Recombinant actin-depolymerizing factor of the apicomplexan Neospora caninum (NcADF) is susceptible to oxidation

Abstract

Neospora caninum is a member of Apicomplexa Phylum and the causative agent of neosporosis, a disease responsible for abortions in cattle. Apicomplexan parasites have a limited set of actin-binding proteins conducting the regulation of the dynamics of nonconventional actin. The parasite actin-based motility is implicated in the parasite invasion process in the host cell. Once no commercial strategy for the neosporosis control is available, the interference in the parasite actin function may result in novel drug targets. Actin-depolymerization factor (ADF) is a member of the ADF/cofilin family, primarily known for its function in actin severing and depolymerization. ADF/cofilins are versatile proteins modulated by different mechanisms, including reduction and oxidation. In apicomplexan parasites, the mechanisms involved in the modulation of ADF function are barely explored and the effects of oxidation in the protein are unknown so far. In this study, we used the oxidants N-chlorotaurine (NCT) and H₂O₂ to investigate the susceptibility of the recombinant N. caninum ADF (NcADF) to oxidation. After exposing the protein to either NCT or H₂O₂, the dimerization status and cysteine residue oxidation were determined. Also, the interference of NcADF oxidation in the interaction with actin was assessed. The treatment of the recombinant protein with oxidants reversibly induced the production of dimers, indicating that disulfide bonds between NcADF cysteine residues were formed. In addition, the exposure of NcADF to NCT resulted in more efficient oxidation of the cysteine residues compared to H₂O₂. Finally, the oxidation of NcADF by NCT reduced the ability of actin-binding and altered the function of NcADF in actin polymerization. Altogether, our results clearly show that recombinant NcADF is sensitive to redox conditions, indicating that the function of this protein in cellular processes involving actin dynamics may be modulated by oxidation.

Keywords: Apicomplexa; N-chlorotaurine; Neospora caninum; actin-binding protein; actin-depolymerizing factor (ADF); redox; taurine chloramine.

Figure 1

Dimerization of oxidized recombinant NcADF. NcADF produced and stored without reductant was incubated with oxidants. (A) NcADF was incubated with 0-150 mM of H₂O₂ for 30 min. (B) NcADF was incubated with 2 mM H₂O₂ and NcADF was detected by western blotting using NcADF antiserum (1:15,000) and anti-mouse secondary antibody conjugated to HRP (1:10,000). (C) NcADF was incubated with 0-50 mM NCT for 30 min. (D) NcADF was incubated with 10 mM NCT, followed by NCT inactivation with sodium thiosulfate (ST) and reduction with TCEP. 15% SDS-PAGE stained with Coomassie G-250 (A, C, D).

Figure 2

Oxidation of NcADF cysteine residues. Recombinant NcADF was reduced with TCEP. After removing TCEP, the protein was oxidized with NCT or H₂O₂. (A) NcADF was incubated with NCT or H₂O₂ and the cysteine residues´ oxidation was detected by three independent DTNB assays performed in duplicate. The bars represent the mean ± SD. The *** indicates p ≤ 0.0001 and the * indicates p ≤ 0.05 in Tukey’s test between the treatments. (B, C) NcADF was incubated with 0-10 mM H₂O₂ (B) or NCT and 25 µM NcADF in 20 µl non-reductant sample buffer was loaded in the gel (C). The oxidants were inactivated with catalase or histidine/methionine, respectively, and alkylated with 0,5 µM bio-IAM. The protein was run in 15% SDS-PAGE and transferred to PVDF membranes. The alkylation was detected by streptavidin-HRP (1:20,000). For protein level control, the PVDF membranes were stained with Direct Blue 71.

Figure 3

Binding and dynamic of oxidized NcADF and actin. Previously reduced NcADF was incubated with H₂O₂ or NCT and the binding with actin was assessed. (A, B) NcADF (16 µM) was treated with 0-2 mM H₂O₂ (A) or NCT (B). After inactivation with catalase (A) or histidine/methionine (B), NcADF was incubated with 16 µM G-actin for 18 h EDC (2 mM) was added to the reaction, which was resolved by 15% SDS-PAGE and stained with Coomassie G-250. (C) G-actin (2.5 µg/ml) was immobilized and incubated with 0-20 µg/ml NcADF oxidized with either 2 mM NCT or H₂O₂. NcADF was detected with NcADF antiserum (1:1,000) and anti-mouse secondary antibody-HRP (1:10,000). Reduced NcADF EC50 (red circles) = 27.6 nM; NCT treated NcADF EC50 (blue squares) = 192.4 nM; H₂O₂ treated NcADF (green triangles) = 29.5 nM. Results from two (2) experimental replicates. (D) PI-G-actin (15% PI-actin; 10 µM) was mixed with 10 µM NcADF previously treated with 0-2 mM NCT. The actin polymerization was induced and the fluorescence was measured over time. Grey line = G-actin control without NcADF; green line = actin polymerization with reduced NcADF; red line = actin polymerization without NcADF; purple line = actin polymerization with NcADF (oxidized with 0.4 mM NCT); blue line = actin polymerization with NcADF (oxidized with 2 mM NCT).

Figure 4

Comparison of cysteines’ position between NcADF and human cofilin 1. (A) Alignment of human cofilin 1 (HsCof1) and NcADF primary sequences. The HsCof1 cysteines (red rectangles) and methionines (pink asterisks), as well as NcADF cysteines (green rectangles) and methionines (blue asterisks) are highlighted. (B) The cartoon representations of NcADF (Baroni et al., 2018) and HsCofilin1 (PDB: 1q8x) tertiary structures were aligned. The two structures are shown independently on the right. The cysteines are represented as green spheres (NcADF) or red spheres (HsCofilin1) and their relative positions are described in the image.