Microenvironment cues involved in melanoma progression are largely unknown. Melanoma is highly influenced in its aggressive phenotype by the changes it determinates in its microenvironment, such as pH decrease, in turn influencing cancer cell invasiveness, progression and tissue remodelling through an abundant secretion of exosomes, dictating cancer strategy to the whole host. A role of exosomes in driving melanoma progression under microenvironmental acidity was never described. We studied four differently staged human melanoma lines, reflecting melanoma progression, under microenvironmental acidic pHs pressure ranging between pH 6.0–6.7. To estimate exosome secretion as a function of tumor stage and environmental pH, we applied a technique to generate native fluorescent exosomes characterized by vesicles integrity, size, density, markers expression, and quantifiable by direct FACS analysis. Functional roles of exosomes were tested in migration and invasion tests. Then we performed a comparative proteomic analysis of acid versus control exosomes to elucidate a specific signature involved in melanoma progression. We found that metastatic melanoma secretes a higher exosome amount than primary melanoma, and that acidic pH increases exosome secretion when melanoma is in an intermediate stage, i.e. metastatic non-invasive. We were thus able to show that acidic pH influences the intercellular cross-talk mediated by exosomes. In fact when exposed to exosomes produced in an acidic medium, pH naïve melanoma cells acquire migratory and invasive capacities likely due to transfer of metastatic exosomal proteins, favoring cell motility and angiogenesis. A Prognoscan-based meta-analysis study of proteins enriched in acidic exosomes, identified 11 genes (HRAS, GANAB, CFL2, HSP90B1, HSP90AB1, GSN, HSPA1L, NRAS, HSPA5, TIMP3, HYOU1), significantly correlating with poor prognosis, whose high expression was in part confirmed in bioptic samples of lymph node metastases. A crucial step of melanoma progression does occur at melanoma intermediate –stage, when extracellular acidic pH induces an abundant release and intra-tumoral uptake of exosomes. Such exosomes are endowed with pro-invasive molecules of clinical relevance, which may provide a signature of melanoma advancement.

Full-sequence data available for Plasmodium falciparumchromosomes 2 and 3 are exploited to perform a statistical analysis of the long tracts of biased amino acid composition that characterize the vast majority of P. falciparum proteins and to make a comparison with similarly defined tracts from other simple eukaryotes. When the relatively minor subset of prevalently hydrophobic segments is discarded from the set of low-complexity segments identified by current segmentation methods in P. falciparum proteins, a good correspondence is found between prevalently hydrophilic low-complexity segments and the species-specific, rapidly diverging insertions detected by multiple-alignment procedures when sequences of bona fide homologs are available. Amino acid preferences are fairly uniform in the set of hydrophilic low-complexity segments identified in the twoP. falciparum chromosomes sequenced, as well as in sequenced genes from Plasmodium berghei, but differ from those observed in Saccharomyces cerevisiae and Dictyostelium discoideum. In the two plasmodial species, amino acid frequencies do not correlate with properties such as hydrophilicity, small volume, or flexibility, which might be expected to characterize residues involved in nonglobular domains but do correlate with A-richness in codons. An effect of phenotypic selection versus neutral drift, however, is suggested by the predominance of asparagine over lysine.

Several fungi secrete lipase isozymes differing in biochemical properties and in some cases in substrate specificity. In the yeast Candida rugosa, a family of related genes encodes for multiple lipase proteins, highly homologous in sequence but partially different in the regions interacting with the substrate molecule. Analysis of these substitutions performed on the basis of multiple alignments and using a 3-D model of the enzyme, allows identification of a restricted number of amino adds possibly involved in substrate specificity of Candida lipases.

Background: Plasmodiumparasites are causative agents of malaria which affects >500 million people and claims ~2 million lives annually. The completion ofPlasmodiumgenome sequencing and availability of PlasmoDB database has provided a platform for systematic study of parasite genome. Aminoacyl-tRNA synthetases (aaRSs) are pivotal enzymes for protein translation and other vital cellular processes. We report an extensive analysis of thePlasmodium falciparumgenome to identify and classifyaaRSsin this organism.Results: Using various computational and bioinformatics tools, we have identified 37aaRSs inP. falciparum. Our key observations are: (i) fraction of proteome dedicated toaaRSs inP. falciparumis very high compared to many other organisms; (ii) 23 out of 37Pf-aaRSsequences contain signal peptides possibly directing them to different cellular organelles; (iii) expression profiles ofPf-aaRSsvary considerably at various life cycle stages of the parasite; (iv) severalPfaaRSsposses very unusual domain architectures; (v) phylogenetic analyses reveal evolutionary relatedness of several parasiteaaRSs to bacterial and plantsaaRSs; (vi) three dimensional structural modelling has provided insights which could be exploited in inhibitor discovery against parasiteaaRSs.Conclusion: We have identified 37Pf-aaRSsbased on our bioinformatics analysis. Our data reveal several unique attributes in this protein family. We have annotated all 37Pf-aaRSsbased on predicted localization, phylogenetics, domain architectures and their overall protein expression profiles. The sets of distinct features elaborated in this work will provide a platform for experimental dissection of this family of enzymes, possibly for the discovery of novel drugs against malaria.

We have built a model of the specificity pocket of the protease of hepatitis C virus on the basis of the known structures of trypsin-like serine proteases and of the conservation pattern of the protease sequences among various hepatitis C strains. The model allowed us to predict that the substrate of this protease should have a cysteine residue in position P1. This hypothesis was subsequently proved by N-terminal sequencing of two products of the protease. The success of this "blind" test increases our confidence in the overall correctness of our proposed alignment of the enzyme sequence with those of other proteases of known structure and constitutes a first step in the construction of a complete model of the viral protease domain.