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The fibroin-based silk fibers of weaver ants are an alternative biomaterial to be investigated and explored for potential biomedical applications. In this context, the silk fibers from the nest of the weaver ant Camponotus textor was solubilized and fractionated by gel permeation. The different fractions were collected, pooled and submitted to analysis with a series of biochemical methods, nuclear magnetic resonance (NMR) spectroscopy, analytical proteomic strategies, and data treatment with bioinformatic tools to perform the structural characterization of the fibroin-based silk fibers produced by the ant. Our data demonstrated the identification of one fibroin proteoform in the ant silk fibers. The protein chracterized as a glycoprotein with MW around 40 kDa and presenting 66% (w/w) of total sugars attached to it through O-linked carbohydrates. The 3D of protein was modeled, revealing a structure predominantly constituted of coiled-coil secondary units in the whole model, featuring at least four superhelices (arrangement with multiple α-helices). The scientific outcomes reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based polymers for biomedical applications. BIOLOGICAL SIGNIFICANCE: The present investigation significantly expanded knowledge regarding to the fibroin-based silk fibers from weaver ants, contributing to improvements in our understanding of the properties and characteristics of these silk fibers. For example, as reported here, carbohydrates were detected in the ants' silk for the first time presenting the fibroin as a glycoprotein. Moreover, the 3D structure provided new insights into the secondary structures considering the whole model of the protein.

Polybia paulista (Hymenoptera: Vespidae) is a neotropical social wasp from southeast Brazil. As most social Hymenoptera, venom from P. paulista comprises a complex mixture of bioactive toxins ranging from low molecular weight compounds to peptides and proteins. Several efforts have been made to elucidate the molecular composition of the P. paulista venom. Data derived from proteomic, peptidomic and allergomic analyses has enhanced our understanding of the whole envenoming process caused by the insect sting. The combined use of bioinformatics, -omics- and molecular biology tools have allowed the identification, characterization, in vitro synthesis and recombinant expression of several wasp venom toxins. Some of these P. paulista - derived bioactive compounds have been evaluated for the rational design of antivenoms and the improvement of allergy specific diagnosis and immunotherapy. Molecular characterization of crude venom extract has enabled the description and isolation of novel toxins with potential biotechnological applications. Here, we review the different approaches that have been used to unravel the venom composition of P. paulista. We also describe the main groups of P. paulista - venom toxins currently identified and analyze their potential in the development of component-resolved diagnosis of allergy, and in the rational design of antivenoms and novel bioactive drugs.

Made available in DSpace on 2020-12-12T01:51:19Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-02-20 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Termite soldiers constitute the defensive frontline of the colonies, despite workers also perform such tasks, especially within the Neotropical Apicotermitinae, in which all species are soldierless. Workers of the genus Ruptitermes display an extreme form of defense, characterized by body rupture and release of a sticky secretion. Previous observations suggested that such behavior may be advantageous against enemies, but the chemical composition of this secretion has been neglected. Here we firstly provide the proteomic profile of the defensive secretion of Ruptitermes reconditus and Ruptitermes pitan workers. Additionally, the mechanisms of action of this behavior was evaluated through different bioassays. A total of 446 proteins were identified in R. reconditus and 391 proteins in R. pitan, which were classified into: toxins, defensins and proteolytic enzymes; sticky components/ alarm communication; proteins related to detoxification processes; proteins involved in folding/conformation and post-translational modifications; housekeeping proteins; and uncharacterized/hypothetical proteins. According to the bioassays, the self-sacrifice is triggered by a physical stimulus, and the defensive secretion may cause immobility and death of the opponents. Assuming that termites are abundant in the tropics and therefore exposed to predators, suicidal behaviors seem to be advantageous, since the loss of an individual benefit the whole colony. Significance: Although recent studies have reported the biochemical composition of different weapons in soldiered species of termites, such efforts had not been applied to sordierless taxa up until now. Thus, this is the first report of the defensive mechanisms in soldierless termite species based on proteomic analysis. The diversity of compounds, which included toxin-like and mucin-like proteins, reflect the mechanisms of action of the defensive secretion released by termite workers, which may cause immobility and death of the opponents. Our findings may contribute to the knowledge regarding the development of defensive strategies in termites, especially in groups which lost the soldier caste during the evolution. Laboratório de Cupins Departamento de Biologia Instituto de Biociências Univ Estadual Paulista UNESP, Campus Rio Claro, Avenida 24A, 1515, Bela Vista Center for the Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro Univ Estadual Paulista UNESP, Rio Claro Laboratório de Cupins Departamento de Biologia Instituto de Biociências Univ Estadual Paulista UNESP, Campus Rio Claro, Avenida 24A, 1515, Bela Vista Center for the Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro Univ Estadual Paulista UNESP, Rio Claro CNPq: 150699/2017-4 FAPESP: 2013/26451-9 FAPESP: 2016/16212-5 FAPESP: 2017/10373-0 CAPES: 206/2018 CNPq: 301656/2013-4 CNPq: 305539/2014-0

Made available in DSpace on 2018-12-11T17:00:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-16 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Dufour's gland is associated with the venom apparatuses of social wasps and bees. This location and its evolutionary adaptations indicate that it could be involved in the production of alarm pheromones in the social wasp Polybia paulista. To investigate this hypothesis, the volatile composition of this gland was analyzed and compared to that in the venom. Eighteen compounds were identified as secreted by Dufour's gland, and 16 of these compounds were also identified in the venom, suggesting that the compounds produced by the gland are secreted and mixed with venom in the venom reservoir of this wasp. These compounds were subjected to a field bioassay to investigate their potential action as alarm pheromones. Alcohols and aldehydes elicited the alert behavior in workers, luring them outside the nest, whereas acids attracted the workers in the direction of the source; fatty acid methyl esters elicited aggression. These results suggest that Dufour's gland produces alarm pheromones. To corroborate this hypothesis the proteomic complement of this gland was assigned using a shot-gun strategy; 59 proteins were identified, and the results indicate specialization of Dufour's gland for the metabolism of fatty acids (elongation, esterification unsaturation, reduction, and decarboxylation) in the biosynthesis of alarm pheromones. Biological significance The present knowledge about the role of Dufour's gland among aculeate Hymenoptera insects suggests that it may have many different roles related to the biosynthesis and secretion of chemical markers for different biological functions, though none are related to the elicitation of alarm behaviors for coordinating a mass attack of the colony against intruders. The present study combined the analysis of secreted volatile compounds (as metabolites) with proteome assignments and a field bioassay with synthetic compounds to clearly demonstrate that Dufour's gland does in fact biosynthesize alarm pheromones in social wasps. This strategy may be reproduced in other investigations related to pheromone production in other insects. Center of Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro São Paulo State University (UNESP) Center of Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro São Paulo State University (UNESP) FAPESP: 2011/51684-1

Made available in DSpace on 2019-10-04T12:37:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-05-30 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The partial proteome of Polybia paulista wasp venom was previously reported elsewhere using a gel-dependent approach and resulted in the identification of a limited number of venom toxins. Here, we reinvestigated the P. paulista venom using a gel-free shotgun proteomic approach; the highly dynamic range of this approach facilitated the detection and identification of 1673 proteins, of which 23 venom proteins presented N-linked glycosylation as a posttranslational modification. Three different molecular forms of PLAT were identified as allergenic proteins, and two of these forms were modified by N-linked glycosylation. This study reveals an extensive repertoire of hitherto undescribed proteins that were classified into the following six different functional groups: (i) typical venom proteins; (ii) proteins related to the folding/conformation and PTMs of toxins; (iii) proteins that protect toxins from oxidative stress; (iv) proteins involved in chemical communication; (v) housekeeping proteins; and (vi) uncharacterized proteins. It was possible to identify venom toxin-like proteins that are commonly reported in other animal venoms, including arthropods such as spiders and scorpions. Thus, the findings reported here may contribute to improving our understanding of the composition of P. paulista venom, its envenoming mechanism and the pathologies experienced by the victim after the wasp stinging accident. Biological significance: The present study significantly expanded the number of proteins identified in P. paulista venom, contributing to improvements in our understanding of the envenoming mechanism produced by sting accidents caused by this wasp. For example, novel wasp venom neurotoxins have been identified, but no studies have assessed the presence of this type of toxin in social wasp venoms. In addition, 23 N-linked glycosylated venom proteins were identified in the P. paulista venom proteome, and some of these proteins might be relevant allergens that are immunoreactive to human IgE. Sao Paulo State Univ, Inst Biosci Rio Claro, Dept Biol, Ctr Study Social Insects, BR-13500 Rio Claro, SP, Brazil Univ Campinas UNICAMP, Fac Med, Lab Translat Immunol, Cidade Univ Zeferino Vaz, BR-13083887 Campinas, SP, Brazil Sao Paulo State Univ, Inst Biosci Rio Claro, Dept Biol, Ctr Study Social Insects, BR-13500 Rio Claro, SP, Brazil FAPESP: 2013/26451-9 FAPESP: 2016/16212-5 FAPESP: 2017/04680-7 FAPESP: 2017/10373-0 FAPESP: 2017/22405-3 CNPq: 301656/2013-4 CNPq: 150699/2017-4

Made available in DSpace on 2018-12-11T17:14:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-06 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Many scorpion accidents occur in the Brazilian Amazonian region and are frequently caused by Tityus obscurus. Approximately 5% of the crude venom of this species is composed of short linear, non-disulfide-bridged peptides, which have not been intensively investigated. As a consequence, only a few of these peptides have been structurally and functionally characterized to date. In the present paper, the peptide fraction of the venom was subjected to peptide profiling using an LCMS-IT-TOF/MS and MSn system. The analysis detected 320 non-disulfide bond-containing peptides (NDBPs), of which twenty-seven had their sequences assigned; among them, thirteen peptides were characterized, constituting novel toxins in T. obscurus venom. Some of the novel peptides showed similarities to hypotensin-like toxins, while other peptides appear to be natural fragments of neurotoxins. The novel peptides were submitted to a series of bioassays, revealing that many are multifunctional toxins that cause, for example, pain, edema formation and hemolysis to potentiate strong inflammatory processes and alterations in the locomotion and lifting activities in the victims of stinging. Knowledge of the complex matrix of peptides composing the venom of T. obscurus will contribute to better understanding of the complex mechanism of envenoming caused by stinging accidents. Significance The scorpion Tityus obscurus causes many envenoming accidents of medical importance in Brazilian Amazon region; despite to this, very few is known about the toxinology of this animal. The knowledge about the venom composition and mechanisms of action is very important to understand the physiopathology processes related to the envenoming caused by this animal. The proteopeptidomic investigations of scorpion venoms in general have focused mainly the neurotoxins (which are disulfide bonds containing peptides) and large proteins. The short, linear, non-disulfide bonds containing peptides (NDBPs) represent up to 5% of scorpion venom compositions; however, they have been few investigated in comparison with the neurotoxins. The present study used a mass spectrometric approach to detect 320 NDBPs and to sequence 27 of them; pharmacological assays permitted to characterize 13 NDBPs as novel toxins involved with inflammation, pain and edema formation. Dept. Biology/CEIS Institute of Biosciences of Rio Claro University of São Paulo State (UNESP) Laboratory of Biological Research Amazon College/Amazon University UNAMA Laboratory of Pharmacology Butantan Institute Dept. Biology/CEIS Institute of Biosciences of Rio Claro University of São Paulo State (UNESP) FAPESP: 2011/51684-1

Made available in DSpace on 2018-12-11T17:03:14Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-01-16 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The proboscis extension reflex (PER) is an unconditioned stimulus (US) widely used to access the ability of honeybees to correlate it with a conditioned stimulus (CS) during learning and memory acquisition. However, little is known about the biochemical/genetic changes in worker honeybee brains induced by the PER alone. The present investigation profiled the proteomic complement associated with the PER to further the understanding of the major molecular transformations in the honeybee brain during the execution of a US. In the present study, a quantitative shotgun proteomic approach was employed to assign the proteomic complement of the honeybee brain. The results were analyzed under the view of protein networking for different processes involved in PER behavior. In the brains of PER-stimulated individuals, the metabolism of cyclic/heterocyclic/aromatic compounds was activated in parallel with the metabolism of nitrogenated compounds, followed by the up-regulation of carbohydrate metabolism, the proteins involved with the anatomic and cytoskeleton; the down-regulation of the anatomic development and cell differentiation in other neurons also occurred. Significance The assay of proboscis extension reflex is frequently used to access honeybees' ability to correlate an unconditioned stimulus with a conditioned stimulus (such as an odor) to establish learning and memory acquisition. The reflex behavior of proboscis extension was associated with various conditioned stimuli, and the biochemical/genetic evaluation of the changes occurring in honeybee brains under these conditions reflect the synergistic effects of both insect manipulations (training to answer to an unconditioned stimulus and training to respond to a conditioned stimulus). Little or no information is available regarding the biochemical changes stimulated by an unconditioned stimulus alone, such as the proboscis extension reflex. The present investigation characterizes the proteomic changes occurring in the brains of honeybee workers submitted to proboscis extension reflex. A series of metabolic and cellular processes were identified to be related to the reflex of an unconditioned stimulus. This strategy may be reproduced to further understand the processes of learning and memory acquisition in honeybees. Center of the Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro São Paulo State University (UNESP) Laboratory for Proteomics and Protein Engineering Carlos Chagas Institute Center of the Study of Social Insects Department of Biology Institute of Biosciences of Rio Claro São Paulo State University (UNESP)

The wasp Agelaia pallipes pallipes is one of the most aggressive species from the neotropical region, causing many stinging accidents every year, characterized by severe envenoming reactions. The identification of peptides is important for understanding the envenoming process; however, the tiny amount of venom produced by these insects makes this task a challenge, using classical analytical approaches. Thus, the venom was previously fractionated, and the sequences were obtained through the use of electrospray ionization with a tridimensional ion-trap and time-of-flight mass analysis under CID conditions. This approach permitted the sequence assignment of nine peptides. The presence of type -d and -w ions generated from the fragmentation of the side chains was used to resolve I/L ambiguity. The distinction between K and Q residues was achieved through esterification of the α- and e-amino groups in the peptides, followed by mass spectrometry analysis. Six of these peptides were short, linear and polycationic, while the three other peptides presented a single disulfide bridge. The use of reduction and alkylation protocols, followed by ESI-IT-TOF/MS analysis under CID conditions, permitted easy sequencing of the three peptides presenting this post-translational modification. These peptides presented activity related to mast cell degranulation, hemolysis, or even the chemotaxis of leukocytes.