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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

SEC elution profile of Myc-Tsa1ΔYF and the effect of overexpressing SRX1on Tsa1 quaternary structure. Crude lysates from Δtsa1 cells expressing Myc-Tsa1ΔYF (A) or Wt overexpressing SRX1 (B) were taken before and after exposure to H2O2 (500 μM) for the indicated time and resolved by SEC. Elution fractions were resolved by non-reducing SDS PAGE, followed by western blot using the anti-Myc or anti-SO2/3 antibody as indicated. The elution fraction of standard molecular weight markers is represented at the top of the gel. The red star indicates non-specific signals revealed by the anti-SO2/3 antibody.
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f0030: SEC elution profile of Myc-Tsa1ΔYF and the effect of overexpressing SRX1on Tsa1 quaternary structure. Crude lysates from Δtsa1 cells expressing Myc-Tsa1ΔYF (A) or Wt overexpressing SRX1 (B) were taken before and after exposure to H2O2 (500 μM) for the indicated time and resolved by SEC. Elution fractions were resolved by non-reducing SDS PAGE, followed by western blot using the anti-Myc or anti-SO2/3 antibody as indicated. The elution fraction of standard molecular weight markers is represented at the top of the gel. The red star indicates non-specific signals revealed by the anti-SO2/3 antibody.

Mentions: We next evaluated Myc-Tsa1ΔYF and the effect of overexpressing SRX1 on the untagged enzyme, both conditions abating enzyme sulfinylation. Myc-Tsa1ΔYF had a unique elution pattern (Fig. 6A). Prior to H2O2 exposure, a large proportion of the protein eluted at the size of the two-stacked decamers, and about a quarter of it in the disulfide-linked dimer form. Then, 15 min after H2O2 exposure, Myc-Tsa1ΔYF was almost totally shifted to the monomer-dimer size, with about more than half of the protein in the disulfide-linked dimer form. At 180 min, the protein had entirely returned to the double decamer form. Elution of untagged Tsa1 from lysates of cells overexpressing SRX1 was very similar to that of Myc-Tsa1ΔYF since prior to H2O2 exposure, a major part of it also eluted in the HMW form, but then was almost completely switched to the monomer-dimer form after 15 min of H2O2 exposure to completely return to the HMW form after 180 min (Fig. 6B). Note that in lysates of SRX1-overexpressing cells exposed for 15 min to H2O2 untagged Tsa1 eluted exclusively as a disulfide-linked dimer, whereas in Wt cells it was about equally distributed between a disulfide-linked dimer and a monomer. As expected from the results of Fig. 2, for both Myc-Tsa1ΔYF and SRX1 overexpression no sulfinylation signal was seen.


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)

SEC elution profile of Myc-Tsa1ΔYF and the effect of overexpressing SRX1on Tsa1 quaternary structure. Crude lysates from Δtsa1 cells expressing Myc-Tsa1ΔYF (A) or Wt overexpressing SRX1 (B) were taken before and after exposure to H2O2 (500 μM) for the indicated time and resolved by SEC. Elution fractions were resolved by non-reducing SDS PAGE, followed by western blot using the anti-Myc or anti-SO2/3 antibody as indicated. The elution fraction of standard molecular weight markers is represented at the top of the gel. The red star indicates non-specific signals revealed by the anti-SO2/3 antibody.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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

f0030: SEC elution profile of Myc-Tsa1ΔYF and the effect of overexpressing SRX1on Tsa1 quaternary structure. Crude lysates from Δtsa1 cells expressing Myc-Tsa1ΔYF (A) or Wt overexpressing SRX1 (B) were taken before and after exposure to H2O2 (500 μM) for the indicated time and resolved by SEC. Elution fractions were resolved by non-reducing SDS PAGE, followed by western blot using the anti-Myc or anti-SO2/3 antibody as indicated. The elution fraction of standard molecular weight markers is represented at the top of the gel. The red star indicates non-specific signals revealed by the anti-SO2/3 antibody.
Mentions: We next evaluated Myc-Tsa1ΔYF and the effect of overexpressing SRX1 on the untagged enzyme, both conditions abating enzyme sulfinylation. Myc-Tsa1ΔYF had a unique elution pattern (Fig. 6A). Prior to H2O2 exposure, a large proportion of the protein eluted at the size of the two-stacked decamers, and about a quarter of it in the disulfide-linked dimer form. Then, 15 min after H2O2 exposure, Myc-Tsa1ΔYF was almost totally shifted to the monomer-dimer size, with about more than half of the protein in the disulfide-linked dimer form. At 180 min, the protein had entirely returned to the double decamer form. Elution of untagged Tsa1 from lysates of cells overexpressing SRX1 was very similar to that of Myc-Tsa1ΔYF since prior to H2O2 exposure, a major part of it also eluted in the HMW form, but then was almost completely switched to the monomer-dimer form after 15 min of H2O2 exposure to completely return to the HMW form after 180 min (Fig. 6B). Note that in lysates of SRX1-overexpressing cells exposed for 15 min to H2O2 untagged Tsa1 eluted exclusively as a disulfide-linked dimer, whereas in Wt cells it was about equally distributed between a disulfide-linked dimer and a monomer. As expected from the results of Fig. 2, for both Myc-Tsa1ΔYF and SRX1 overexpression no sulfinylation signal was seen.

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