RESUMEN
BACKGROUND: Mutations within the DNA binding domain (DBD) of the tumor suppressor p53 are found in >50% of human cancers and may significantly modify p53 secondary structure impairing its function. p28, an amphipathic cell-penetrating peptide, binds to the DBD through hydrophobic interaction and induces a posttranslational increase in wildtype and mutant p53 restoring functionality. We use mutation analyses to explore which elements of secondary structure may be critical to p28 binding. METHODS: Molecular modeling, Raman spectroscopy, Atomic Force Spectroscopy (AFS) and Surface Plasmon Resonance (SPR) were used to identify which secondary structure of site-directed and naturally occurring mutant DBDs are potentially altered by discrete changes in hydrophobicity and the molecular interaction with p28. RESULTS: We show that specific point mutations that alter hydrophobicity within non-mutable and mutable regions of the p53 DBD alter specific secondary structures. The affinity of p28 was positively correlated with the ß-sheet content of a mutant DBD, and reduced by an increase in unstructured or random coil that resulted from a loss in hydrophobicity and redistribution of surface charge. CONCLUSIONS: These results help refine our knowledge of how mutations within p53-DBD alter secondary structure and provide insight on how potential structural alterations in p28 or similar molecules improve their ability to restore p53 function. GENERAL SIGNIFICANCE: Raman spectroscopy, AFS, SPR and computational modeling are useful approaches to characterize how mutations within the p53DBD potentially affect secondary structure and identify those structural elements prone to influence the binding affinity of agents designed to increase the functionality of p53.
Asunto(s)
Péptidos de Penetración Celular/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía de Fuerza Atómica/métodos , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Unión Proteica/genética , Estructura Secundaria de Proteína , Resonancia por Plasmón de Superficie/métodos , Proteína p53 Supresora de Tumor/genéticaRESUMEN
ß2-Microglobulin (B2M) is a human protein involved in the regulation of immune response and represents a useful biomarker for several diseases. Recently, anti-B2M monoclonal antibodies have been introduced as innovative therapeutic agents. A deeper understanding of the molecular interaction between the two partners could be of utmost relevance for both designing array-based analytical devices and improving current immunotherapies. A visualization at the nanoscale performed by Atomic Force Microscopy revealed that binding of B2M to the antibody occurred according to two preferred interaction geometries. Additionally, Atomic Force Spectroscopy and Surface Plasmon Resonance provided us with detailed information on the binding kinetics and the energy landscape of the complex, both at the single molecule level and in bulk conditions. Combination of these complementary techniques contributed to highlight subtle differences in the kinetics behaviour characterizing the complexes. Collectively, the results may deserve significant interest for designing, development and optimization of novel generations of nanobiosensor platforms.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Reacciones Antígeno-Anticuerpo , Microscopía de Fuerza Atómica , Resonancia por Plasmón de Superficie , Microglobulina beta-2/química , Microglobulina beta-2/inmunología , Sitios de Unión de Anticuerpos/inmunología , Humanos , CinéticaRESUMEN
BACKGROUND: TP53 tumor suppressor gene is mutated in more than 50% of human tumors. Mutated p53 proteins could sequestrate and inactivate p73 reducing the apoptotic and anti-proliferative effects of the transcription factor, and yielding cancer cells more aggressive and chemoresistant. The possibility of using drugs to prevent the mutant p53/p73 complex formation preserving the p73 function, calls for a deeper insight into the molecular and biochemical mechanisms of mutant p53/p73 protein interaction. METHODS: The kinetics of the mutant p53R175H/p73 complex was investigated with innovative and complementary techniques, operating in real time, in near physiological conditions and without any labeling. Specifically, Atomic Force Spectroscopy and Surface Plasmon Resonance working at single-molecule level and in bulk condition, respectively, were used. RESULTS: The two techniques revealed that a stable complex is formed between mutant p53R175H and p73 proteins; the complex being characterized by a high interaction force and a dissociation equilibrium constant in the order of 10(-7)M, as expected for specific interactions. No binding was instead observed between p73 and wild type p53. CONCLUSIONS: Mutant p53R175H protein, unlike wild type p53, can form a stable complex with p73. The mutant p53R175H/p73 protein complex could be a target for innovative pharmaceutical drugs that, by dissociating it or preventing biomolecule interaction thus preserving the p73 function, could enhance the response of cancerous cells carrying mutant p53R175H protein to common chemotherapeutic agents. GENERAL SIGNIFICANCE: The kinetic information obtained in vitro may help to design specific pharmaceutical drugs directed against cancerous cells carrying mutant p53 proteins.
Asunto(s)
Proteínas de Unión al ADN/química , Microscopía de Fuerza Atómica/métodos , Proteínas Nucleares/química , Resonancia por Plasmón de Superficie/métodos , Proteína p53 Supresora de Tumor/química , Proteínas Supresoras de Tumor/química , Proteínas de Unión al ADN/ultraestructura , Humanos , Mutación , Proteínas Nucleares/ultraestructura , Proteína Tumoral p73 , Proteína p53 Supresora de Tumor/ultraestructura , Proteínas Supresoras de Tumor/ultraestructuraRESUMEN
The interaction between azurin (Az) and cytochrome c 551 (CytC551) from Pseudomonas aeruginosa deserves particular interest for both its physiological aspects and their possible applications in bionano devices. Here, the kinetics of the interaction has been studied by surface plasmon resonance and fluorescence quenching. Surface plasmon resonance data have been successfully interpreted by the heterogeneous ligand model, which predicts the existence of two binding sites on the immobilized Az for CytC551 molecules in solution. On the other hand, the fluorescence study indicates the formation of a complex, with the involvement of the lone Az tryptophan (Trp) at position 48. The two different techniques point out the occurrence of an encounter complex between Az and CytC551 that evolves toward the formation of a more stable complex characterized by an equilibrium dissociation constant KD typical of transient interactions.
Asunto(s)
Azurina/química , Proteínas Bacterianas/química , Grupo Citocromo c/química , Modelos Moleculares , Pseudomonas aeruginosa/química , Azurina/genética , Proteínas Bacterianas/genética , Sitios de Unión , Grupo Citocromo c/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Cinética , Unión Proteica , Pseudomonas aeruginosa/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Espectrometría de Fluorescencia , Resonancia por Plasmón de Superficie , TermodinámicaRESUMEN
We have used femtosecond pump-probe spectroscopy to investigate the excited-state dynamics of the anticancer blue copper protein rusticyanin, by exciting its ligand to metal charge-transfer band with 25 fs pump pulses centered at 585 nm. The charge-transfer excited state decays exponentially to the ground state with a time constant of about 230 fs, and its recovery is modulated by coherent oscillations. The Fourier transform of the oscillatory component of the signal provides most of the vibrational modes obtained by means of conventional resonance Raman studies, in addition to the low frequency modes below 80 cm(-1) believed to reflect collective motions of biological relevance.
Asunto(s)
Azurina/química , Cobre/química , Antineoplásicos/química , Azurina/metabolismo , Ceruloplasmina/química , Color , Electrones , Humanos , Simulación de Dinámica Molecular , Plastocianina/química , Estructura Terciaria de Proteína , Spinacia oleracea/metabolismoRESUMEN
p28 is a 28-amino acid peptide fragment of the cupredoxin azurin derived from Pseudomonas aeruginosa that preferentially penetrates cancerous cells and arrests their proliferation in vitro and in vivo. Its antitumor activity reportedly arises from post-translational stabilization of the tumor suppressor p53 normally downregulated by the binding of several ubiquitin ligases. This would require p28 to specifically bind to p53 to inhibit specific ligases from initiating proteosome-mediated degradation. In this study, atomic force spectroscopy, a nanotechnological approach, was used to investigate the interaction of p28 with full-length p53 and its isolated domains at the single molecule level. Analysis of the unbinding forces and the dissociation rate constant suggest that p28 forms a stable complex with the DNA-binding domain of p53, inhibiting the binding of ubiquitin ligases other than Mdm2 to reduce proteasomal degradation of p53.
Asunto(s)
Antineoplásicos/metabolismo , Azurina/metabolismo , Fragmentos de Péptidos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antineoplásicos/química , Azurina/química , Humanos , Proteínas Inmovilizadas/química , Proteínas Inmovilizadas/metabolismo , Microscopía de Fuerza Atómica , Fragmentos de Péptidos/química , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Pseudomonas aeruginosa/química , Análisis Espectral , Proteína p53 Supresora de Tumor/químicaRESUMEN
Recent experimental data reveal that the peptide fragment of Azurin called p28, constituted by the amino acid residues from 50 to 77 of the whole protein, retains both the Azurin cellular penetration ability and antiproliferative activity. p28 is hypothesized to act by stabilizing the well-known tumour suppressor p53 via a pathway independent from the oncogene Mdm2, which is the main p53 down-regulator, with its anticancer potentiality being probably connected with the binding of its amino acid residues 11 to 18 to p53. However, the p28 mode of action has not been completely elucidated yet, mostly because the details of the p28 interaction with p53 are still unknown. In the present study, computational docking modelling supported by cluster analysis, molecular dynamics simulations and binding free energy calculations have been performed to model the interaction between the DNA-binding domain (DBD) of p53 and the p28 fragment. Since the folding state of p28 when interacting with p53 inside the cell is not known, both the folded and the unfolded structures of this peptide have been taken into consideration. In both the cases, we have found that p28 is able to form with DBD a complex characterized by favourable negative binding free energy, high shape complementarity, and the presence of several hydrogen bonds at the interface. These results suggest that p28 might exert its anticancer action by hampering the binding of ubiquitin ligases to DBD, susceptible to promoting the p53 proteasomal degradation.
Asunto(s)
Antineoplásicos/química , Azurina/química , Fragmentos de Péptidos/química , Proteína p53 Supresora de Tumor/química , Modelos Químicos , Simulación de Dinámica Molecular , Estructura Terciaria de ProteínaRESUMEN
The Lamprologini are the most species-rich and diverse tribe of Lake Tanganyika cichlids, comprising around 90 described species. We reconstruct the most complete ( approximately 70 species) mtDNA phylogeny to date for this tribe, based on NADH dehydrogenase subunit 2 (ND2 approximately 1047 bp) and the non-coding control region ( approximately 874 bp) and examine the degree to which mtDNA trees are good proxies for species trees. Phylogenetic relationships are assessed using Bayesian inference, maximum likelihood and maximum parsimony to determine the robustness of relationships. The resulting topologies are largely congruent and only the tree produced by an unpartitioned BI analysis is rejected using the non-parametric likelihood-based AU test. The trees are remarkably balanced, with two major clades consistently recovered in all analyses and with reasonable support. A smaller clade of deep-water species is also recovered. Overall support is good, when compared to some groups that have undergone adaptive radiation and rapid lineage formation. The much-expanded phylogeny of the group helps resolve the placement of some previously problematic taxa, such as Neolamprologus moori, highlighting the importance of greater taxonomic sampling. The results include a number of divergent placements of closely related species, and the following genera Neolamprologus, Lamprologus, Julidiochromis, Telmatochromis are not monophyletic, with alternative hypotheses consistent with traditional taxonomy providing a significantly worse fit to the data. We find several examples of divergent mtDNA taxa sequences of presumed closely related species. This could be due to incorrect taxonomy or to the failure of the mtDNA to reflect species relationships and may support the hypothesis that speciation within this group has been facilitated by introgressive hybridisation.
Asunto(s)
Cíclidos/clasificación , Cíclidos/genética , ADN Mitocondrial/análisis , Agua Dulce , Filogenia , Animales , Huesos/anatomía & histología , Burundi , Cartílago/anatomía & histología , Cíclidos/anatomía & histología , Congo , Evolución Molecular , Modelos Biológicos , Tanzanía , ZambiaRESUMEN
Anemonefish are a group of 28 species of coral reef fish belonging to the family Pomacentridae, subfamily Amphiprioninae, all characterized by living in symbiosis with sea anemones of several genera. Some anemonefish are specialized to cooperate with a single or few species of sea anemone, being immune to their poisonous tentacles but sensible to those of other species of sea anemones, while other anemonefish are more generalist and able to live together with a number of different species of sea anemone hosts. Despite the common life style, anemonefish species occur in a variety of colors, body shapes and degree of dependence from the host. To understand the evolutionary mechanisms responsible for the anemonefish diversification, we studied 23 out of 28 species of anemonefish by analyzing three mitochondrial regions: the cytochrome b gene, the 16S ribosomal RNA gene and the first half of the D-loop, a non-coding, regulatory region to reconstruct their molecular phylogeny through Bayesian and maximum parsimony approaches. The evolution of specialization was studied by means of character reconstruction methods. This work includes the highest number of anemonefish so far analyzed and particularly some species that had never been studied before. The results support a monophyletic origin for the subfamily Amphiprioninae, in contrast to the current taxonomy, based on morphological characters, that divides anemonefish into two separate genera. Moreover, we formulate some hypotheses concerning the life style and origin of the ancestral anemonefish.
Asunto(s)
Evolución Molecular , Perciformes/clasificación , Perciformes/genética , Animales , Secuencia de Bases , Cartilla de ADN/genética , ADN Mitocondrial/genética , Modelos Genéticos , Datos de Secuencia Molecular , Perciformes/fisiología , Filogenia , Anémonas de Mar/fisiología , Especificidad de la Especie , SimbiosisRESUMEN
Hox genes encode DNA binding proteins that specify cell fate in the anterior-posterior axis of metazoan animal embryos. While each Hox cluster contains the same genes among the different mammalian species, this does not happen in ray-finned fish, in which both the number and organization of Hox genes and even Hox clusters are variables. Ray-finned fish are believed to have undergone an additional genome duplication that led to the presence of 8 Hox clusters (four twin pairs) in their ancestor. Here we describe the Tilapia (Oreochromis niloticus) Hox genes set in terms of gene content, clusters organization and base composition and compare it with those of pufferfish and zebrafish. We observed that in all these fish, when paralogous genes are conserved in both the twin clusters, the gene which has a lower GC level generally: (i) belongs to the less gene-rich (less conserved) cluster; (ii) has a reduced field of embryonic expression; or (iii) is a pseudogene. The relationship between the decrease of GC level and the loss of conservation and function of one of the paralogous genes from twin clusters is discussed.
Asunto(s)
Evolución Molecular , Genes Homeobox , Familia de Multigenes , Tilapia/genética , Animales , Composición de Base , Datos de Secuencia Molecular , Filogenia , Reacción en Cadena de la PolimerasaRESUMEN
Comparisons of DNA sequences among evolutionarily distantly related genomes permit identification of conserved functional regions in noncoding DNA. Hox genes are highly conserved in vertebrates, occur in clusters, and are uninterrupted by other genes. We aligned (PipMaker) the nucleotide sequences of the HoxA clusters of tilapia, pufferfish, striped bass, zebrafish, horn shark, human, and mouse, which are separated by approximately 500 million years of evolution. In support of our approach, several identified putative regulatory elements known to regulate the expression of Hox genes were recovered. The majority of the newly identified putative regulatory elements contain short fragments that are almost completely conserved and are identical to known binding sites for regulatory proteins (Transfac database). The regulatory intergenic regions located between the genes that are expressed most anteriorly in the embryo are longer and apparently more evolutionarily conserved than those at the other end of Hox clusters. Different presumed regulatory sequences are retained in either the Aalpha or Abeta duplicated Hox clusters in the fish lineages. This suggests that the conserved elements are involved in different gene regulatory networks and supports the duplication-deletion-complementation model of functional divergence of duplicated genes.