Introduction and main objective: Snake toxins have arisen from recruitment of genes from various vertebrate protein families. This is illustrated by the families of P-III class snake venom metalloproteinases (SVMPs), A Disintegrin And Metalloproteinases (ADAMs) , snake venom serine proteinases (SVSPs) and the mammalian coagulation serine proteinases . Numerous experimental observations have indicated that plasma proteins, cells and tissues are affected by the venom proteinases in a complex way [3-7]. We hypothesize that under this complex phenomenon venom proteinases trigger precise and still unknown molecular cascades. To tackle the challenge of uncovering these putative molecular cascades we have adopted a systemic approach by proteomics based on mass spectrometry.
Specific aims: a) Hemorrhage induced by HF3. HF3 is an extremely hemorrhagic P-III class Bothrops jararaca SVMP. Recently we used high-throughput proteomic approaches to assess the complex in vivo effects of HF3 and our results underscored a scenario characterized by the interplay between the hydrolysis of intracellular, extracellular, and plasma proteins and the increase of plasma inhibitors in the hemorrhagic process . In this project we wish: i) to identify the alterations in the proteome of cultured fibroblasts incubated with HF3 as well as to evaluate in a high throughput fashion the substrate repertoire (degradome) of HF3 in cultured fibroblasts; ii) to analyze the effects of HF3 on a endothelial cell line via mass spectrometric approaches to evaluate the general proteome alterations with focus on the phosphorylation of cell proteins and the possible triggering of cell signaling by the proteinase; iii) to analyze the interaction of HF3 with cell surface receptors, especially integrins, by interactome approaches and Surface Plasmon Resonance; b) Platelet aggregation induced by PA-BJ. PA-BJ is a B. jararaca SVSP that triggers platelet-aggregation in a thrombin-like fashion by cleaving the GPCR receptors PAR1 and PAR4 on platelets [5,6]. We wish: i) to understand the signaling events caused by the proteolytic activation of PAR1 by PA-BJ and thrombin via mass spectrometric analysis of the proteome and secretome of platelets incubated with the proteinases; ii) to identify the proteins that undergo phosphorylation under the effects of PA-BJ and thrombin; iii) to analyze the in vivo effects of PA-BJ on platelets using the mouse as an experimental model.
Expected results. We expect to gain a competitive edge in high throughput proteomic analyses of cells and tissues, enabling us to understand complex signaling triggered by proteinases.
Moreover, the project will yield protocols and tools to elucidate proteolytic mechanisms (degradomics) upon platelets and components of the extracellular matrix. Furthermore, the expertise acquired by the group should be shared with other investigators of the Center to address the scientific and technical issues associated with their projects.
References - 1. Fox and Serrano, FEBS J. 275:3016, 2008; 2. Serrano and Maroun, Toxicon 45:1115, 2005; 3. Oliveira et al., Thromb. Haemost. 104:485, 2010; 4. Paes Leme et al., J. Proteome Res. 11:279, 2012; 5. Serrano et al., Biochemistry 34:7186, 1995; 6. Santos et al., FEBS Lett. 477:199, 2000; 7. Paes Leme et al., J. Thromb. Haemost. 6:1363-72, 2008.