PATRICK JACK SPENCER

PATRICK JACK SPENCER

(Fonte: Lattes)

Resumo

Possui graduação em Ciências Biológicas pela Universidade Presbiteriana Mackenzie (1991), mestrado em Tecnologia Nuclear pela Universidade de São Paulo (1995) e doutorado em Tecnologia Nuclear pela Universidade de São Paulo (2000) tendo sido bolsista sandwich no US Army Medical Research Institute for Infeccious Diseases (98-99). É responsável pelo Biotério de criação e manutenção de animais de laboratório do IPEN. Tem experiência na área de Bioquímica, com ênfase em Proteínas, atuando principalmente nos seguintes temas: veneno, proteínas, bothrops, irradiação e miotoxina.(Texto extraído do Currículo Lattes em 22 dez. 2021)

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Proteomic analysis of soluble proteins retrieved from Duttaphrynus melanostictus skin secretion by IEx-batch sample preparation
2019 - MARIANO, DOUGLAS O.C.; PREZOTTO-NETO, JOSE P.; SPENCER, PATRICK J.; SCIANI, JULIANA M.; PIMENTA, DANIEL C.
Amphibians display a toxic secretion that works as chemical defenses against predators and/or microorganisms that is stored in specialized glands located in the tegument. For some animals, such glands have accumulated in specific regions of the body and formed prominent structures known as macroglands. The Bufonidae family displays conspicuous macroglands in a post-orbital position, termed parotoids, which secretions are known to be extremely viscous and rich in alkaloids and steroids. Few proteins have been described in this material, though. Mainly, because of the difficulties to handle such biological matrix. In this context, we have performed a proteomic study on the parotoid macrogland secretion of the Asian bufonid Duttaphrynus melanostictus. By employing the Ion-Exchange (IEx)-batch chromatography (anionic, cationic and both) we obtained six fractions - bound and unbound – that were submitted to an in solution-trypsin digestion followed by LC-MS/MS. Proteins related to: antioxidant activity, binding processes (carbohydrate/lipid/protein), energy metabolism, hydrolases, lipid metabolism and membrane traffic were identified. Moreover, IEx was able to preserve the biological activity of the retrieved proteins (peptidasic). The current study increases the knowledge on the proteins present in the bufonids parotoid macrogland secretion, providing a better understanding of the physiological role played by such molecules. Significance: The current approach allowed a detailed proteomic analysis of the several proteins synthesized in the D. melanostictus parotoid macrogland (Bufonidae) that are secreted into the skins, but embedded within a complex viscous biological matrix. Moreover, our results aim to increase the knowledge on the biological role played by such proteins at the skin.
Protein identification from the parotoid macrogland secretion of Duttaphrynus melanostictus
2019 - MARIANO, DOUGLAS O.C.; MESSIAS, MARCELA D.G.; SPENCER, PATRICK J.; PIMENTA, DANIEL C.
Background: Bufonid parotoid macrogland secretion contains several low molecular mass molecules, such as alkaloids and steroids. Nevertheless, its protein content is poorly understood. Herein, we applied a sample preparation methodology that allows the analysis of viscous matrices in order to examine its proteins. Methods: Duttaphrynus melanostictus parotoid macrogland secretion was submitted to ion-exchange batch sample preparation, yielding two fractions: salt-displaced fraction and acid-displaced fraction. Each sample was then fractionated by anionic-exchange chromatography, followed by in-solution proteomic analysis. Results: Forty-two proteins could be identified, such as acyl-CoA-binding protein, alcohol dehydrogenase, calmodulin, galectin and histone. Moreover, de novo analyses yielded 153 peptides, whereas BLAST analyses corroborated some of the proteomicidentified proteins. Furthermore, the de novo peptide analyses indicate the presence of proteins related to apoptosis, cellular structure, catalysis and transport processes. Conclusions: Proper sample preparation allowed the proteomic and de novo identification of different proteins in the D. melanostictus parotoid macrogland secretion. These results may increase the knowledge about the universe of molecules that compose amphibian skin secretion, as well as to understand their biological/physiological role in the granular gland.
The amphibian diacylglycerol O-acyltransferase 2 (DGAT2)
2019 - SCIANI, JULIANA M.; NEVES, ADRIANA; VASSÃO, RUTH C.; SPENCER, PATRICK; ANTONIAZZI, MARTA M.; JARED, CARLOS; PIMENTA, DANIEL C.
Amphibians are, currently, considered the first vertebrates that had performed the aquatic to terrestrial transition during evolution; therefore, water balance and dehydration control were prerequisites for such environment conquering. Among anurans, Phyllomedusa is a well-studied genus, due to its peptide-rich skin secretion. Here, we have analyzed the skin secretion of Phyllomedusa distincta targeting the proteins present in the skin secretion. The major soluble protein was chromatographically isolated and utilized to immunize rabbits. Through proteomics approaches, we were able to identify such protein as being the diacylglycerol O-acyltransferase 2 (DGAT2), a crucial enzyme involved in lipid synthesis and in the skin water balance. Immunohistochemistry assays revealed the protein tissular distribution for different animal species, belonging to different branches of the phylogenetic tree. Specifically, there was positivity to the anti-DGAT2 on Amphibians’ skin, and no antibody recognition on fish and mammals’ skins. The DGAT2 multiple sequence alignment reveals some degree of conservation throughout the genera; however, there is a different cysteine pattern among them. Molecular modeling analyses corroborate that the different cysteine pattern leads to distinct 3D structures, explaining the different antibody recognition. Moreover, the protein phylogenetic analyses place the Xenopus DGAT2 (the available amphibian representative) next to the Coelacanthus enzyme, which have led the authors to term this a ‘paleo-protein’. DGAT2 would be, therefore, an ancient protein, crucial to the terrestrial environment conquest, with a unique folding—as indicated by the molecular models and immunohistochemistry analyses—a consequence of the different cysteine pattern but with conserved biological function.
Biochemical analyses of proteins from duttaphrynus melanostictus (Bufo melanostictus) skin secretion
2018 - MARIANO, DOUGLAS O.C.; MESSIAS, MARCELA Di G.; PREZOTTO-NETO, JOSE P.; SPENCER, PATRICK J.; PIMENTA, DANIEL C.
A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.