RESUMO
Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.
Assuntos
Cisteína Endopeptidases/genética , Proteína SUMO-1/genética , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/toxicidade , Plaquetas/efeitos dos fármacos , Bothrops , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/toxicidade , Cisteína Endopeptidases/metabolismo , Desintegrinas/genética , Desintegrinas/toxicidade , Escherichia coli/genética , Humanos , Fosfolipase D/genética , Fosfolipase D/toxicidade , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/toxicidade , Proteínas Recombinantes de Fusão/toxicidade , Proteína SUMO-1/metabolismo , Venenos de Aranha , AranhasRESUMO
Cell migration is a key process for the defense of pluricellular organisms against pathogens, and it involves a set of surface receptors acting in an ordered fashion to contribute directionality to the movement. Among these receptors are the integrins, which connect the cell cytoskeleton to the extracellular matrix components, thus playing a central role in cell migration. Integrin clustering at focal adhesions drives actin polymerization along the cell leading edge, resulting in polarity of cell movement. Therefore, small integrin-binding proteins such as the snake venom disintegrins that inhibit integrin-mediated cell adhesion are expected to inhibit cell migration. Here we review the current knowledge on disintegrin and disintegrin-like protein effects on cell migration and their potential use as pharmacological tools in anti-inflammatory therapy as well as in inhibition of metastatic invasion.
Assuntos
Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Desintegrinas/toxicidade , Neutrófilos/efeitos dos fármacos , Venenos de Serpentes/química , Animais , Anoikis/efeitos dos fármacos , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Desintegrinas/antagonistas & inibidores , Humanos , Metástase Neoplásica/tratamento farmacológico , Neovascularização Fisiológica/efeitos dos fármacos , Venenos de Serpentes/farmacologiaRESUMO
The isolation and study of individual snake venom components paves the way for a deeper understanding of the pathophysiology of envenomings--thus potentially contributing to improved therapeutic modalities in the clinical setting--and also opens possibilities for the discovery of novel toxins that might be useful as tools for dissecting cellular and molecular processes of biomedical importance. This review provides a summary of the different toxins that have been isolated and characterized from the venom of Bothrops asper, the snake species causing the majority of human envenomings in Central America. This venom contains proteins belonging to at least eight families: metalloproteinase, serine proteinase, C-type lectin-like, L-amino acid oxidase, disintegrin, DC-fragment, cystein-rich secretory protein, and phospholipase A(2). Some 25 venom proteins within these families have been isolated and characterized. Their main biochemical properties and toxic actions are described, including, in some cases, their possible relationships to the pathologic effects induced by B. asper venom.
Assuntos
Bothrops , Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/toxicidade , Animais , Venenos de Crotalídeos/enzimologia , Desintegrinas/química , Desintegrinas/metabolismo , Desintegrinas/toxicidade , L-Aminoácido Oxidase/química , L-Aminoácido Oxidase/metabolismo , L-Aminoácido Oxidase/toxicidade , Lectinas Tipo C/química , Lectinas Tipo C/metabolismo , Metaloproteases/química , Metaloproteases/metabolismo , Metaloproteases/toxicidade , Modelos Moleculares , Fosfolipases A2/química , Fosfolipases A2/metabolismo , Fosfolipases A2/toxicidade , Serina Proteases/química , Serina Proteases/metabolismo , Serina Proteases/toxicidadeRESUMO
Viperine and crotaline snake venoms contain one or more hemorrhagic metalloproteases called hemorrhagins. The most potent hemorrhagins belong to the P-III class and have, in addition to the protease domain, disintegrin-like and cysteine-rich domains. Although proteolytic degradation of vascular endothelium basement membrane has been established to be the main factor responsible for hemorrhage, several studies reveal other factors that actually do facilitate this process. Recent evidence has shown that the nonprotease domains of the P-III class hemorrhagins are able to inhibit the platelet aggregation by blocking essential procoagulant integrins on platelets. In this study we report the identification of a hemorrhagin from Bothrops atrox venom. This enzyme, a P-III class metalloprotease, undergoes an apparent spontaneous degradation, releasing a proteic fragment containing the disintegrin-like/cysteine-rich domains. This fragment shows the capability to induce an edematogenic process, suggesting the existence of a still unknown nonenzymatic mechanism of vascular permeability increase.