Early response of C2C12 myotubes to a sub-cytotoxic dose of hemorrhagic metalloproteinase HF3 from Bothrops jararaca venom

Autor(es)

Menezes, Milene C. ^[1] ; Kitano, Eduardo S. ^[1] ; Bauer, Verena C. ^[1] ; Oliveira, Ana K. ^{[1, 2]} ; Cararo-Lopes, Eduardo ^{[1, 3]} ; Nishiyama, Jr., Milton Y. ^[1] ; Zelanis, Andre ^[4] ; Serrano, Solange M. T. ^[1]

Afiliação do(s) autor(es)

^[1] Inst Butantan, Lab Especial Toxinol Aplicada, Ctr Toxins Immune Response & Cell Signaling CeTIC, Sao Paulo - Brazil

^[2] Brazilian Ctr Res Energy & Mat CNPEM, Brazilian Biosci Natl Lab LNBio, Sao Paulo - Brazil

^[3] Rutgers Canc Inst New Jersey, New Brunswick, NJ - USA

^[4] Fed Univ Sao Paulo ICT UNIFESP, Dept Sci & Technol, Sao Jose Dos Campos, SP - Brazil

• Tipo de documento: Artigo Científico
• Fonte: JOURNAL OF PROTEOMICS; v. 198, p. 163-176, APR 30 2019.
• Citações Web of Science: 0

Resumo

Manifestations of local tissue damage, such as hemorrhage and myonecrosis, are among the most dramatic effects of envenomation by viperid snakes. Snake venom metalloproteinases (SVMPs) of the P-III class are main players of the hemorrhagic effect due to their activities in promoting blood vessel disruption. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca, shows a minimum hemorrhagic dose of 240 fmol on rabbit skin. The aim of this study was to assess the effects of a sub-cytotoxic dose of HF3 (50 nM) on the proteomic profile of C2C12 differentiated cells (myotubes) in culture, and on the peptidomic profile of the culture supernatant. Quantitative proteomic analysis using stable-isotope dimethyl labeling showed differential abundance of various proteins including enzymes involved in oxidative stress and inflammation responses. Identification of peptides in the supernatant of HF3-treated myotubes revealed proteolysis and pointed out potential new substrates of HF3, including glyceraldehyde-3-phosphate dehydrogenase, and some damage-associated molecular patterns (DAMPs). These experiments demonstrate the subtle effects of HF3 on muscle cells and illustrate for the first time the early proteolytic events triggered by HF3 on myotubes. Moreover, they may contribute to future studies aimed at explaining the inflammation process, hemorrhage and myonecrosis caused by SVMPs. Significance: One of the main features of viperid snake envenomation is myotoxicity at the bite site, which, in turn is often associated with edema, blistering and hemorrhage, composing a complex pattern of local tissue damage. In this scenario, besides muscle cells, other types of cells, components of the extracellular matrix and blood vessels may also be affected, resulting in an outcome of deficient muscle regeneration. The main venom components participating in this pathology are metalloproteinases and phospholipases A2. Muscle necrosis induced by metalloproteinases is considered as an indirect effect related to ischemia, due to hemorrhage resulted from damage to the microvasculature. The pathogenesis of local effects induced by Bothrops venoms or isolated toxins has been studied by traditional methodologies. More recently, proteomic and peptidomic approaches have been used to study venom-induced pathogenesis. Here, in order to investigate the role of metalloproteinase activity in local tissue damage, we asked whether the hemorrhagic metalloproteinase HF3, at sub-cytotoxic levels, could alter the proteome of C2C12 myotubes in culture, thereby providing an insight into the mechanisms for the development of myonecrosis. Our results from mass spectrometric analyses showed subtle, early changes in the cells, including differential abundance of some proteins and proteolysis in the culture supernatant. The data illustrate the potential ability of metalloproteinases to trigger early systemic responses progressing from local cells and up to tissues. (AU)

• Processo FAPESP: 13/07467-1 - CeTICS - Centro de Toxinas, Imuno-Resposta e Sinalização Celular
• Beneficiário: Hugo Aguirre Armelin
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 15/23691-4 - Ativ pró-inflamatória de proteinases de venenos de serpentes: i) obtenção de proteinases recombinantes em sistema livre de CEL; ii) geração de microvesículas por monócitos estimulados com proteinases nativas e recomb e sua interação com células endoteliais
• Beneficiário: Milene Cristina Menezes dos Santos
• Modalidade de apoio: Bolsas no Brasil - Pós-Doutorado
• Processo FAPESP: 11/11308-0 - Estudo do proteoma de folhas de cultivares e genótipos de cana-de-açúcar
• Beneficiário: Eduardo Shigueo Kitano
• Modalidade de apoio: Bolsas no Brasil - Doutorado Direto