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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-Tsa1 and the effect of inactivating SRX1 on the enzyme quaternary structure. Crude lysates from Δtsa1 (A) or in Δtsa1Δsrx1 (B) cells expressing Myc-Tsa1 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. (C) Representative chromatogram of standard molecular weight markers, thyroglobulin (670 kDa), apoferitin (443 kDa), β-amylase (200 kDa) to help resolve the size of the two-stacked decamers.
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f0015: SEC elution profile of Myc-Tsa1 and the effect of inactivating SRX1 on the enzyme quaternary structure. Crude lysates from Δtsa1 (A) or in Δtsa1Δsrx1 (B) cells expressing Myc-Tsa1 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. (C) Representative chromatogram of standard molecular weight markers, thyroglobulin (670 kDa), apoferitin (443 kDa), β-amylase (200 kDa) to help resolve the size of the two-stacked decamers.

Mentions: To analyze the in vivo oligomeric state of Myc-Tsa1, we performed size exclusion chromatography on N-ethylmaleimide (NEM)-treated crude cell extracts from Myc-Tsa1-expressing Δtsa1 cells that were left untreated or were exposed to H2O2 (500 μM) for 15 and 180 min. Collected fractions were resolved by SDS-PAGE in which reducing agents were omitted in order to simultaneously evaluate disulfide-linked homodimer formation. Western blots were probed with anti-Myc and anti-PrxSO2/3 antibodies (Fig. 3A). In untreated cells lysates, Myc-Tsa1 largely eluted at a size between that of a monomer (theoretical molecular weight of 21.5 kDa) and dimer (43 kDa), with traces of it equally distributed in all fractions, up to the column size exclusion (670 kDa). Myc-Tsa1 was mostly in the disulfide-linked dimer form, but these disulfides had probably formed after lysis, due to incomplete free sulfhydryls quenching, since Myc-Tsa1 from trichloroacetic acid (TCA)-precipitated lysates from the same cells largely migrated as a reduced monomer (not shown). As expected, probing membranes with the anti-PrxSO2/3 did not yield any signal. In lysates from the 15 min H2O2 exposure sample, half of Myc-Tsa1 now eluted at about the 27 kDa elution control, close to the size of monomeric enzyme (21.5 kDa), and the other half at a size of about 500 kDa, which is compatible with that of two-stacked Myc-Tsa1 decamers, also referred from now on to the high molecular weight (HMW) form. A chromatogram of standard molecular weight markers is shown in Fig. 3C. Disulfide-linked dimers were here almost totally absent, as a consequence of enzyme sulfinylation, as also shown by probing membranes with the anti-PrxSO2/3 antibody that revealed intense signals. Sulfinylation was equally distributed in fractions corresponding to the monomeric and HMW forms. Remarquably, in lysates from the 180 min H2O2 exposure sample, the Myc-Tsa1 elution pattern resembled now to that of untreated cells. Still, a strong sulfinylation signal persisted in Myc-Tsa1 monomers. We similarly analyzed the elution of Myc-Tsa1 from Δsrx1 lysates (Fig. 3B). Elution of Myc-Tsa1 from lysates of untreated cells resembled the one observed in WT untreated cells, except for a smaller proportion of monomeric enzyme and the presence of a small proportion of the enzyme in the HMW form. In lysates of the 15 min H2O2 exposure sample, elution of Myc-Tsa1 had also a wild type pattern, except for a higher abundance of the HMW species. In lysates of the 180 min sample however, the major fraction of Myc-Tsa1 now remained in the sulfinylated HMW form, in contrast to what was observed in WT cells.


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-Tsa1 and the effect of inactivating SRX1 on the enzyme quaternary structure. Crude lysates from Δtsa1 (A) or in Δtsa1Δsrx1 (B) cells expressing Myc-Tsa1 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. (C) Representative chromatogram of standard molecular weight markers, thyroglobulin (670 kDa), apoferitin (443 kDa), β-amylase (200 kDa) to help resolve the size of the two-stacked decamers.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4556779&req=5

f0015: SEC elution profile of Myc-Tsa1 and the effect of inactivating SRX1 on the enzyme quaternary structure. Crude lysates from Δtsa1 (A) or in Δtsa1Δsrx1 (B) cells expressing Myc-Tsa1 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. (C) Representative chromatogram of standard molecular weight markers, thyroglobulin (670 kDa), apoferitin (443 kDa), β-amylase (200 kDa) to help resolve the size of the two-stacked decamers.
Mentions: To analyze the in vivo oligomeric state of Myc-Tsa1, we performed size exclusion chromatography on N-ethylmaleimide (NEM)-treated crude cell extracts from Myc-Tsa1-expressing Δtsa1 cells that were left untreated or were exposed to H2O2 (500 μM) for 15 and 180 min. Collected fractions were resolved by SDS-PAGE in which reducing agents were omitted in order to simultaneously evaluate disulfide-linked homodimer formation. Western blots were probed with anti-Myc and anti-PrxSO2/3 antibodies (Fig. 3A). In untreated cells lysates, Myc-Tsa1 largely eluted at a size between that of a monomer (theoretical molecular weight of 21.5 kDa) and dimer (43 kDa), with traces of it equally distributed in all fractions, up to the column size exclusion (670 kDa). Myc-Tsa1 was mostly in the disulfide-linked dimer form, but these disulfides had probably formed after lysis, due to incomplete free sulfhydryls quenching, since Myc-Tsa1 from trichloroacetic acid (TCA)-precipitated lysates from the same cells largely migrated as a reduced monomer (not shown). As expected, probing membranes with the anti-PrxSO2/3 did not yield any signal. In lysates from the 15 min H2O2 exposure sample, half of Myc-Tsa1 now eluted at about the 27 kDa elution control, close to the size of monomeric enzyme (21.5 kDa), and the other half at a size of about 500 kDa, which is compatible with that of two-stacked Myc-Tsa1 decamers, also referred from now on to the high molecular weight (HMW) form. A chromatogram of standard molecular weight markers is shown in Fig. 3C. Disulfide-linked dimers were here almost totally absent, as a consequence of enzyme sulfinylation, as also shown by probing membranes with the anti-PrxSO2/3 antibody that revealed intense signals. Sulfinylation was equally distributed in fractions corresponding to the monomeric and HMW forms. Remarquably, in lysates from the 180 min H2O2 exposure sample, the Myc-Tsa1 elution pattern resembled now to that of untreated cells. Still, a strong sulfinylation signal persisted in Myc-Tsa1 monomers. We similarly analyzed the elution of Myc-Tsa1 from Δsrx1 lysates (Fig. 3B). Elution of Myc-Tsa1 from lysates of untreated cells resembled the one observed in WT untreated cells, except for a smaller proportion of monomeric enzyme and the presence of a small proportion of the enzyme in the HMW form. In lysates of the 15 min H2O2 exposure sample, elution of Myc-Tsa1 had also a wild type pattern, except for a higher abundance of the HMW species. In lysates of the 180 min sample however, the major fraction of Myc-Tsa1 now remained in the sulfinylated HMW form, in contrast to what was observed in WT cells.

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