[Show abstract][Hide abstract] ABSTRACT: Proteins are found in solution as ensembles of conformations in dynamic equilibrium. Exploration of functional motions occurring on μs-ms time scales by Molecular Dynamics (MD) simulations still remains computationally challenging. Alternatively, normal mode (NM) analysis is a well suited method to characterize intrinsic slow collective motions, often associated to protein function, but the absence of anharmonic effects preclude a proper characterization of conformational distributions in a multidimensional NM space. Using both methods jointly appears as an attractive approach that allows an extended sampling of the conformational space. In line with this view the MDeNM (Molecular Dynamics with excited Normal Modes) method presented here consists in multiple-replicas short MD simulations in which motions described by a given subset of low frequency NMs are kinetically excited. This is achieved by adding additional atomic velocities along several randomly determined linear combinations of NM vectors, thus allowing an efficient coupling between slow and fast motions. The relatively high-energy conformations generated with MDeNM are further relaxed with standard MD simulations enabling to determine free energy landscapes. Two widely studied proteins were selected as examples: hen egg lysozyme and HIV-1 protease. In both cases, MDeNM provides a larger extent of sampling in a few nanoseconds, outperforming long standard MD simulations. It was noticed a high degree of correlation with motions inferred from experimental sources (X-ray, EPR and NMR), and with free energy estimations obtained by metadynamics. Finally, the large sets of conformations obtained with MDeNM can be used in a better characterization of relevant dynamical populations, allowing a better interpretation of experimental data such as SAXS and NMR spectra.
Full-text · Article · Apr 2015 · Journal of Chemical Theory and Computation
[Show abstract][Hide abstract] ABSTRACT: Background
Over the last decades, a vast structural knowledge has been gathered on the HIV-1 protease (PR). Noticeably, most of the studies focused the B-subtype, which has the highest prevalence in developed countries. Accordingly, currently available anti-HIV drugs target this subtype, with considerable benefits for the corresponding patients.
However, in developing countries, there is a wide variety of HIV-1 subtypes carrying PR polymorphisms related to reduced drug susceptibility. The non-active site mutation, M36I, is the most frequent polymorphism, and is considered as a non-B subtype marker.
Yet, the structural impact of this substitution on the PR structure and on the interaction with natural substrates remains poorly documented.
Herein, we used molecular dynamics simulations to investigate the role of this polymorphism on the interaction of PR with six of its natural cleavage-sites substrates.
Free energy analyses by MMPB/SA calculations showed an affinity decrease of M36I-PR for the majority of its substrates. The only exceptions were the RT-RH, with equivalent affinity, and the RH-IN, for which an increased affinity was found. Furthermore, molecular simulations suggest that, unlike other peptides, RH-IN induced larger structural fluctuations in the wild-type enzyme than in the M36I variant.
With multiple approaches and analyses we identified structural and dynamical determinants associated with the changes found in the binding affinity of the M36I variant. This mutation influences the flexibility of both PR and its complexed substrate. The observed impact of M36I, suggest that combination with other non-B subtype polymorphisms, could lead to major effects on the interaction with the 12 known cleavage sites, which should impact the virion maturation.
[Show abstract][Hide abstract] ABSTRACT: In the study of cyanobacterial communities, molecular methods can be used to detect and quantify toxin-producing strains and assess genotype diversity. These are promising methods for estimating the risk of exposure to the cyanobacterial toxin microcystin (MC), assuming that the abundance of toxic genotypes is related to the MC concentration. This assumption has been tested in many studies, most of which were conducted in temperate and subtropical aquatic environments, and has not always been met. In this study, we collected cyanobacterial samples from a tropical reservoir and tested whether the MC concentration could be explained by variations in the number or ratio of toxin-producing genotypes or by the presence of certain Microcystis genotypes. The genetic diversity of cyanobacteria was determined by cpcBA sequencing, and the proportion of potential MC-producing genotypes was quantified by qPCR using mcyB as the target gene. Cyanobacteria dominated during the entire sampling period (88.4–99.8% of the total phytoplankton biomass), with Microcystis, Anabaena and Cylindrospermopsis occurring as the main genera. Three major Microcystis genotypes were recognized along with other rare or unique genotypes. MCs were detected in all samples (MC-LR, RR and YR), with most concentrations being less than 200 ng L−1. Microcystis was the most abundant group during all the study period and was the main MC-producing genus. Potentially toxic Microcystis cells were present in all samples, varying from 10% to 100% of the total Microcystis. MC concentrations were not correlated with the number or ratio of toxic cells of Microcystis determined by qPCR or with the predominance of certain genotypes. These results indicate the limitations of using molecular methods such as qPCR in MC risk assessment.
[Show abstract][Hide abstract] ABSTRACT: Introduction: Despite efforts to control malaria, around 10% of the world population is at risk of acquiring this disease. Plasmodium falciparum accounts for the majority of severe cases and deaths. Malaria control programs have failed due to the therapeutic failure of first-line antimalarials and to parasite resistance. Thus, new and better therapeutic alternatives are required. Proteomic analysis allows determination of protein expression levels under drug pressure, leading to the identification of new therapeutic drug targets and their mechanisms of action. Objective: The aim of this study was to analyze qualitatively the expression of P.falciparum trophozoite proteins (strain ITG2), after exposure to antimalarial drugs, through a proteomic approach. Materials and methods: In vitro cultured synchronized parasites were treated with quinine, mefloquine and the natural antiplasmodial diosgenone. Protein extracts were prepared and analyzed by two-dimensional electrophoresis. The differentially expressed proteins were selected and identified by MALDI-TOF mass spectrometry. Results: The following proteins were identified among those differentially expressed in the parasite in the presence of the drugs tested: enolase (PF10_0155), calcium-binding protein (PF11_0098), chaperonin (PFL0740c), the host cell invasion protein (PF10_0268) and proteins related to redox processes (MAL8P1.17). These findings are consistent with results of previous studies where the parasite was submitted to pressure with other antimalarial drugs. Conclusion: The observed changes in the P. falciparum trophozoite protein profile induced by antimalarial drugs involved proteins mainly related to the general stress response.
Full-text · Article · Jun 2014 · Biomédica: revista del Instituto Nacional de Salud
[Show abstract][Hide abstract] ABSTRACT: The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.
[Show abstract][Hide abstract] ABSTRACT: PhoB/PhoR is a two-component system originally described as involved in inorganic phosphate (Pi) transport and metabolism under Pi limitation. In order to disclose other roles of this system, a proteomic analysis of Vibrio cholerae 569BSR and its phoB/phoR mutant under high Pi levels was performed. Most of the proteins downregulated by the mutant have roles in energy production and conversion and in amino acid transport and metabolism. In contrast, the phoB/phoR mutant upregulated genes mainly involved in adaptation to atypical conditions, indicating that the absence of a functional PhoB/PhoR caused increased expression of a number of genes from distinct stress response pathways. This might be a strategy to overcome the lack of RpoS, whose expression in the stationary phase cells of V. cholerae seems to be controlled by PhoB/PhoR. Moreover, compared to the wild-type strain the phoB/phoR mutant presented a reduced cell density at stationary phase of culture in Pi abundance, lower resistance to acid shock, but higher tolerance to thermal and osmotic stresses. Together our findings show, for the first time, the requirement of PhoB/PhoR for full growth under high Pi level and for the accumulation of RpoS, indicating that PhoB/PhoR is a fundamental system for the biology of V. cholerae.
Full-text · Article · May 2013 · Journal of proteomics
[Show abstract][Hide abstract] ABSTRACT: Trypanosoma cruzi proteins with molecular weight between 30-34kDa have shown high reactivity in western blot assays with serum samples from chagasic individuals. However, in-depth analysis of the constituents of these protein fractions has not been performed. This is the first report of an immunoaffinity proteomic approach to identify the immunodominant 30-34kDa proteins of T. cruzi that could eventually be used for the diagnosis of Chagas disease. We used two different sample preparation protocols for protein digestion coupled to mass spectrometry to identify proteins in the protein fraction. The immunodominant proteins and their respective epitopes were then identified by co-immunoprecipitation and excision-epitope mapping/mass spectrometry, using human sera followed by the prediction and three-dimensional structural modeling of reactive epitopes. The use of different sample preparation methods allowed the identification of a relatively high number of proteins, some of which were only identified after one or multiple sample preparation and digestion protocols. Seven immunodominant proteins were identified by co-immunoprecipitation with purified IgGs from chagasic serum samples. Moreover, six reactive peptide epitopes were detected in four of these proteins by excision-epitope mapping/mass spectrometry. Three-dimensional structural models were obtained for the immunoreactive peptides, which correlated well with the linear B-cell epitope prediction tools.
Full-text · Article · Nov 2012 · Journal of proteomics
[Show abstract][Hide abstract] ABSTRACT: Phytomonas species are plant parasites of the family Trypanosomatidae, which are transmitted by phytophagous insects. Some Phytomonas species cause major agricultural damages. The hemipteran Oncopeltus fasciatus is natural and experimental host for several species of trypanosomatids, including Phytomonas spp. The invasion of the insect vectors' salivary glands is one of the most important events for the life cycle of Phytomonas species. In the present study, we show the binding of Phytomonas serpens at the external face of O. fasciatus salivary glands by means of scanning electron microscopy and the in vitro interaction of living parasites with total proteins from the salivary glands in ligand blotting assays. This binding occurs primarily through an interaction with a 130 kDa salivary gland protein. The mass spectrometry of the trypsin-digest of this protein matched 23% of human laminin-5 β3 chain precursor sequence by 16 digested peptides. A protein sequence search through the transcriptome of O. fasciatus embryo showed a partial sequence with 51% similarity to human laminin β3 subunit. Anti-human laminin-5 β3 chain polyclonal antibodies recognized the 130 kDa protein by immunoblotting. The association of parasites with the salivary glands was strongly inhibited by human laminin-5, by the purified 130 kDa insect protein, and by polyclonal antibodies raised against the human laminin-5 β3 chain. This is the first report demonstrating that a laminin-like molecule from the salivary gland of O. fasciatus acts as a receptor for Phytomonas binding. The results presented in this investigation are important findings that will support further studies that aim at developing new approaches to prevent the transmission of Phytomonas species from insects to plants and vice-versa.
[Show abstract][Hide abstract] ABSTRACT: Zinc finger proteins are widely found in eukaryotes, representing an important class of DNA-binding proteins frequently involved in transcriptional regulation. Zinc finger motifs are composed by two antiparallel β-strands and one α-helix, stabilized by a zinc ion coordinated by conserved histidine and cysteine residues. In Schistosoma mansoni, these regulatory proteins are known to modulate morphological and physiological changes, having crucial roles in parasite development. A previously described C(2)H(2) zinc finger protein, SmZF1, was shown to be present in cell nuclei of different life stages of S. mansoni and to activate gene transcription in a heterologous system. A high-quality SmZF1 tridimensional structure was generated using comparative modeling. Molecular dynamics simulations of the obtained structure revealed stability of the zinc fingers motifs and high flexibility on the terminals, comparable to the profile observed on the template X-ray structure based on thermal b-factors. Based on the protein tridimensional features and amino acid composition, we were able to characterize four C(2)H(2) zinc finger motifs, the first involved in protein-protein interactions while the three others involved in DNA binding. We defined a consensus DNA binding sequence using three distinct algorithms and further carried out docking calculations, which revealed the interaction of fingers 2-4 with the predicted DNA. A search for S. mansoni genes presenting putative SmZF1 binding sites revealed 415 genes hypothetically under SmZF1 control. Using an automatic annotation and GO assignment approach, we found that the majority of those genes code for proteins involved in developmental processes. Taken together, these results present a consistent base to the structural and functional characterization of SmZF1.
No preview · Article · Oct 2012 · Journal of molecular graphics & modelling
[Show abstract][Hide abstract] ABSTRACT: Heat shock protein (HSP) 104 is a highly conserved molecular chaperone that catalyzes protein unfolding, disaggregation and degradation under stress conditions. We characterized HSP104 gene structure and expression in Trypanosoma cruzi, a protozoan parasite that causes Chagas' disease. The T. cruzi HSP104 is an 869 amino-acid protein encoded by a single-copy gene that has the highest sequence similarity (76%) with that of T. brucei and the lowest (23%) with that of the human protein. HSP104 transcripts were detected at room temperature, and levels increased after incubation at 37° or 40°C. The HSP104 protein was found at low levels in non-heat-shocked cells, and accumulated continuously up to 24 h at elevated temperatures. We developed a predicted structural model of hexameric T. cruzi HSP104, which showed some conserved features.
Full-text · Article · Aug 2012 · Genetics and molecular research: GMR
[Show abstract][Hide abstract] ABSTRACT: The polymorphism of species A rotavirus genotype G1 strains (RVA-G1) circulating in Rio de Janeiro between 1996 and 2004 was evaluated. The VP7 encoding gene of 36 G1 isolates was sequenced and compared to references strains. The deduced amino acid sequences were used as basis for in silico analysis of the VP7 protein. We observed the circulation of two major G1 lineages and five sublineages during the studied period. Comparison between the VP7 trimeric structures of a rotavirus vaccine strain and Brazilian G1 strains showed mutations at amino acid residues located at the calcium binding site and at several neutralizing antibody recognition sites. Although the rotavirus vaccine program has clearly been successful in Brazil, these results suggest the possibility of the emergence of G1 strains that could evade the immune response elicited by a RVA vaccine and cause a vaccine breakthrough. Consequently, continuous monitoring of rotavirus intragenotypes diversity is critical to understand how it could affect vaccine effectiveness.
Full-text · Article · May 2012 · Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases
[Show abstract][Hide abstract] ABSTRACT: The control of Vibrio cholerae phoBR expression by PhoB involves its binding to Pho boxes at −35 (box 1), −60 (box 2), and −80 (box 3) from the putative phoB translation start site. These loci were located in the sense (box 1) and antisense (boxes 2 and 3) strands of the phoBR regulatory region, and PhoB binds to these individual boxes with distinct affinities. Fusions of sequences containing different
combinations of these boxes upstream of the lacZ reporter in a plasmid demonstrated that only those carrying boxes 1, 2, and 3, or 1 alone, activated transcription under
inorganic phosphate (Pi) limitation. When a fragment, including only boxes 1 and 2, was fused to lacZ, expression was no longer induced by low Pi, suggesting a repressive role for PhoB∼box2 (PhoB bound to box 2) over the transcriptional activity induced by PhoB∼box1.
The similarity between lacZ expression levels from promoter fragments containing the three boxes or box 1 alone showed that PhoB∼box3 eliminated the
repressive effect imposed by PhoB∼box2 on phoBR transcription. Complementation assays with a phoBR-containing plasmid demonstrated that the 234-bp promoter fragment carrying the three boxes is absolutely required for operon
expression in Vibrio cholerae ΔphoBR cells. This was observed under Pi abundance, when phoBR was expressed at a basal level and, also in low Pi conditions, when Pho regulon genes were fully expressed. Thus, under Pi limitation, PhoB exerts dual regulatory functions by binding sequentially distinct Pho boxes to enable the fine-tuning and
precise control of phoBR expression in V. cholerae cells.
Full-text · Article · Dec 2011 · Journal of bacteriology
[Show abstract][Hide abstract] ABSTRACT: Cylindrospermopsis raciborskii (Woloszynska) Seenaya & Subba Raju is a freshwater cyanobacterium of worldwide distribution. In the North-eastern region of Brazil many eutrophic water reservoirs are characterized by the dominance of C. raciborskii, with recurrent occurrence of blooms. These water bodies have high conductivity due to a high ionic concentration, and are defined as hard (with high values of CaCO3). In this study, we investigated the long-term effect (12 days) of high calcium concentration (8 mM Ca(2+)) on C. raciborskii (T3 strain) growth, morphology, toxin content, and metabolism. Changes in protein expression profiles were investigated by proteomic analysis using 2D gel electrophoresis and mass spectrometry. A continued exposure to calcium had a pronounced effect on C. raciborskii (T3): it limited growth, decreased thricome length, increased chlorophyll-a content, altered toxin profile (although did not affect PST content, saxitoxin + neosaxitoxin), and inhibited the expression of proteins related to primary metabolism.
Full-text · Article · Oct 2011 · Brazilian Journal of Microbiology
[Show abstract][Hide abstract] ABSTRACT: The Amazon catfish genus Pterygoplichthys (Loricariidae, Siluriformes) is closely related to the loricariid genus Hypostomus, in which at least two species lack detectable ethoxyresorufin-O-deethylase (EROD) activity, typically catalyzed by cytochrome P450 1 (CYP1) enzymes. Pterygoplichthys sp. liver microsomes also lacked EROD, as well as activity with other substituted resorufins, but aryl hydrocarbon receptor agonists induced hepatic CYP1A mRNA and protein suggesting structural/functional differences in Pterygoplichthys CYP1s from those in other vertebrates. Comparing the sequences of CYP1As of Pterygoplichthys sp. and of two phylogenetically related siluriform species that do catalyze EROD (Ancistrus sp., Loricariidae and Corydoras sp., Callichthyidae) showed that these three proteins share amino acids at 17 positions that are not shared by any fish in a set of 24 other species. Pterygoplichthys and Ancistrus (the loricariids) have an additional 22 amino acid substitutions in common that are not shared by Corydoras or by other fish species. Pterygoplichthys has six exclusive amino acid substitutions. Molecular docking and dynamics simulations indicate that Pterygoplichthys CYP1A has a weak affinity for ER, which binds infrequently in a productive orientation, and in a less stable conformation than in CYP1As of species that catalyze EROD. ER also binds with the carbonyl moiety proximal to the heme iron. Pterygoplichthys CYP1A has amino acid substitutions that reduce the frequency of correctly oriented ER in the AS preventing the detection of EROD activity. The results indicate that loricariid CYP1As may have a peculiar substrate selectivity that differs from CYP1As of most vertebrate.