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In vivo parameters influencing 2-Cys Prx oligomerization: The role of enzyme sulfinylation.

Noichri Y, Palais G, Ruby V, D'Autreaux B, Delaunay-Moisan A, Nyström T, Molin M, Toledano MB - Redox Biol (2015)

Bottom Line: The critical molecular event allowing the peroxidase to chaperone switch is thought to be the enzyme assembly into high molecular weight (HMW) structures brought about by enzyme hyperoxidation.How hyperoxidation promotes HMW assembly is not well understood and Prx mutants allowing disentangling its peroxidase and chaperone functions are lacking.Our data confirm the strict causative link between H2O2-induced hyperoxidation and HMW formation/stabilization, also raising the question of whether CP hyperoxidation triggers the assembly of HMW structures by the stacking of decamers, which is the prevalent view of the literature, or rather, the stabilization of preassembled stacked decamers.

View Article: PubMed Central - PubMed

Affiliation: Oxidative Stress and Cancer, IBITECS, SBIGEM, CEA-Saclay, 91191 Gif-sur-Yvette, France.

No MeSH data available.


Related in: MedlinePlus

Evaluation of the sensitivity to sulfinylation of Tsa1 and derivative mutants. TCA-precipitated lysates from, Δtsa1 (A–C, G and H) or in Δtsa1Δsrx1 (E) or in Wt (D and F) yeast cells expressing Myc-Tsa1, MycTsa1C48S, Myc-Tsa1C171S, Myc-Tsa1ΔYF or overexpressing SRX1, as indicated, which were exposed to H2O2 (500 μM) (A–F), or to the indicated amount of H2O2 during 15 min (G and H) were resolved by reducing SDS PAGE, followed by western blot using the anti-Myc, anti-Tsa1 or anti-SO2/3 antibodies, as indicated. The red arrow indicates the sulfinylation signal band.
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f0010: Evaluation of the sensitivity to sulfinylation of Tsa1 and derivative mutants. TCA-precipitated lysates from, Δtsa1 (A–C, G and H) or in Δtsa1Δsrx1 (E) or in Wt (D and F) yeast cells expressing Myc-Tsa1, MycTsa1C48S, Myc-Tsa1C171S, Myc-Tsa1ΔYF or overexpressing SRX1, as indicated, which were exposed to H2O2 (500 μM) (A–F), or to the indicated amount of H2O2 during 15 min (G and H) were resolved by reducing SDS PAGE, followed by western blot using the anti-Myc, anti-Tsa1 or anti-SO2/3 antibodies, as indicated. The red arrow indicates the sulfinylation signal band.

Mentions: We first inspected how Tsa1 mutations and the modulation of SRX1 expression impacted enzyme sulfinylation by western blots with an anti-PrxSO2/3 antibody (Fig. 2). Lysates were prepared before and at different times after cell exposure to 500 μM H2O2, a concentration causing full Tsa1 sulfinylation (see Fig. 2G). Sulfinylation of Myc-Tsa1 was maximal at 15 min, started to decrease at 60 min and disappeared at 180 min (Fig. 2A). Myc-Tsa1C48S did not produce any sulfinylation signal, due to the absence of CP (see Fig. 4B), thus confirming the high specificity of the antibody towards CP sulfinylation. Myc-Tsa1C171S produced a sulfinylation signal that was 1.5–2 fold lower than Myc-Tsa1 (Fig. 2B), and required twice as much H2O2 to reach the levels of sulfinylation seen in Myc-Tsa1 (Fig. 2compare G and H). The lower sensitivity to sulfinylation of Myc-Tsa1C171S is consistent with the effect of the same mutation in A. thaliana 2-Cys Prx and human ER Prdx4 [10,23]. Myc-Τsa1ΔYF also produced a sulfinylation signal in response to H2O2, but again its intensity was strongly decreased and disappeared much faster than that of Myc-tsa1 (60 vs 180 min, compare Fig. 2A and C), thus confirming data obtained with S. pombe Tpx1. As the C-Ter domain contributes to the stability of the FF active site conformation, and hence to H2O2 reactivity, deleting it affects the sensitivity to sulfinylation by decreasing H2O2 reactivity [22]. The same rationale can be made for Myc-Tsa1C171S if we consider that the CR substitution alters the C-Ter structure. Untagged Wt Tsa1 produced a sulfinylation signal of intensity similar to Myc-Tsa1, but which surprisingly disappeared much faster than that of the tagged enzyme (60 vs 180 min, compare Fig. 2A and D). This difference in the enzyme-recycling rate might be explained by an effect of the tag of decreasing Srx enzyme binding or Srx accessibility to the sulfinylated residue. In Δsrx1, sulfinylation of untagged Tsa1 at 15 min was indistinguishable form the one seen in Wt cells, but as previously shown [6], the signal remained up to 180 min by lack of enzyme recycling (Fig. 2 compare D and E). In contrast, in cells carrying pCM190-SRX1, the sulfinylation signal was barely visible (Fig. 2 compare D and F), which indicate a much faster rate of enzyme recycling by virtue of Srx1 overexpression.


In vivo parameters influencing 2-Cys Prx oligomerization: The role of enzyme sulfinylation.

Noichri Y, Palais G, Ruby V, D'Autreaux B, Delaunay-Moisan A, Nyström T, Molin M, Toledano MB - Redox Biol (2015)

Evaluation of the sensitivity to sulfinylation of Tsa1 and derivative mutants. TCA-precipitated lysates from, Δtsa1 (A–C, G and H) or in Δtsa1Δsrx1 (E) or in Wt (D and F) yeast cells expressing Myc-Tsa1, MycTsa1C48S, Myc-Tsa1C171S, Myc-Tsa1ΔYF or overexpressing SRX1, as indicated, which were exposed to H2O2 (500 μM) (A–F), or to the indicated amount of H2O2 during 15 min (G and H) were resolved by reducing SDS PAGE, followed by western blot using the anti-Myc, anti-Tsa1 or anti-SO2/3 antibodies, as indicated. The red arrow indicates the sulfinylation signal band.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4556779&req=5

f0010: Evaluation of the sensitivity to sulfinylation of Tsa1 and derivative mutants. TCA-precipitated lysates from, Δtsa1 (A–C, G and H) or in Δtsa1Δsrx1 (E) or in Wt (D and F) yeast cells expressing Myc-Tsa1, MycTsa1C48S, Myc-Tsa1C171S, Myc-Tsa1ΔYF or overexpressing SRX1, as indicated, which were exposed to H2O2 (500 μM) (A–F), or to the indicated amount of H2O2 during 15 min (G and H) were resolved by reducing SDS PAGE, followed by western blot using the anti-Myc, anti-Tsa1 or anti-SO2/3 antibodies, as indicated. The red arrow indicates the sulfinylation signal band.
Mentions: We first inspected how Tsa1 mutations and the modulation of SRX1 expression impacted enzyme sulfinylation by western blots with an anti-PrxSO2/3 antibody (Fig. 2). Lysates were prepared before and at different times after cell exposure to 500 μM H2O2, a concentration causing full Tsa1 sulfinylation (see Fig. 2G). Sulfinylation of Myc-Tsa1 was maximal at 15 min, started to decrease at 60 min and disappeared at 180 min (Fig. 2A). Myc-Tsa1C48S did not produce any sulfinylation signal, due to the absence of CP (see Fig. 4B), thus confirming the high specificity of the antibody towards CP sulfinylation. Myc-Tsa1C171S produced a sulfinylation signal that was 1.5–2 fold lower than Myc-Tsa1 (Fig. 2B), and required twice as much H2O2 to reach the levels of sulfinylation seen in Myc-Tsa1 (Fig. 2compare G and H). The lower sensitivity to sulfinylation of Myc-Tsa1C171S is consistent with the effect of the same mutation in A. thaliana 2-Cys Prx and human ER Prdx4 [10,23]. Myc-Τsa1ΔYF also produced a sulfinylation signal in response to H2O2, but again its intensity was strongly decreased and disappeared much faster than that of Myc-tsa1 (60 vs 180 min, compare Fig. 2A and C), thus confirming data obtained with S. pombe Tpx1. As the C-Ter domain contributes to the stability of the FF active site conformation, and hence to H2O2 reactivity, deleting it affects the sensitivity to sulfinylation by decreasing H2O2 reactivity [22]. The same rationale can be made for Myc-Tsa1C171S if we consider that the CR substitution alters the C-Ter structure. Untagged Wt Tsa1 produced a sulfinylation signal of intensity similar to Myc-Tsa1, but which surprisingly disappeared much faster than that of the tagged enzyme (60 vs 180 min, compare Fig. 2A and D). This difference in the enzyme-recycling rate might be explained by an effect of the tag of decreasing Srx enzyme binding or Srx accessibility to the sulfinylated residue. In Δsrx1, sulfinylation of untagged Tsa1 at 15 min was indistinguishable form the one seen in Wt cells, but as previously shown [6], the signal remained up to 180 min by lack of enzyme recycling (Fig. 2 compare D and E). In contrast, in cells carrying pCM190-SRX1, the sulfinylation signal was barely visible (Fig. 2 compare D and F), which indicate a much faster rate of enzyme recycling by virtue of Srx1 overexpression.

Bottom Line: The critical molecular event allowing the peroxidase to chaperone switch is thought to be the enzyme assembly into high molecular weight (HMW) structures brought about by enzyme hyperoxidation.How hyperoxidation promotes HMW assembly is not well understood and Prx mutants allowing disentangling its peroxidase and chaperone functions are lacking.Our data confirm the strict causative link between H2O2-induced hyperoxidation and HMW formation/stabilization, also raising the question of whether CP hyperoxidation triggers the assembly of HMW structures by the stacking of decamers, which is the prevalent view of the literature, or rather, the stabilization of preassembled stacked decamers.

View Article: PubMed Central - PubMed

Affiliation: Oxidative Stress and Cancer, IBITECS, SBIGEM, CEA-Saclay, 91191 Gif-sur-Yvette, France.

No MeSH data available.


Related in: MedlinePlus