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- Stasyk, Taras, et al.
(författare)
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A new highly toxic protein isolated from the death cap Amanita phalloides is an L-amino acid oxidase
- 2010
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 277:5, s. 1260-1269
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Tidskriftsartikel (refereegranskat)abstract
- A new highly cytotoxic protein, toxophallin, was recently isolated from the fruit body of the death cap Amanita phalloides mushroom [Stasyk et al. (2008) Studia Biologica 2, 21-32]. The physico-chemical, chemical and biological characteristics of toxophallin differ distinctly from those of another death cap toxic protein, namely phallolysin. The interaction of toxophallin with target cells is not mediated by a specific cell surface receptor. It induces chromatin condensation, as well as DNA and nucleus fragmentation, which are typical for apoptosis. However, caspase III inhibitor [benzyloxycarbonyl-Asp(OMe)-fluoromethylketone] did not stop toxophallin-induced DNA fragmentation. Thus, toxophallin uses a caspase-independent pathway of apoptosis induction. In the present study, we applied a complementary approach based on a combination of proteomics and molecular biology tools for the protein identification of toxophallin. The primary structure of toxophallin was partially studied via direct sequencing of its tryptic peptides, followed by PCR-based cloning of the corresponding cDNA. A subsequent bioinformatic search revealed a structural homology of toxophallin with the l-amino acid oxidase of the Laccaria bicolor mushroom. This demonstrates the usefulness of our approach for the identification of proteins in organisms with unknown genomes. We also found a broad substrate specificity of toxophallin with respect to oxidizing selected amino acids. Ascorbic acid inhibited the cytotoxic effect of toxophallin, most likely as a result of scavenging hydrogen peroxide, which is the product of oxidase catalysis. Thus, in addition to highly toxic cyclopeptides and toxic lectin phallolysin, the death cap fruit body contains another cytotoxic protein in the form of an enzyme, namely l-amino acid oxidase.
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| 2. |
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| 3. |
- Stasyk, Taras, et al.
(författare)
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Phosphoproteome profiling of transforming growth factor (TGF)-beta signaling : abrogation of TGFbeta1-dependent phosphorylation of transcription factor-II-I (TFII-I) enhances cooperation of TFII-I and Smad3 in transcription
- 2005
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Ingår i: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 16:10, s. 4765-4780
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Tidskriftsartikel (refereegranskat)abstract
- Transforming growth factor-beta (TGFbeta) signaling involves activation of a number of signaling pathways, several of which are controlled by phosphorylation events. Here, we describe a phosphoproteome profiling of MCF-7 human breast epithelial cells treated with TGFbeta1. We identified 32 proteins that change their phosphorylation upon treatment with TGFbeta1; 26 of these proteins are novel targets of TGFbeta1. We show that Smad2 and Smad3 have different effects on the dynamics of TGFbeta1-induced protein phosphorylation. The identified proteins belong to nine functional groups, e.g., proteins regulating RNA processing, cytoskeletal rearrangements, and proteasomal degradation. To evaluate the proteomics findings, we explored the functional importance of TGFbeta1-dependent phosphorylation of one of the targets, i.e., transcription factor-II-I (TFII-I). We confirmed that TGFbeta1 stimulated TFII-I phosphorylation at serine residues 371 and 743. Abrogation of the phosphorylation by replacement of Ser371 and Ser743 with alanine residues resulted in enhanced complex formation between TFII-I and Smad3, and enhanced cooperation between TFII-I and Smad3 in transcriptional regulation, as evaluated by a microarray-based measurement of expression of endogenous cyclin D2, cyclin D3, and E2F2 genes, and by a luciferase reporter assay. Thus, TGFbeta1-dependent phosphorylation of TFII-I may modulate TGFbeta signaling at the transcriptional level.
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