Chemical Proteomics of the Tumor Suppressor Fhit Covalently Bound to the Cofactor Ap(3)A Elucidates Its Inhibitory Action on Translation

[Image: see text] The tumor suppressor protein fragile histidine triad (Fhit) is known to be associated with genomic instability and apoptosis. The tumor-suppressive function of Fhit depends on the interaction with the alarmone diadenosine triphosphate (Ap(3)A), a noncanonical nucleotide whose concentration increases upon cellular stress. How the Fhit–Ap(3)A complex exerts its signaling function is unknown. Here, guided by a chemical proteomics approach employing a synthetic stable Fhit–Ap(3)A complex, we found that the Fhit–Ap(3)A complex, but not Fhit or Ap(3)A alone, impedes translation. Our findings provide a mechanistic model in which Fhit translocates from the nucleolus into the cytosol upon stress to form an Fhit–Ap(3)A complex. The Fhit–Ap(3)A complex impedes translation both in vitro and in vivo, resulting in reduced cell viability. Overall, our findings provide a mechanistic model by which the tumor suppressor Fhit collaborates with the alarmone Ap(3)A to regulate cellular proliferation.