Biochemical and Biophysical Research Communications
Aggresome-like structure induced by isothiocyanates is novel proteasome-dependent degradation machinery
Section snippets
Materials and methods
Antibodies. Monoclonal anti-α-tubulin (clone B-5-1-2), monoclonal anti-β-tubulin (clone D66), monoclonal anti-ubiquitin (clone 6C1), monoclonal anti-vimentin (clone V9), and monoclonal anti-β-actin (clone AC-15) antibodies were purchased from Sigma–Aldrich (St. Louis, MO). Monoclonal anti-γ-tubulin (clone TU-30) antibody, rabbit polyclonal anti-β-tubulin (H235), and Protein G conjugated agarose beads were purchased from Santa Cruz Biotechnology. Monoclonal anti-HSP90 (clone 9D2), monoclonal
ITC treatment induces aggresome-like structure formation
After BITC treatment, tubulin became punctated throughout the cytoplasm (Fig. 1A) with a brighter dot neighboring the nucleus, suggesting that tubulin had formed aggregates. In fact, the tubulin-containing aggregates remained insoluble in buffers with up to 1% of non-ionic detergents, either NP-40, Triton X-100, or Tween 20. The aggregates could be spun down at 300g (Fig. 1B).
The known aggresome components include vimentin, γ-tubulin, dynein, HSP90, HSP70, HSP27, 20S proteasome, mitochondria,
Discussion
In this study, we found that small molecular ITCs binding to tubulin may trigger the formation of protein aggregates in HeLa cells. Similar results were obtained using human non-small lung cancer A549 cells (data not shown), suggesting that the phenomenon may not be cell line-specific. Based on these results, we postulate that ITCs covalently modify specific cysteine residues in both α- and β-tubulins and other yet unidentified target proteins, causing conformational changes and misfolding.
Acknowledgments
We thank Dr. Elizabeth S. Sztul of Department of Cell Biology at University of Alabama, Dr. David Yang of Department of Chemistry at Georgetown University, Drs. Leslie Wilson and Mary Ann Jordan at University of California Santa Barbara, Dr. Saadi Khochbin at Equipe Epigénétique et Signalisation Cellulaire in France, Dr. Jacques Landry at Centre de recherche en cancérologie l’Hôtel-Dieu de Québec and Université Laval in Canada, and Dr. Changcheng Song of Laboratory of Cancer Prevention at
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