Caracterización matemática de la unión de la proteína PfNBP-1 al eritrocito mediante la aplicación de la probabilidad y entropía / Mathematical characterization of binding between PfNBP-1 protein and erythrocyite by applying probability and entropy

Objetivo: Estudiar la proteína Plasmodium falciparum Normocyte Binding Protein-1 (PfNBP-1), partiendo de un método de caracterización físico-matemática desarrollado previamente para péptidos de alta unión del merozoito de malaria al eritrocito. Materiales y métodos: Se tomaron 21 péptidos con tamaño de 20 aminoácidos no sobrelapados de los cuales dos son de alta unión, se cuantificó la frecuencia de aparición de los 20 aminoácidos esenciales en cada posición y se calculó la probabilidad, sumatoria de probabilidad y la entropía con el objetivo de diferenciar matemáticamente los péptidos de alta y baja unión. Posteriormente se calcularon los mismos valores para péptidos teóricos análogos, en los que fueron cambiados por glicinas los aminoácidos críticos confirmados experimentalmente. Resultados: Los péptidos de PfNBP-1 comprobados experimentalmente de alta unión, presentaron valores de probabilidad, sumatoria de probabilidad y entropía ubicados dentro del macroestado de unión y sus péptidos teóricos análogos presentaron resultados que se diferenciaban cada vez más del macroestado de unión a medida que se reemplazaban aminoácidos críticos por glicinas. En cuanto a las secuencias de no unión de PfNBP-1, se encontró que los valores calculados son diferentes a los asociados al macroestado de unión, comprobando que en el 100 % de casos estudiados es posible diferenciar los péptidos de no unión y alta unión matemáticamente. Conclusiones: La probabilidad y la entropía permiten caracterizar adecuadamente los péptidos de alta unión de PfNBP-1, y evidenciar el orden matemático subyacente al proceso de unión de proteínas de malaria.

Objective: To study the Plasmodium falciparum Normocyte Binding Protein-1 (PfNBP-1) based on a of physicalmathematical characterization method previously developed for high binding peptides of malaria merozoite to erythrocyte. Materials and methods: 21 non overlapped peptides with size of 20 amino acids, including two of high binding were taken; the frequency of occurrence of the 20 essential amino acids in each position was quantified and probability, summation of probability and entropy were calculated in order to mathematically differentiate high and low binding peptides. Later the same values were calculated for theoretical analogs peptides, where the critical amino acids confirmed experimentally were changed by glycine. Results: The experimentally validated high binding peptides of PfNBP-1 showed values of probability, summation of probability and entropy located within the binding macrostate peptides and their theoretical analogues peptides presented results that differed increasingly of the binding macrostate as critical amino acids were replaced by glycine. For the PfNBP-1 sequences of non-binding, it was found that the calculated values are different from those associated with the macrostate of binding and it was verified that in 100% of studied cases it is possible to mathematically differentiate binding and non-binding peptides. Conclusions: The probability and entropy allow to adequately characterize the high-binding peptides of PfNBP-1 and show the mathematical order underlying the process of protein binding of malaria to the erythrocyte.

Interacciones moleculares de un flavonoide prenilado con transportadores de antimicóticos dependientes de ATP / Molecular interactions of a prenylated flavonoid with ATP-depending antimycotic transporters

Uno de los principales mecanismos para la resistencia de Candida albicans a azoles es la sobreexpresión de transportadores que extruyen los fármacos antimicóticos y disminuyen su concentración intracelular. Anteriormente nuestros laboratorios habían informado que el flavonoide prenilado 2', 4'-dihidroxi - 5'-(1'''-dimetilalil)-6-prenil pinocembrina (6PP) inhibe en forma competitiva los transportadores cdr, dependientes del ATP, además de ser antimicrobiano per se. En este trabajo realizamos estudios bioinformáticos para analizar los posibles sitios de interacción molecular estereoespecífica del 6PP, fluconazol y adenosín trifosfato (ATP) con los transportadores cdr1 y cdr2. Se confeccionaron los modelos tridimensionales de los ligandos y de los transportadores a través de estudios de homología de datos respecto de otras macromoléculas. Se calculó la variación de energía libre de Gibbs ( G) asociada a 45 modelos de interacción molecular y se estudiaron cdr1 y cdr2 en formacomparativa, dada su similitud estructural. Se seleccionaron los modelos más probables desde el punto de vista termodinámico. El 6PP y el fluconazol podrían competir de diversas maneras por un sitio estereoespecífico que atraviesa la membrana de la levadura. Este sitio, numerado como 1, fue estudiado exhaustivamente, es común a los transportadores cdr1 y cdr2 y aporta selectivamente distintosaminoácidos. Entre estos últimos, los de mayor participación en los modelos de interacción fueron, en cdr1 y cdr2, respectivamente: PHE 754,756 y TYR 747,749 . Se seleccionaron los 9 mejores complejos formados por cada ligando, de acuerdo a su energía de afinidad decreciente (en KJ/mol en mejor modelo de cdr2: 6PP = -9,3; fluconazol = -6,8). Además el flavonoide prenilado competiría con el ATP en el dominio citosólico y con menor probabilidad lo imitaría el fluconazol ( G en KJ/mol en mejor modelo de cdr2: 6P P= -8,2, ATP = -7,6; fluconazol = -6,7)...

Mecanismos celulares y moleculares de la neurotoxicidad por plomo

resumen está disponible en el texto completo

Abstract: Lead, a heavy metal, has been used by humans for many technological aims, a fact that has determined its actual widespread distribution. Although various actions have been taken to diminish the use and distribution of lead in the environment, it remains a significant health problem. The evolution of technological processes applied in the industry has followed economic interest. Its only in recent times that criteria related to health and ecology have been considered while designing new industries. Particularly susceptible groups are children and workers involved in mining, metallurgy, paint manufacturing and battery recycling. The communities living in areas where those industries are settled have also a higher lead exposure risk. Its high biological toxicity has determined lead to become one of the most significant environmental contaminants with pathogenic potential for humans. The toxic mechanism of lead is essentially due to its capability to substitute other polyvalent cations (particularly divalent cations such as calcium and zinc) into the molecular machinery of living organisms. Thanks to its ionic structure, lead establishes very favorable interactions, usually with higher affinity, with chemical groups that normally coordinate divalent cations in proteins. The coordination of cations in proteins is usually achieved by negatively charged acidic residues. These residues establish ionic interactions with the positively charged ion, resulting in a change in the structure and electric charge of the protein. These interactions determine that lead may affect different biologically significant processes, including metal transporting proteins, ionic channels, cell adhesion molecules, diverse enzymes which have metallic cofactors, signaling molecules such as calmodulin and protein kinase C and DNA binding proteins, among other molecular targets. Lead interactions with the coordinating amino acid residues in proteins may induce an abnormal conformational configuration of proteins, as compared to the conformational structure acquired when interacting with commonly active cations, thus significantly altering its functional properties in the very complex molecular machinery. Among the biologically active sites usually occupied by lead, those related to calcium seem to have the most significant pathological importance for lead toxicity due to their widespread distribution and highly significant functional relevance for the normal cell function. Two of the principal calcium binding motifs in proteins, the EF-hand motif and the C2 motif, have an intrinsic high affinity for lead. In the case of EF-hand motifs, calmodulin is one of the most remarkable targets for lead due its importance in regulating cellular processes, being activated by lead at lower concentrations than required for calcium and displaying an abnormal activity. The C2 motif is expressed mainly in calcium dependent membrane associated proteins such as protein kinase C (PKC) or synaptotagmin. The principal characteristic in these motifs is an electrical change in the protein after the calcium binding, allowing its interaction with biological membranes. In synaptotagmin, according with the electrical characteristics of lead, the interaction of the complex lead-synaptotagmin with biological membranes is similar to the interaction calcium-synaptotagmin with membranes, which is eminently electrical. Hence, the conformation of this complex is probably different to the conformation with calcium, fact evidenced by the failure of lead-synaptotagmin to interact with other proteins of the exocitic machinery. In relation to lead neurotoxicity, membrane ionic channels seem to be among the most relevant molecular targets of lead. In particular, calcium and potassium channel function may be significantly impaired by lead, affecting the activation of calcium activated potassium channels, the inactivation process of calcium channels, and the ionic conductance of calcium channels. As occurs with other heavy metals, lead is capable of blocking the calcium channel, probably at the selectivity filter. The high affinity lead binding to the acidic residues of the filter provokes a slow flux of the metal trough the channel pore, blocking the calcium conductance. The regulation of ionic channels will be significantly altered also. Calmodulin is a common calcium sensing protein for many ionic channels and its alteration by lead could affect the channel operation. Abnormal functioning of regulatory and signaling proteins such as calmodulin, protein kinase C and synaptotagmins, which normally require calcium for its activity, may also display an abnormal functioning, thus determining a widespread metabolic influence of lead poisoning. Lead distributes evenly into the cell thus reaching intracellular organelles, including the endoplasmic reticulum, mitochondria and the cell nucleus. This results in significant alterations of intracellular calcium metabolism and regulation due in part to the malfunctioning of calcium channels and ionic pumps in plasma membrane, endoplasmic reticulum and mitochondria. Inadequate energy generation due to mitochondrial damage and malfunctioning in cation dependent enzymes, alterations in protein folding due to the direct binding of lead to calcium activated reticular chaperones, or indirectly, altering the intrareticular calcium levels, and the disruption of the structure of DNA binding motifs such as zinc fingers, among others, promotes alterations in gene expression and DNA reparation. Lead poisoning is one of the most important chronic environmental illnesses affecting children in modern life. Developing central nervous system is particularly susceptible to lead toxicity. At critical times in development, lead may have a disorganizing influence with long-lasting effects which may continue into teenage and beyond. Mechanisms originating this disorganizing influence in the central nervous system are a consequence of the interaction of lead with various targets as previously described; alterations of cell molecular machinery, at the systems level induce excitotoxic phenomena, interferes with neurotransmission at neurotransmitter synthesis, release and receptor activation levels, alters intracellular signaling and produce cell membrane peroxidative damage. Compared to adult lead poisoning, pediatric lead is most common and its effects may occur at reduced blood levels with subclinical symptoms; thus a high index of suspicion is necessary for physicians when dealing with pediatric patients. Long-term effects of lead may produce cognitive and motor impairment, with behavioral alterations. The particular vulnerability of the immature nervous system to the lead poisoning is probably due to the fact that in this stage of development the establishment of appropriate neural networks is highly dependent on the synaptic activity, which in turn could be altered by lead. Lead poisoning has been considered as a potential co-factor in complex neuropsychological alterations such as schizophrenia. In this sense it is worth to note the possibility that the physical and psychic symptoms of Vincent Van Gogh may have been due to chronic lead poisoning. The following are among the clinical symptoms described by Van Gogh in his autographed letters: initial debilitation, stomatitis with loss of teeth, recurring abdominal pains, anemia (with a "plumbic" skin tone), neuropathy of the radial and saturnine encephalopathy, including epileptic crises, progressive character changes and periods of delirium, all of which meet present criteria for diagnosis of Organic Mental Disorder due to cerebral lesion or somatic illness, and Organic Character Disorder (DSM-IV-R). Apha-thujone, found in absinthe and in many popular herbal medicines, may also have contributed to Van Gogh symptoms since he was a well-known absynthe drinker. Many countries, including Mexico, have implemented politics aimed to eliminate lead from the majority of their industrial processes. This has been carried out with considerable effort, and in some cases, with open confrontations between the scientific community and industrial sector. Although there have been actually significant advances to eliminate lead from many products (gasoline, painting manufacturing, etc.), lead is not degradable, thence once it is released in the environment it remains there for long periods of time. This implies that we should have to deal with lead poisoning in the years to come and to be aware of this diagnostic possibility in any suspicious case. This review is centered in the description of the molecular mechanisms of lead toxicity and its repercussion in the cellular excitability and central nervous system function.