RESUMO
The interaction of the porphyrin derivative H2TCPPSpm4, having spermine pendants in the four meso positions, with the G-quadruplex (GQ) structure formed by the DNA aptamer TGGGAG has been investigated by means of UV, electronic circular dichroism and PAGE studies. The results reported here demonstrate that the porphyrin derivative is capable of stabilizing or destabilizing the higher-ordered structures of parallel GQs, depending on the method used to reach their relative stoichiometry (titration vs. single addition). Noteworthily, when two equivalents of H2TCPPSpm4 were mixed directly with one equivalent of the (TGGGAG)4 GQ to reach a 2 : 1 H2TCPPSpm4 : GQ ratio T1/2 higher than 80 °C was also observed confirming the presence of higher-ordered GQ structures.
Assuntos
Quadruplex G , Porfirinas/química , Espermina/química , Aptâmeros de Nucleotídeos/química , Sequência de Bases , Dicroísmo Circular , Eletroforese em Gel de Campo Pulsado , Oligonucleotídeos/química , Transição de Fase , Espectrofotometria UltravioletaRESUMO
Ubiquitin Proteasome System (UPS) is an adaptable and finely tuned system that sustains proteostasis network under a large variety of physiopathological conditions. Its dysregulation is often associated with the onset and progression of human diseases; hence, UPS modulation has emerged as a promising new avenue for the development of treatments of several relevant pathologies, such as cancer and neurodegeneration. The clinical interest in proteasome inhibition has considerably increased after the FDA approval in 2003 of bortezomib for relapsed/refractory multiple myeloma, which is now used in the front-line setting. Thereafter, two other proteasome inhibitors (carfilzomib and ixazomib), designed to overcome resistance to bortezomib, have been approved for treatment-experienced patients, and a variety of novel inhibitors are currently under preclinical and clinical investigation not only for haematological malignancies but also for solid tumours. However, since UPS collapse leads to toxic misfolded proteins accumulation, proteasome is attracting even more interest as a target for the care of neurodegenerative diseases, which are sustained by UPS impairment. Thus, conceptually, proteasome activation represents an innovative and largely unexplored target for drug development. According to a multidisciplinary approach, spanning from chemistry, biochemistry, molecular biology to pharmacology, this review will summarize the most recent available literature regarding different aspects of proteasome biology, focusing on structure, function and regulation of proteasome in physiological and pathological processes, mostly cancer and neurodegenerative diseases, connecting biochemical features and clinical studies of proteasome targeting drugs.
Assuntos
Neoplasias/fisiopatologia , Doenças Neurodegenerativas/fisiopatologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Ubiquitina/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Resistência a Medicamentos/fisiologia , Fator de Transcrição E2F4/metabolismo , Holoenzimas , Humanos , Gotículas Lipídicas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Musculares/metabolismo , NF-kappa B/metabolismo , Neoplasias/tratamento farmacológico , Doenças Neurodegenerativas/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Inibidores de Proteassoma/uso terapêutico , Proteostase/fisiologia , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mature microRNAs are short non-coding RNA sequences which upon incorporation into the RISC ribonucleoprotein complex, play a crucial role in regulation of gene expression. However, miRNAs can exist within the cell also as free molecules fulfilling their biological activity. Therefore, it is emerging that in addition to sequence even the structure adopted by mature miRNAs might play an important role to reach the target. Indeed, we analysed by several spectroscopic techniques the secondary structures of two artificial miRNAs selected by computational tool (miR-Synth) as best candidates to silence c-MET and EGFR genes and of two endogenous miRNAs (miR-15a and miR-15b) having the same seed region, but different biological activity. Our results demonstrate that both endogenous and artificial miRNAs can arrange in several 3D-structures which affect their activity and selectivity toward the targets.
Assuntos
MicroRNAs/química , MicroRNAs/genética , Sequência de Bases , Receptores ErbB/deficiência , Receptores ErbB/genética , Inativação Gênica , Conformação de Ácido Nucleico , Proteínas Proto-Oncogênicas c-met/deficiência , Proteínas Proto-Oncogênicas c-met/genética , Análise de Sequência de RNARESUMO
Graphene oxide (GO) is one of the most appealing bidimensional materials able to interact non-covalently with achiral molecules and to act as chiral inducers. Vortexes can tune chirality and, consequently transfer a specific handedness to non-covalent host molecules, either when dispersed in water or when deposited on a solid surface.
RESUMO
Thermodynamic parameters pertinent to the equilibria of ternary system copper(II), adenosine 5'-monophosphate (AMP), and 4,9-diazadodecane-1,12-diamine (Spermine, Spe) were determined by means of potentiometric and calorimetric techniques, together with the corresponding parent binary complex parameters. This system could be considered as a model in the study of more complicated biological systems. In particular, it could give useful evidence in the investigation of the interaction between tetramine and DNA. The experimental results seem to suggest that the major contribution in DNA stabilization by means of tetramines is due to the electrostatic interactions occurring between the protonated nitrogen atoms of the amine molecules and the phosphate groups of nucleotides in the nucleic chains.
Assuntos
Monofosfato de Adenosina , Quelantes/análise , Cobre , Espermina , Calorimetria , Fenômenos Químicos , Química , Potenciometria , Prótons , TermodinâmicaRESUMO
Thermodynamic characteristics pertinent to the formation equilibria of two ternary systems: 1) Copper(II), 4,9-diazadodecane-1,12-diamine (spermine, Spe), and adenosine 5'-triphosphate (ATP) and 2) Copper(II), Spe, and tripolyphosphate (TPP) have been determined by means of potentiometric and calorimetric techniques, together with the parent binary complex characteristics. Ternary complexes involving ATP can give information useful in the interpretation of bioenergetic reactions and of biological interactions between nucleic acids and polyamines. As a model system, the TPP-containing ternary complexes have been studied, together with the parent binary complexes. The thermodynamic study of these systems is very important because it can give information about the structural environment of the complexes; moreover, it can help in outlining different noncovalent interactions such as coulombic forces and hydrogen bonds.
Assuntos
Trifosfato de Adenosina/metabolismo , Cobre/metabolismo , Polifosfatos/metabolismo , Espermina/metabolismo , Calorimetria , Ligantes , Potenciometria , TermodinâmicaRESUMO
The conformation changes in solution of three fungal laccases in different environmental conditions were examined by circular dichroism (CD) and electron paramagnetic resonance (EPR) spectroscopy. CD measurements indicate that the secondary structure of proteins depends slightly on the pH or ionic strength, though the presence of salt could interfere in the molecular recognition process between substrates and enzymes. The enzymes, however, are highly destabilized by prolonged exposure to low pH or high temperature. The observed unfolding of the proteins coincides with their inactivation and, in some cases, with precipitation. On the other hand, these conditions do not determine the disruption of the geometric arrangement of their metal centres, and this fact suggests that these centres represent the more stable core of the proteins.
Assuntos
Oxirredutases/química , Oxirredutases/metabolismo , Pleurotus/enzimologia , Polyporales/enzimologia , Catálise , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Lacase , Concentração Osmolar , Conformação Proteica , TemperaturaRESUMO
The interaction between monomeric insulin and monosaccharides has been investigated through circular dichroism, fluorescence spectroscopy and two dimensional nuclear magnetic resonance. CD spectra indicate that D-glucose interacts with monomeric insulin whereas D-galactose, D-mannose and 2-deoxy-D-glucose have a lower effect. Fluorescence emission was quenched at sugar concentrations of 5-10 mM. Titration with the different sugars produces a quenching of the tyrosine spectrum from which a binding free energy value for the insulin-sugar complexes has been evaluated. Transfer nuclear Overhauser enhancement NMR experiments indicate the existence of dipolar interactions at short interatomic distances between C-1 proton of D-glucose in the beta form and the monomeric insulin. Further, NMR total correlation spectra experiments revealed that the hormone is in the monomeric form and that upon addition of glucose no aggregation occurs. The interaction does not involve relevant changes in the secondary structure of insulin suggesting that the interaction occur at the side chain level. Molecular dynamics simulations and modeling studies, based on the dynamic fluctuations of potential binding moiety sidechains, argued from results of NMR spectroscopy, provide additional informations to locate the putative binding sites of D-glucose to insulin.
Assuntos
Glucose/química , Insulina/química , Sítios de Ligação , Dicroísmo Circular , Glucose/metabolismo , Humanos , Insulina/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Ligação Proteica , Prótons , Software , Espectrometria de Fluorescência/métodosRESUMO
Due to their ballistic precision, apoptosis induction by protons could be a strategy to specifically eliminate neoplastic cells. To characterize the cellular and molecular effects of these hadrons, we performed dose-response and time-course experiments by exposing different cell lines (PC3, Ca301D, MCF7) to increasing doses of protons and examining them with FACS, RT-PCR, and electron spin resonance (ESR). Irradiation with a dose of 10 Gy of a 26,7 Mev proton beam altered cell structures such as membranes, caused DNA double strand breaks, and significantly increased intracellular levels of hydroxyl ions, are active oxygen species (ROS). This modified the transcriptome of irradiated cells, activated the mitochondrial (intrinsic) pathway of apoptosis, and resulted in cycle arrest at the G2/M boundary. The number of necrotic cells within the irradiated cell population did not significantly increase with respect to the controls. The effects of irradiation with 20 Gy were qualitatively as well as quantitatively similar, but exposure to 40 Gy caused massive necrosis. Similar experiments with photons demonstrated that they induce apoptosis in a significantly lower number of cells and in a temporally delayed manner. These data advance our knowledge on the cellular and molecular effects of proton irradiation and could be useful for improving current hadrontherapy protocols.
Assuntos
Apoptose/efeitos da radiação , Neoplasias/radioterapia , Terapia com Prótons , Apoptose/genética , Sequência de Bases , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Dano ao DNA , Primers do DNA/genética , Espectroscopia de Ressonância de Spin Eletrônica , Feminino , Citometria de Fluxo , Humanos , Masculino , Necrose , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Fótons/uso terapêutico , RNA Mensageiro/genética , RNA Neoplásico/genética , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CD and uv-visible absorption studies with several tetracationic water-soluble porphyrin derivatives show that some of these species can serve as probes to discriminate between A- and B-conformational forms of single-stranded polynucleotides. It is also observed that these porphyrins can participate in the formation of double helices by forming transient intermediate complexes en-route to duplex formation.
Assuntos
Polirribonucleotídeos/química , Porfirinas/química , Água/química , Fenômenos Químicos , Físico-Química , DNA/química , Conformação de Ácido Nucleico , RNA de Cadeia Dupla/química , SolubilidadeRESUMO
Since copper [Cu(II)] is a necessary cofactor for both intra-mitochondrial enzymes involved in energy production and hydroxyl scavenger enzymes, two hypothesised mechanisms for action of interleukin-I beta (IL-1 beta), we studied whether Cu(II) addition could prevent the inhibitory effect of IL-1 beta on insulin release and glucose oxidation in rat pancreatic islets. Islets were incubated with or without 50 U/ml IL-1 beta, in the presence or absence of various concentrations of Cu(II)-GHL (Cu(II) complexed with glycyl-L-histidyl-L-lysine, a tripeptide known to enhance copper uptake into cultured cells). CuSO4 (1-1000 ng/ml) was used as a control for Cu(II) effect when present as an inorganic salt. At the end of the incubation period, insulin secretion was evaluated in the presence of either 2.8 mmol/l (basal insulin secretion) or 16.7 mmol/l glucose (glucose-induced release). In control islets basal insulin secretion was 92.0 +/- 11.4 pg.islet-1 h-1 (mean +/- SEM, n = 7) and glucose-induced release was 2824.0 +/- 249.0 pg.islet-1 h-1. In islets pre-exposed to 50 U/ml IL-1 beta, basal insulin release was not significantly affected but glucose-induced insulin release was greatly reduced (841.2 +/- 76.9, n = 7, p < 0.005). In islets incubated with IL-1 beta and Cu-GHL (0.4 mumol/l, maximal effect) basal secretion was 119.0 +/- 13.1 pg.islet-1 h-1 and glucose-induced release was 2797.2 +/- 242.2, (n = 7, p < 0.01 in respect to islets exposed to IL-1 beta alone).(ABSTRACT TRUNCATED AT 250 WORDS)