SEQUENCE SLIDER: integration of structural and genetic data to characterize isoforms from natural sources.

Proteins isolated from natural sources can be composed of a mixture of isoforms with similar physicochemical properties that coexist in the final steps of purification. Yet, even where unverified, the assumed sequence is enforced throughout the structural studies. Herein, we propose a novel perspective to address the usually neglected sequence heterogeneity of natural products by integrating biophysical, genetic and structural data in our program SEQUENCE SLIDER. The aim is to assess the evidence supporting chemical composition in structure determination. Locally, we interrogate the experimental map to establish which side chains are supported by the structural data, and the genetic information relating sequence conservation is integrated into this statistic. Hence, we build a constrained peptide database, containing most probable sequences to interpret mass spectrometry data (MS). In parallel, we perform MS de novo sequencing with genomic-based algorithms to detect point mutations. We calibrated SLIDER with Gallus gallus lysozyme, whose sequence is unequivocally established and numerous natural isoforms are reported. We used SLIDER to characterize a metalloproteinase and a phospholipase A2-like protein from the venom of Bothrops moojeni and a crotoxin from Crotalus durissus collilineatus. This integrated approach offers a more realistic structural descriptor to characterize macromolecules isolated from natural sources.

Variant expression signatures of microRNAs and protein related to growth in a crossbreed between two strains of Nile tilapia (Oreochromis niloticus).

Nile tilapia (Oreochromis niloticus) is a species of worldwide importance for aquaculture. A crossbred lineage was developed through introgressive backcross breeding techniques and combines the high growth performance of the Chitralada (CHIT) lwith attractive reddish color of the Red Stirling (REDS) strains. Since the crossbreed has an unknown genetically improved background, the objective of this work was to characterize expression signatures that portray the advantageous phenotype of the crossbreeds. We characterized the microRNA transcriptome by high throughput sequencing (RNA-seq) and the proteome through mass spectrometry (ESI-Q-TOF-MS) and applied bioinformatics for the comparative analysis of such molecular data on the three strains. Crossbreed expressed a distinct set of miRNAs and proteins compared to the parents. They comprised several microRNAs regulate traits of economic interest. Proteomic profiles revealed differences between parental and crossbreed in expression of proteins associated with glycolisis. Distinctive miRNA and protein signatures contribute to the phenotype of crossbreed.

Biochemical, pharmacological and structural characterization of BmooMP-I, a new P-I metalloproteinase from Bothrops moojeni venom.

Snakebite envenoming is still a worrying health problem in countries under development, being recognized as a neglected disease by the World Health Organization. In Latin America, snakes from the genus Bothrops are widely spread and in Brazil, the Bothrops moojeni is a medically important species. The pharmacological effects of bothropic snake venoms include pain, blisters, bleeding, necrosis and even amputation of the affected limb. Snake venom metalloproteinases are enzymes abundantly present in venom from Bothrops snakes. These enzymes can cause hemorrhagic effects and lead to myonecrosis due to ischemia. Here, we present BmooMP-I, a new P-I class of metalloproteinase (this class only has the catalytic domain in the mature form) isolated from B. moojeni venom. This protein is able to express fibrinogenolytic and gelatinase activities, which play important roles in the prey's immobilization and digestion, and also induces weak hemorrhagic effect. The primary sequence assignment was done by a novel method, SEQUENCE SLIDER, which combines crystallographic, bioinformatics and mass spectrometry data. The high-resolution crystal structure reveals the monomeric assembly and the conserved metal binding site H141ExxH145xxG148xxH151 with the natural substitution Gly148Asp that does not interfere in the zinc coordination. The presence of a structural calcium ion on the surface of the protein, which can play an important role in the stabilization of hemorrhagic toxins, was observed in the BmooMP-I structure. Due to the relevant local and systemic effects of snake venom metalloproteinases, studies involving these proteins help to better understand the pathological effects of snakebite envenoming.

Quantitative Proteomic Profiling Reveals That Diverse Metabolic Pathways Are Influenced by Melatonin in an in Vivo Model of Ovarian Carcinoma.

To obtain more information into the molecular mechanisms underlying ovarian cancer (OC), we proposed a comparative proteomic analysis in animals receiving long-term melatonin as therapy or only vehicle using multidimensional protein identification combined with mass spectrometry. To induce tumor, a single dose of 100 µg 7,12-dimethylbenz(a)anthracene (DMBA) dissolved in 10 µL of sesame oil was injected under the left ovarian bursa of 20 Fischer 344 rats. The right ovaries were injected with sesame oil only. After tumors were developed, half of the animals received intraperitoneal administration of melatonin (200 µg/100g body weight/day) for 60 days. Melatonin therapy promoted down-regulation in numerous proteins involved in OC signaling pathways. The most significant portion of these proteins are involved in several metabolic processes, mainly those associated with mitochondrial systems, generation of metabolites and energy, hypoxia-inducible factor-1 signaling, antigen processing and presentation, endoplasmic reticulum stress-associated pathways, and cancer-related proteoglycans. A small number of proteins that were overexpressed by melatonin therapy included ATP synthase subunit ß, fatty acid-binding protein, and 10-kDa heat shock protein. Taken together, our findings suggest that melatonin therapy efficiently modulated important signaling pathways involved in OC, and these proteins might be further targets that should be explored in new therapeutic opportunities for OC.

Brown recluse spider venom: proteomic analysis and proposal of a putative mechanism of action.

Loxosceles intermedia spider venom was subjected to proteomic analysis through a MudPIT shot-gun approach to identify the protein composition. Were identified 39 proteins which seem to responsible by the lesion of different types of tissues, to some physiopathological actions and by the prevention of structural damage to the toxin structures.

The venomous secrets of the web droplets from the viscid spiral of the orb-weaver spider Nephila clavipes (Araneae, Tetragnatidae).

The capture web of N. clavipes presents viscous droplets, which play important roles in web mechanics and prey capture. By using scanning and transmission electron microscopy, it was demonstrated that the web droplets are constituted of different chemical environments, provided by the existence both of an aqueous and a lipid layer, which, in turn, present a suspension of tenths of vesicles containing polypeptides and/or lipids. GC/EI-MS Analysis of the contents of these vesicles led to the identification of some saturated fatty acids, such as decanoic acid, undecanoic acid, dodecanoic acid, tetradecanoic acid, octadecanoic acid, and icosanoic acid, while other components were unsaturated fatty acids, such as (Z)-tetradec-9-enoic acid, (Z)-octadec-9-enoic acid, and (Z)-icosa-11-enoic acid; and polyunsaturated fatty acids like (9Z,12Z)-octadeca-9,12-dienoic acid, (9Z,12Z,15Z)-octadeca-9,12,15-trienoic acid, and (11Z,14Z)-icosa-11,14-dienoic acid. Toxic proteins such as calcium-activated proteinase and metalloproteinase jararhagin-like precursor were also identified by using a proteomic approach, indicating the possible involvement of these enzymes in the pre-digestion of spiders' preys web-captured. Apparently, the mixture of fatty acids are relatively toxic to insects by topical application (LD50 64.3+/-7.6 ng mg(-1) honeybee), while the proteins alone present no topical effect; however, when injected into the prey-insects, these proteins presented a moderate toxicity (LD50 40.3+/-4.8 ng mg(-1) honeybee); the mixture of fatty acids and proteins is very toxic to the preys captured by the web droplets of the viscid spiral of Nephila clavipes when topically applied on them (LD50 14.3+/-1.8 ng mg(-1) honeybee).