We have analyzed the proteome of MCF-7 cells exposed to palytoxin (PlTX), to characterize protein components involved in the death response induced by the toxin. The protein profiles of cell lysates were obtained by 2D-electrophoresis, and we found that four components were increased by PlTX treatment. By tryptic digestion of protein spots in the gels and LC-ESI-MS/MS analysis of resulting peptides, those four components were found to include three isoforms of the heat shock protein hsp 27 differing with regard to their phosphrylation state, as well as DJ-1/PARK7. The effects exerted by PlTX on hsp 27 and DJ-1 proteins was further quantified by immunoblotting analyses of proteins separated by mono-dimensional SDS-PAGE and using antibodies recognizing total hsp 27, the hsp 27 forms phosphorylated in Ser82, as well as DJ-1 protein. Dose-response and time-course experiments yielded results that only partially confirmed those found by protein staining after 2D-electrophoresis. These findings were further checked by immunoblotting of proteins after fractionation by 2D-electrophoresis, and we found that only some forms of those co-migrating in a single band upon mono-dimensional SDS-PAGE were actually increased in extracts from PlTX-treated cells. We obtained evidence that the three hsp 27 isoforms whose relative abundance was increased in MCF-7 cells exposed to PlTX comprised two proteins phosphorylated in Ser82, whereas the third form most likely contains a phosphorylated amino-acid residue other than Ser82. Moreover, we could show that PlTX treatment determined the accumulation of an oxidized isoform of DJ-1 in MCF-7 cells. We conclude that the toxicity pathway of PlTX in MCF-7 cells involves post-translational modifications of hsp 27 and DJ-1 stress response proteins, comprising a shift in the equilibria among hsp 27 isoforms toward those phosphorylated in Ser82, as well as the oxidation of DJ-1.

The cytotoxicity pathway triggered by palytoxin involves a change in the cellular pool of stress response proteins / Sala, GIAN LUCA; Bellocci, Mirella; Rossini, Gian Paolo. - In: CHEMICAL RESEARCH IN TOXICOLOGY. - ISSN 0893-228X. - STAMPA. - 22:(2009), pp. 2009-2016. [10.1021/tx900297g]

The cytotoxicity pathway triggered by palytoxin involves a change in the cellular pool of stress response proteins

SALA, GIAN LUCA;BELLOCCI, MIRELLA;ROSSINI, Gian Paolo
2009-01-01

Abstract

We have analyzed the proteome of MCF-7 cells exposed to palytoxin (PlTX), to characterize protein components involved in the death response induced by the toxin. The protein profiles of cell lysates were obtained by 2D-electrophoresis, and we found that four components were increased by PlTX treatment. By tryptic digestion of protein spots in the gels and LC-ESI-MS/MS analysis of resulting peptides, those four components were found to include three isoforms of the heat shock protein hsp 27 differing with regard to their phosphrylation state, as well as DJ-1/PARK7. The effects exerted by PlTX on hsp 27 and DJ-1 proteins was further quantified by immunoblotting analyses of proteins separated by mono-dimensional SDS-PAGE and using antibodies recognizing total hsp 27, the hsp 27 forms phosphorylated in Ser82, as well as DJ-1 protein. Dose-response and time-course experiments yielded results that only partially confirmed those found by protein staining after 2D-electrophoresis. These findings were further checked by immunoblotting of proteins after fractionation by 2D-electrophoresis, and we found that only some forms of those co-migrating in a single band upon mono-dimensional SDS-PAGE were actually increased in extracts from PlTX-treated cells. We obtained evidence that the three hsp 27 isoforms whose relative abundance was increased in MCF-7 cells exposed to PlTX comprised two proteins phosphorylated in Ser82, whereas the third form most likely contains a phosphorylated amino-acid residue other than Ser82. Moreover, we could show that PlTX treatment determined the accumulation of an oxidized isoform of DJ-1 in MCF-7 cells. We conclude that the toxicity pathway of PlTX in MCF-7 cells involves post-translational modifications of hsp 27 and DJ-1 stress response proteins, comprising a shift in the equilibria among hsp 27 isoforms toward those phosphorylated in Ser82, as well as the oxidation of DJ-1.
22
2009
2016
The cytotoxicity pathway triggered by palytoxin involves a change in the cellular pool of stress response proteins / Sala, GIAN LUCA; Bellocci, Mirella; Rossini, Gian Paolo. - In: CHEMICAL RESEARCH IN TOXICOLOGY. - ISSN 0893-228X. - STAMPA. - 22:(2009), pp. 2009-2016. [10.1021/tx900297g]
Sala, GIAN LUCA; Bellocci, Mirella; Rossini, Gian Paolo
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11380/637578
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