Photoaffinity labeling and mass spectrometry identify ribosomal protein S3as a potential target for hybrid polar cytodifferentiation agents

The ability of a novel class of hybrid polar compounds (HPCs) to induce dif ferentiation and consequent cessation of proliferation of transformed cells has led to their development as potential chemotherapeutic agents in the t reatment of cancer. Suberoylanilide hydroxamic acid (SAHA) is a prototype o f a family of hydroxamic acid based compounds (SAHA-like HPCs) that can, at micromolar concentrations, induce a variety of transformed cell lines to d ifferentiate. The mechanism of action of the HPCs is not entirely understoo d. Searching for a cellular target of the SAHA-like HPCs, we synthesized a photoaffinity labeling reagent structurally based on SAHA, and probed for S AHA-binding proteins in murine erythroleukemia (MEL) cells. Photoaffinity l abeling in cell free extracts identified a 32-kDa protein (p32) that was sp ecifically labeled by the photoaffinity reagent. Cell fractionation assays localized p32 to the P100 fraction, p32 was partially purified and identifi ed by mass spectrometry as the 40 S ribosomal protein S3. Expression of epi tope-tagged S3 in bacterial lysates followed by photoaffinity labeling conf irmed its specific labeling. Identification of a cytodifferentiation agent target may shed light on the mechanism by which the SAHA-like HPCs exert th eir antitumor effects.