Glutathione reductase: A cytoplasmic antioxidant enzyme and a potential target for phenothiazinium dyes in Neospora caninum

Author(s)

Venancio-Brochi, Jade Cabestre ^[1] ; Pereira, Luiz Miguel ^[1] ; Calil, Felipe Antunes ^{[2, 3]} ; Teixeira, Olivia ^[2] ; Baroni, Luciana ^[1] ; Abreu-Filho, Pericles Gama ^[1] ; Braga, Gilberto Ubida Leite ^[1] ; Nonato, Maria Cristina ^[2] ; Yatsuda, Ana Patricia ^[1]

Affiliation

^[1] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Ave Cafe, Ribeirao Preto - Brazil

^[2] Univ Sao Paulo, Fac Ciencias Farmaceut Ribeirao Preto, Lab Cristal Proteinas, BR-14040903 Ribeirao Preto - Brazil

^[3] Univ Calif La Jolla, Ludwig Inst Canc Res, Sch Med, La Jolla, CA 92093 - USA

• Document type: Journal article
• Source: International Journal of Biological Macromolecules; v. 187, p. 964-975, SEP 30 2021.
• Web of Science Citations: 0

Abstract

Neospora caninum causes heavy losses related to abortions in bovine cattle. This parasite developed a complex defense redox system, composed of enzymes as glutathione reductase (GR). Methylene blue (MB) impairs the activity of recombinant form of Plasmodium GR and inhibits the parasite proliferation in vivo and in vitro. Likewise, MB and its derivatives inhibits Neospora caninum proliferation, however, whether the MB mechanism of action is correlated to GR function remains unclear. Therefore, here, N. caninum GR (NcGR) was characterized and its potential inhibitors were determined. NcGR was found in the tachyzoite cytosol and has a similar structure and sequence compared to its homologs. We verified the in vitro activity of rNcGR (875 nM) following NADPH absorbance at 340 nM (100 mM KH2PO4, pH 7.5, 1 mM EDTA, ionic strength: 600 mM, 25 degrees C). rNcGR exhibited a Michaelian behavior (Km(GSSG):0.10 +/- 0.02 mM; kcat(GSSG):0.076 +/- 0.003 s-1; Km(NADPH):0.006 +/- 0.001 mM; kcat(NADPH): 0.080 +/- 0.003 s-1). The IC50 of MB,1,9-dimethyl methylene blue, new methylene blue, and toluidine blue O on rNcGR activity were 2.1 +/- 0.2 mu M, 11 +/- 2 mu M, 0.7 +/- 0.1 mu M, and 0.9 +/- 0.2 mu M, respectively. Our results suggest the importance of NcGR in N. caninum biology and antioxidant mechanisms. Moreover, data presented here strongly suggest that NcGR is an important target of phenothiazinium dyes in N. caninum proliferation inhibition. (AU)

• FAPESP's process: 19/05758-5 - Redox control in Neospora caninum: investigation of antioxidant system enzymes
• Grantee: Jade Cabestre Venancio Brochi
• Support Opportunities: Scholarships in Brazil - Doctorate
• FAPESP's process: 18/21020-3 - Actin-depolymerization factor (NcADF): oxidation as regulator of NcADF activity on programmed cell death of Neospora caninum
• Grantee: Ana Patricia Yatsuda Natsui
• Support Opportunities: Regular Research Grants