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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 untagged Tsa1. Crude lysates from Wt cells 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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f0025: SEC elution profile of untagged Tsa1. Crude lysates from Wt cells 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: The presence of an N-terminal tag has previously been shown to modify the function of 2-Cys Prxs [8]. That the Myc tag also alters the function of Tsa1 was already suggested by the inability of Myc-Tsa1 to fully rescue the H2O2 tolerance of a Δtsa1 strain (unpublished data), and by the slower recycling of the sulfinylated enzyme (see Fig. 1). We thus assayed the elution profile of untagged Tsa1 (Fig. 5). Untagged Tsa1 elution pattern was quite different from that of the Myc-tagged enzyme, since more than half of it was in the HMW form (70%), and the remainder in the monomer-dimer form. At 15 min after H2O2 exposure, the HMW form switched to the monomer-dimer form, which resulted in equal amounts of enzymes in these two forms; at 180 min, the elution pattern had returned to that of untreated cells, but with a slightly higher amount of protein, as a probable result of gene induction. Traces of the sulfinylation signal were seen at 15 min.


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 untagged Tsa1. Crude lysates from Wt cells 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

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

f0025: SEC elution profile of untagged Tsa1. Crude lysates from Wt cells 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: The presence of an N-terminal tag has previously been shown to modify the function of 2-Cys Prxs [8]. That the Myc tag also alters the function of Tsa1 was already suggested by the inability of Myc-Tsa1 to fully rescue the H2O2 tolerance of a Δtsa1 strain (unpublished data), and by the slower recycling of the sulfinylated enzyme (see Fig. 1). We thus assayed the elution profile of untagged Tsa1 (Fig. 5). Untagged Tsa1 elution pattern was quite different from that of the Myc-tagged enzyme, since more than half of it was in the HMW form (70%), and the remainder in the monomer-dimer form. At 15 min after H2O2 exposure, the HMW form switched to the monomer-dimer form, which resulted in equal amounts of enzymes in these two forms; at 180 min, the elution pattern had returned to that of untreated cells, but with a slightly higher amount of protein, as a probable result of gene induction. Traces of the sulfinylation signal were seen at 15 min.

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