , Timosina/análogos & derivados , Espectroscopia de Ressonância Magnética , Ligação Proteica , Timalfasina , Timosina/químicaRESUMO
Natural organosulfur compounds (OSCs) from Allium sativum L. display antioxidant and chemo-sensitization properties, including the in vitro inhibition of tumor cell proliferation through the induction of apoptosis. Garlic water- and oil-soluble allyl sulfur compounds show distinct properties and the capability to inhibit the proliferation of tumor cells. In the present study, we optimized a new protocol for the extraction of water-soluble compounds from garlic at low temperatures and the production of glutathionyl-OSC conjugates during the extraction. Spontaneously, Cys/GSH-mixed-disulfide conjugates are produced by in vivo metabolism of OSCs and represent active molecules able to affect cellular metabolism. Water-soluble extracts, with (GSGaWS) or without (GaWS) glutathione conjugates, were here produced and tested for their ability to release hydrogen sulfide (H2S), also in the presence of reductants and of thiosulfate:cyanide sulfurtransferase (TST) enzyme. Thus, the TST catalysis of the H2S-release from garlic OSCs and their conjugates has been investigated by molecular in vitro experiments. The antiproliferative properties of these extracts on the human T-cell lymphoma cell line, HuT 78, were observed and related to histone hyperacetylation and downregulation of GAPDH expression. Altogether, the results presented here pave the way for the production of a GSGaWS as new, slowly-releasing hydrogen sulfide extract for potential therapeutic applications.
Assuntos
Alho/química , Glutationa/metabolismo , Sulfeto de Hidrogênio/metabolismo , Compostos de Enxofre/uso terapêutico , Enxofre/metabolismo , Biocatálise/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia de Fase Reversa , Temperatura Baixa , Humanos , Linfoma de Células T/patologia , Microscopia de Fluorescência , Peso Molecular , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Substâncias Redutoras/farmacologia , Solubilidade , Compostos de Enxofre/farmacologia , Tiorredoxinas/metabolismo , Tiossulfato Sulfurtransferase/antagonistas & inibidores , Tiossulfato Sulfurtransferase/metabolismo , Água/químicaRESUMO
Both in epithelial development as well as in epithelial cancers, the p53 family member p63 plays a crucial role acting as a master transcriptional regulator. P63 steady state protein levels are regulated by the E3 ubiquitin ligase Itch, via a physical interaction between the PPxY consensus sequence (PY motif) of p63 and one of the 4 WW domains of Itch; this substrate recognition process leads to protein-ubiquitylation and p63 proteasomal degradation. The interaction of the WW domains, a highly compact protein-protein binding module, with the short proline-rich sequences is therefore a crucial regulatory event that may offer innovative potential therapeutic opportunity. Previous molecular studies on the Itch-p63 recognition have been performed in vitro using the Itch-WW2 domain and the peptide interacting fragment of p63 (pep63), which includes the PY motif. Itch-WW2-pep63 interaction is also stabilized in vitro by the conformational constriction of the S-S cyclization in the p63 peptide. The PY motif of p63, as also for other proteins, is characterized by the nearby presence of a (T/S)P motif, which is a potential recognition site of the WW domain of the IV group present in the prolyl-isomerase Pin1. In this study, we demonstrate, by in silico and spectroscopical studies using both the linear pep63 and its cyclic form, that the threonine phosphorylation of the (T/S)PPPxY motif may represent a crucial regulatory event of the Itch-mediated p63 ubiquitylation, increasing the Itch-WW domains-p63 recognition event and stabilizing in vivo the Itch-WW-p63 complex. Moreover, our studies confirm that the subsequently trans/cis proline isomerization of (T/S)P motif by the Pin1 prolyl-isomerase, could modulate the E3-ligase interaction, and that the (T/S)pPtransPPxY motif represent the best conformer for the ItchWW-(T/S)PPPxY motif recognition.
Assuntos
Proteínas de Membrana/metabolismo , Treonina/química , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Proteínas de Membrana/genética , Fosforilação , Ligação Proteica , Ubiquitina-Proteína Ligases/genéticaRESUMO
The structural characterization of [W8S]contryphan Vn, an analogue of Contryphan Vn with tryptophan 8 substituted with a serine residue (W8S), was performed by NMR spectroscopy, molecular dynamics simulations and fluorescence spectroscopy. Contryphan Vn, a bioactive cyclic peptide from the venom of the cone snail Conus ventricosus, contains an S-S bridge between two cysteines and a D-tryptophan. Like other Contryphans, [W8S]contryphan Vn has proline 7 isomerized trans, while the proline 4 has nearly equivalent populations of cis and trans configurations. The thermodynamic and kinetic parameters of the trans-cis isomerization of proline 4 were measured. The isomers of [W8S]contryphan Vn with proline 4 in cis and trans show structural differences. The absence of the salt bridge between the same Asp2 and Lys6, present in Contryphan Vn, may be attributed to the lack of the hydrophobic side chain of Trp8 where it likely protects the electrostatic interactions. These results may contribute to identifying, in these cyclic peptides, the structural determinants of the mechanism of proline trans-cis isomerization, this being also an important step in protein folding.
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Peptídeos Cíclicos/química , Prolina/química , Serina/química , Triptofano/química , Sequência de Aminoácidos , Isomerismo , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos Cíclicos/síntese químicaRESUMO
Increased glycolytic flux is a common feature of many cancer cells, which have adapted their metabolism to maximize glucose incorporation and catabolism to generate ATP and substrates for biosynthetic reactions. Indeed, glycolysis allows a rapid production of ATP and provides metabolic intermediates required for cancer cells growth. Moreover, it makes cancer cells less sensitive to fluctuations of oxygen tension, a condition usually occurring in a newly established tumor environment. Here, we provide evidence for a dual role of MAPK14 in driving a rearrangement of glucose metabolism that contributes to limiting reactive oxygen species (ROS) production and autophagy activation in condition of nutrient deprivation. We demonstrate that MAPK14 is phosphoactivated during nutrient deprivation and affects glucose metabolism at 2 different levels: on the one hand, it increases SLC2A3 mRNA and protein levels, resulting in a higher incorporation of glucose within the cell. This event involves the MAPK14-mediated enhancement of HIF1A protein stability. On the other hand, MAPK14 mediates a metabolic shift from glycolysis to the pentose phosphate pathway (PPP) through the modulation of PFKFB3 (6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase 3) degradation by the proteasome. This event requires the presence of 2 distinct degradation sequences, KEN box and DSG motif Ser273, which are recognized by 2 different E3 ligase complexes. The mutation of either motif increases PFKFB3 resistance to starvation-induced degradation. The MAPK14-driven metabolic reprogramming sustains the production of NADPH, an important cofactor for many reduction reactions and for the maintenance of the proper intracellular redox environment, resulting in reduced levels of ROS. The final effect is a reduced activation of autophagy and an increased resistance to nutrient deprivation.
Assuntos
Autofagia/fisiologia , Glucose/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Apoptose/fisiologia , Linhagem Celular , Humanos , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Estresse OxidativoRESUMO
RATIONALE: A number of observations suggest that iron accumulates in the lungs of patients with idiopathic pulmonary fibrosis (IPF) with vascular abnormalities, including pulmonary hypertension. OBJECTIVES: The aim of this study was to determine the prevalence and intensity of accumulation of alveolar epithelial lining fluid (ELF) iron and of alveolar macrophage hemosiderin in IPF and its relationship with disease severity. METHODS: Forty seven IPF patients and 14 healthy controls were retrospectively evaluated for iron accumulation in the lower respiratory tract using total iron spectrophotometric measures and for hemosiderin accumulation using the Perls' stain with the Golde score. MEASUREMENTS AND MAIN RESULTS: Total iron levels in ELF were significantly increased in IPF patients compared to non-smoking controls (p < 0.05); there were no differences with healthy smokers (p = 0.2). Hemosiderin accumulation in alveolar macrophages was similar in never smoking and ever smoking IPF patients (p = 0.5), was significantly higher in IPF patients than in both smoking and non-smoking healthy controls (p < 0.05, all comparisons) and was positively correlated with echocardiographic estimates of pulmonary artery systolic pressure (p < 0.05) and with increasing disease severity scores (p < 0.05). CONCLUSIONS: The data show exaggerated accumulation of iron in IPF broncho-alveolar ELF and alveolar cells with no association with tobacco smoke, thus suggesting, occult pulmonary hemorrhage as a likely cause.
Assuntos
Hemorragia/diagnóstico , Fibrose Pulmonar Idiopática/fisiopatologia , Ferro/metabolismo , Macrófagos Alveolares/metabolismo , Adulto , Idoso , Estudos de Casos e Controles , Feminino , Hemossiderina/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Alvéolos Pulmonares/metabolismo , Estudos Retrospectivos , Índice de Gravidade de Doença , Fumar/metabolismo , EspectrofotometriaRESUMO
The HECT-containing E3 ubiquitin ligase Itch mediates the degradation of several proteins, including p63 and p73, involved in cell specification and fate. Itch contains four WW domains, which are essential for recognition on the target substrate, which contains a short proline-rich sequence. Several signaling complexes containing these domains have been associated with human diseases such as muscular dystrophy, Alzheimer's or Huntington's diseases. To gain further insight into the structural determinants of the Itch-WW2 domain, we investigated its interaction with p63. We assigned, by 3D heteronuclear NMR experiments, the backbone and side chains of the uniformly (13)C-(15)N-labeled Itch-WW2. In vitro interaction of Itch-WW2 domain with p63 was studied using its interactive p63 peptide, pep63. Pep63 is an 18-mer peptide corresponding to the region from 534-551 residue of p63, encompassing the PPxY motif that interacts with the Itch-WW domains, and we identified the residues involved in this molecular recognition. Moreover, here, a strategy of stabilization of the conformation of the PPxY peptide has been adopted, increasing the WW-ligand binding. We demonstrated that cyclization of pep63 leads to an increase of both the biological stability of the peptide and of the WW-ligand complex. Stable metal-binding complexes of the pep63 have been also obtained, and localized oxidative damage on Itch-WW2 domain has been induced, demonstrating the possibility of use of metal-pep63 complexes as models for the design of metal drugs to inhibit the Itch-WW-p63 recognition in vivo. Thus, our data suggest a novel strategy to study and inhibit the recognition mechanism of Itch E3-ligase.
Assuntos
Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Ciclização , Humanos , Espectroscopia de Ressonância Magnética , Metais/metabolismo , Dados de Sequência Molecular , Oxirredução , Estresse Oxidativo , Ligação Proteica , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas Repressoras/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Ubiquitina-Proteína Ligases/químicaRESUMO
Anticancer drug efficacy might be leveraged by strategies to target certain biochemical adaptations of tumors. Here we show how depriving cancer cells of glutamine can enhance the anticancer properties of 3-bromopyruvate, a halogenated analog of pyruvic acid. Glutamine deprival potentiated 3-bromopyruvate chemotherapy by increasing the stability of the monocarboxylate transporter-1, an effect that sensitized cells to metabolic oxidative stress and autophagic cell death. We further elucidated mechanisms through which resistance to chemopotentiation by glutamine deprival could be circumvented. Overall, our findings offer a preclinical proof-of-concept for how to employ 3-bromopyruvate or other monocarboxylic-based drugs to sensitize tumors to chemotherapy.
Assuntos
Antineoplásicos/farmacologia , Glutamina/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Piruvatos/farmacologia , Animais , Antineoplásicos/administração & dosagem , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Complexo II de Transporte de Elétrons/metabolismo , Glutamato-Amônia Ligase/metabolismo , Humanos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Estresse Oxidativo , Estabilidade Proteica/efeitos dos fármacos , Proteólise , Piruvatos/administração & dosagemRESUMO
Natural allyl sulfur compounds show antiproliferative effects on tumor cells, but the biochemical mechanisms underlying the antitumorigenic properties of the organ sulfur compounds are not yet fully understood. Sodium 2-propenyl-thiosulfate is a garlic water-soluble organo-sulfane sulfur compound able to promote apoptosis in cancer cells, affecting the 'managing' of the redox state in the cell. Our studies show that sodium 2-propenyl-thiosulfate reacts spontaneously with reduced glutathione at physiological pH, leading to the formation of S-allyl-mercapto-glutathione, radicals and peroxyl species, which are able to induce inhibition of enzymes with cysteine in the catalytic site, such as sulfurtransferases. S-Allyl-mercapto-glutathione was purified and characterized by NMR and MS, and its cytotoxic effect at 500 µm on HuT 78 cells was analyzed, showing activation of the p38-MAPK pathway. Many allyl sulfur compounds are also able to promote chemoprevention by induction of xenobiotic-metabolizing enzymes, inducing down-activation or detoxification of the carcinogens. Thus, the effects of the S-allyl-mercapto-glutathione on proteins involved in the cellular detoxification system, such as glutathione S-transferase, have been evaluated both in vitro and in HuT 78 cells. Although the antitumor properties of water-soluble sulfur compounds may arise from several mechanisms and it is likely that more cellular events occur simultaneously, a relevant role is played by the formation of both reduced glutathione conjugates and radical species that affect the activity of the thiol-proteins involved in fundamental cellular processes.
Assuntos
Compostos Alílicos/farmacologia , Apoptose/efeitos dos fármacos , Glutationa/química , Glutationa/metabolismo , Linfoma de Células T/patologia , Ésteres do Ácido Sulfúrico/farmacologia , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fluorescência , Humanos , Linfoma de Células T/metabolismo , Espectroscopia de Ressonância Magnética , Oxirredução , Células Tumorais CultivadasRESUMO
RTN1-C protein is a membrane protein localized in the ER and expressed in the nervous system, and its biological role is not completely clarified. Our previous studies have shown that the C-terminal region of RTN1-C, corresponding to the fragment from residues 186 to 208, was able to bind the nucleic acids and to interact with histone deacetylase (HDAC) enzymes. In the present work the properties of the synthetic RTN1-C(CT) peptide corresponding to this region were studied with relation to its ability to bind the metal ions in its N-terminal region. RTN1-C(CT) peptide is characterized by the presence of high-affinity copper and nickel ion sites. The nuclease activity of the metal-peptide complex was observed due to the presence of an ATCUN-binding motif. Moreover, the effect of the Cu/Ni-RTN1-C(CT) complexes on the HDAC activity was investigated. The histone deacetylase inhibitors are a new class of antineoplastic agents currently being evaluated in clinical trials. Our data show that the acetylated form of the metal-peptide complex is able to inhibit the HDAC activity at micromolar concentrations. These results allow to propose the Cu/Ni-RTN1-C(CT) complexes as models for the design of antitumor agents.
Assuntos
Histona Desacetilases/metabolismo , Proteínas do Tecido Nervoso/química , Acetilação , Sequência de Aminoácidos , Animais , Sítios de Ligação , Dicroísmo Circular , Sequência Consenso , DNA/metabolismo , Retículo Endoplasmático/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Humanos , Metais/metabolismo , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/farmacologia , Peptídeos/síntese química , Peptídeos/química , Plasmídeos , Ratos , Espectrometria de FluorescênciaRESUMO
DNA sequence recognition by the homodimeric C-terminal domain of the human papillomavirus type 16 E2 protein (E2C) is known to involve both direct readout and DNA-dependent indirect readout mechanisms, while protein-dependent indirect readout has been deduced but not directly observed. We have investigated coupling between specific DNA binding and the dynamics of the unusual E2C fold, using pH as an external variable. Nuclear magnetic resonance and isothermal titration calorimetry show that pH titration of His318 in the complex interface and His288 in the core of the domain is coupled to both binding and the dynamics of the beta-barrel core of E2C, with a tradeoff between dimer stability and function. Specific DNA binding is, in turn, coupled to the slow dynamics and amide hydrogen exchange in the entire beta-barrel, reaching residues far apart from the DNA recognition elements but not affecting the two helices of each monomer. The changes are largest in the dimerization interface, suggesting that the E2C beta-barrel acts as a hinge that regulates the relative position of the DNA recognition helices. In conclusion, the cooperative dynamics of the human papillomavirus type 16 E2C beta-barrel is coupled to sequence recognition in a protein-dependent indirect readout mechanism. The patterns of residue substitution in genital papillomaviruses support the importance of the protonation states of His288 and His318 and suggest that protein-dependent indirect readout and histidine pH titration may regulate DNA binding in the cell.
Assuntos
Proteínas de Ligação a DNA/química , DNA/metabolismo , Papillomavirus Humano 16/química , Proteínas Oncogênicas Virais/química , DNA/química , Proteínas de Ligação a DNA/metabolismo , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Movimento (Física) , Proteínas Oncogênicas Virais/metabolismo , Maleabilidade , Ligação Proteica , Conformação ProteicaRESUMO
The binding of lipids (free fatty acids as well as acidic phospholipids) to cytochrome c (cyt c) induces conformational changes and partial unfolding of the protein, strongly influencing cyt c oxidase/peroxidase activity. ATP is unique among the nucleotides in being able to turn non-native states of cyt c back to the native conformation. The peroxidase activity acquired by lipid-bound cyt c turns out to be very critical in the early stages of apoptosis. Nucleotide specificity is observed for apoptosome formation and caspase activation, the cleavage occurring only in the presence of dATP or ATP. In this study, we demonstrate the connection between peroxidase activity and oleic acid-induced conformational transitions of cyt c and show how ATP is capable of modulating such interplay. By NMR measurement, we have demonstrated that ATP interacts with a site (S1) formed by K88, R91, and E62 and such interaction was weakened by mutation of E62, suggesting the selective role in the interaction played by the base moiety. Interestingly, the interactions of ATP and GTP with cyt c are significantly different at low nucleotide concentrations, with GTP being less effective in perturbing the S1 site and in eliciting apoptotic activity. To gain insights into the structural features of cyt c required for its pro-apoptotic activity and to demonstrate a regulatory role for ATP (compared to the effect of GTP), we have performed experiments on cell lysates by using cyt c proteins mutated on amino acid residues that, as suggested by NMR measurements, belong to S1. Thus, we provide evidence that ATP acts as an allosteric effector, regulating structural transitions among different conformations and different oxidation states of cyt c, which are endowed with apoptotic activity or not. On this basis, we suggest a previously unrecognized role for ATP binding to cyt c at low millimolar concentrations in the cytosol, beyond the known regulatory role during the oxidative phosphorylation in mitochondria.
Assuntos
Trifosfato de Adenosina/fisiologia , Proteínas Reguladoras de Apoptose/química , Proteínas Reguladoras de Apoptose/metabolismo , Citocromos c/química , Citocromos c/metabolismo , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/genética , Animais , Proteínas Reguladoras de Apoptose/genética , Sítios de Ligação/genética , Citocromos c/genética , Cavalos , Humanos , Mutação , Ácido Oleico/metabolismo , Peroxidase/metabolismo , Ligação Proteica/genética , Conformação Proteica , Relação Estrutura-Atividade , Células U937RESUMO
Nucleic acid recognition is often mediated by alpha-helices or disordered regions that fold into alpha-helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical alpha-helix is stabilized at the N-terminus, while the PII forms at the C-terminus half of the peptide. Re-examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N-terminus half is a canonical alpha-helix, while the C-terminal half adopts a 3(10) helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 10(4)-fold lower affinity, and 500-fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained alpha-helix conformation, "presented" by this unique architecture. (c) 2009 Wiley Periodicals, Inc. Biopolymers 91: 432-443, 2009.
Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Sítios de Ligação , Dicroísmo Circular , DNA/química , Proteínas de Ligação a DNA/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/química , Conformação Proteica , Dobramento de ProteínaRESUMO
Sodium 2-propenyl thiosulfate, a water-soluble organo-sulfane sulfur compound isolated from garlic, induces apoptosis in a number of cancer cells. The molecular mechanism of action of sodium 2-propenyl thiosulfate has not been completely clarified. In this work we investigated, by in vivo and in vitro experiments, the effects of this compound on the expression and activity of rhodanese. Rhodanese is a protein belonging to a family of enzymes widely present in all phyla and reputed to play a number of distinct biological roles, such as cyanide detoxification, regeneration of iron-sulfur clusters and metabolism of sulfur sulfane compounds. The cytotoxic effects of sodium 2-propenyl thiosulfate on HuT 78 cells were evaluated by flow cytometry and DNA fragmentation and by monitoring the progressive formation of mobile lipids by NMR spectroscopy. Sodium 2-propenyl thiosulfate was also found to induce inhibition of the sulfurtransferase activity in tumor cells. Interestingly, in vitro experiments using fluorescence spectroscopy, kinetic studies and MS analysis showed that sodium 2-propenyl thiosulfate was able to bind the sulfur-free form of the rhodanese, inhibiting its thiosulfate:cyanide-sulfurtransferase activity by thiolation of the catalytic cysteine. The activity of the enzyme was restored by thioredoxin in a concentration-dependent and time-dependent manner. Our results suggest an important involvement of the essential thioredoxin-thioredoxin reductase system in cancer cell cytotoxicity by organo-sulfane sulfur compounds and highlight the correlation between apoptosis induced by these compounds and the damage to the mitochondrial enzymes involved in the repair of the Fe-S cluster and in the detoxification system.
Assuntos
Compostos Alílicos/farmacologia , Ésteres do Ácido Sulfúrico/farmacologia , Tiorredoxinas/metabolismo , Tiossulfato Sulfurtransferase/metabolismo , Apoptose/efeitos dos fármacos , Catálise , Ciclo Celular , Linhagem Celular , Proliferação de Células , Hidrólise , Lipídeos/biossíntese , Espectroscopia de Ressonância Magnética , Espectrometria de FluorescênciaRESUMO
Transmitted by the Aedes aegypti mosquito, the dengue virus is the etiological agent of dengue fever, dengue hemorrhagic fever and dengue shock syndrome, and, as such, is a significant factor in the high death rate found in most tropical and subtropical areas of the world. Dengue diseases are not only a health burden to developing countries, but pose an emerging problem worldwide. The immunopathological mechanisms appear to include a complex series of immune responses. A rapid increase in the levels of cytokines and chemical mediators during dengue disease plays a key role in inducing plasma leakage, shock and hemorrhagic manifestations. Currently, there are no vaccines available against dengue virus, although several tetravalent live-attenuated dengue vaccines are in clinical phases I or II, and prevention through vaccination has become a major priority on the agendas of the World Health Organization and of national ministries of health and military organizations. An alternative to vaccines is found in therapeutic-based approaches. Understanding the molecular mechanisms of viral replication has led to the development of potential drugs, and new molecular viral targets for therapy are emerging. The NS3 protease domain of the NS3 protein is responsible for processing the viral polyprotein and its inhibition is one of the principal aims of pharmacological therapy. This review is an overview of the progress made against dengue virus; in particular, it examines the unique properties--structural and functional--of the NS3 protease for the treatment of dengue virus infections by the inhibition of viral polyprotein processing.
Assuntos
Antivirais/uso terapêutico , Vírus da Dengue/enzimologia , Dengue/tratamento farmacológico , Serina Endopeptidases/metabolismo , Proteínas não Estruturais Virais/antagonistas & inibidores , Sequência de Aminoácidos , Antivirais/farmacologia , Dengue/epidemiologia , Dengue/imunologia , Vacinas contra Dengue , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/imunologia , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Dengue Grave/tratamento farmacológico , Dengue Grave/epidemiologia , Dengue Grave/imunologia , Especificidade por Substrato , Replicação Viral , Organização Mundial da SaúdeRESUMO
Calmodulin, regulatory, and essential myosin light chain are evolutionary conserved proteins that, by binding to IQ motifs of target proteins, regulate essential intracellular processes among which are efficiency of secretory vesicles release at synapsis, intracellular signaling, and regulation of cell division. The yeast Saccharomyces cerevisiae calmodulin Cmd1 and the essential myosin light chain Mlc1p share the ability to interact with the class V myosin Myo2p and Myo4 and the class II myosin Myo1p. These myosins are required for vesicle, organelle, and mRNA transport, spindle orientation, and cytokinesis. We have used the budding yeast model system to study how calmodulin and essential myosin light chain selectively regulate class V myosin function. NMR structural analysis of uncomplexed Mlc1p and interaction studies with the first three IQ motifs of Myo2p show that the structural similarities between Mlc1p and the other members of the EF-hand superfamily of calmodulin-like proteins are mainly restricted to the C-lobe of these proteins. The N-lobe of Mlc1p presents a significantly compact and stable structure that is maintained both in the free and complexed states. The Mlc1p N-lobe interacts with the IQ motif in a manner that is regulated both by the IQ motifs sequence as well as by light chain structural features. These characteristic allows a distinctive interaction of Mlc1p with the first IQ motif of Myo2p when compared with calmodulin. This finding gives us a novel view of how calmodulin and essential light chain, through a differential binding to IQ1 of class V myosin motor, regulate this activity during vegetative growth and cytokinesis.