Effect of ajoene, a natural antitumor small molecule, on human 20S proteasome activity in vitro and in human leukemic HL60 cells.

The pharmacologic properties of ajoene, the major sulfur-containing compound purified from garlic, and its possible role in the prevention and treatment of cancer has received increasing attention. Several studies demonstrated that induction of apoptosis and cell cycle blockade are typical biologic effects observed in tumor cells after proteasome inhibition. The proteasome is responsible for the degradation of a variety of intracellular proteins and plays a key role in the regulation of many cellular processes. The aim of the present work was therefore to explore the effects of ajoene on the proteasome activities. In vitro activities of 20S proteasome purified from human erythrocytes on fluorogenic peptide substrates specific for trypsin-like, chymotrypsin-like and peptidylglutamyl peptide hydrolyzing activities revealed that ajoene inhibited the trypsin-like activity in a dose- and time-dependent manner. Further, the ability of 20S proteasome to degrade the OVA(51-71) peptide, a model proteasomal substrate, was partially but significantly inhibited by ajoene. In addition, when human leukemia cell line HL60 was treated with ajoene, both trypsin- and chymotrypsin-like activities were affected, cells arrested in G2/M phase and total amount of cytosolic proteasome increased. All these data clearly indicate that ajoene may affect proteasome function and activity both in vitro and in the living cell. This is a novel aspect in the biologic profile of this garlic compound giving new insights into the understanding of the molecular mechanisms of its potential antitumor action.

Proteomics of loosely bound cell wall proteins of Arabidopsis thaliana cell suspension cultures: a critical analysis.

The complete sequencing of the Arabidopsis thaliana genome allows the use of the recently developed mass spectrometry techniques to identify the cell wall proteins (CWPs). Most proteomic approaches depend on the quality of sample preparation. Extraction of CWPs is particularly complex since the proteins may be free in the apoplast or are embedded in a polysaccharide matrix where they are retained by Van der Waals interactions, hydrogen bonds, hydrophobic or ionic interactions, or cross-linked by covalent bonds. Specific and sequential extraction procedures thus need to be developed. We report on the sequential extraction of loosely bound CWPs from living A. thaliana cells in culture. Different salts and chelating agents were used for releasing the proteins from the wall. Their effects on the extraction of CWPs and on the integrity of the plasma membrane were evaluated. Bioinformatic software was used to identify proteins and to predict their sub-cellular localization. The obtained data show that the plasma membrane of cells in culture was easily damaged by some steps of the extraction procedure, leading to the release of increasing amounts of intracellular proteins. Nevertheless, we identified fifty CWPs among which thirteen were new proteins for the cell wall. In addition, 76% of these CWPs were basic proteins not resolved in two-dimensional (2-D) gel electrophoresis. The existence of two hypothetical proteins was confirmed. The structure of three proteins could be confirmed using mass spectrometry data.