Viability of TcNTH1 transfected epimastigotes submitted to oxidative stress.

<p>TcNTH1 overexpressing epimastigotes and its control (parasites transfected with empty vector) were treated for 30 min with different H₂O₂ initial concentrations (A) or with a glucose-glucose oxidase system producing sustained H₂O₂ concentrations for 24 hours (B). Viability was determined by AlamarBlue assays. *p value: 0,01</p

Expression of TcNTH1 DNA glycosylase in <i>T</i>. <i>cruzi</i> cellular forms.

<p><b>A:</b> TcNTH1 polyclonal antibodies prepared in mice specifically identifies a purified recombinant TcNTH1 (lane 1) and a TcNTH1 expressed in recombinant bacterial homogenates (lane 2). This protein was not recognized in non-expressing bacteria (lane 3). <b>B:</b> Western blot detection of TcNTH1 in total protein homogenates from epimastigotes (lane 1), trypomastigotes (lane 2) and amastigotes (lane 3). Lanes 4 and 5 correspond to TcNTH1 purified from transformed <i>E</i>. <i>coli</i> and to the same protein from recombinant over-expressing <i>T</i>. <i>cruzi</i> epimastigote homogenate, respectively. <b>C:</b> Same as B but using an anti-HIS antibody. <b>D:</b> Loading control for epimastigotes (lane 1), trypomastigotes (lane 2) and amastigotes (lane 3) using an alpha-tubulin antibody. All electrophoretic separations were performed in 12%SDS-PAGE.</p

The catalytic residues disposition and its effects on lesion recognition by TcNTH1.

<p><b>A:</b> Protein-DNA complexes of <i>G</i>. <i>stearothermophilus</i> Endonuclease III (PDB 1P59, cyan) as determined by X-ray crystallography and TcNTH1 from <i>T</i>. <i>cruzi</i> (magenta) as determined by molecular docking. Only protein backbones are shown. <b>B:</b> The same protein-DNA complexes in a different view and depicting the protein surface. Structurally divergent residues are labeled to suggest regions of interest for further analyses. The DNA-interacting region is circulated and the lesion site is shown inside the rectangle. The DNA molecule represented is from 1P59, which is in a similar position when compared to the TcNTH1-DNA complex, as shown in A.</p

Expression and the Peculiar Enzymatic Behavior of the <i>Trypanosoma cruzi</i> NTH1 DNA Glycosylase - Fig 1

<p><b>Multiple amino acid sequences alignment (A) and deduced cladogram (B) of <i>T</i>. <i>cruzi</i> NTH1 with NTH1 from <i>Escherichia coli</i>, <i>Geobacillus stearothermophilus</i>, <i>Homo sapiens</i>, <i>Schizosaccharomyces cerevisiae</i> and <i>Leishmania infantum</i>.</b> (*) are identical residues match and (“:” and “.”) are chemically similar residues. Black highlighted are the residues involved in DNA recognition and binding. Gray highlighted are the residues that generates the lesion DNA base recognition pocket. Boxes are critical residues for catalysis. Arrow-heads (▼) are [4Fe-4S]²⁺ clusters union residues. Overline indicates the helix-hairpin-helix (HhH) motif.</p

TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity.

<p><b>A, B and C:</b> A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with <i>E</i>. <i>coli</i> Endo III (bacterial NTH1, positive control, lane 2). <b>A:</b> Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. <b>B:</b> Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. <b>C:</b> Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. <b>D:</b> A [γ-³²P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with <i>E</i>. <i>coli</i> Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with <i>E</i>. <i>coli</i> Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.</p

Viability of TcNTH1 transfected epimastigotes submitted to oxidative stress.

<p>TcNTH1 overexpressing epimastigotes and its control (parasites transfected with empty vector) were treated for 30 min with different H₂O₂ initial concentrations (A) or with a glucose-glucose oxidase system producing sustained H₂O₂ concentrations for 24 hours (B). Viability was determined by AlamarBlue assays. *p value: 0,01</p