Fig. 1.

Inactivation of DOHH in vivo by an iron chelator and by PCBP1 or PCBP2 depletion. (A) Two-step modification of eIF5A to form Hpu. Dhp synthase (DHS) catalyzes the formation of deoxyhypusine (Dhp) on eIF5A. DOHH, a diiron enzyme, catalyzes the hydroxylation of Dhp to Hpu. (B) Progressive inactivation of DOHH after treatment with Dfo. HEK293T cells were incubated with [³H]spermidine and increasing concentrations of Dfo. Labeled Dhp and Hpu were separated by ion-exchange chromatography. (C and D) Loss of DOHH activity in cells depleted of PCBPs using siRNA. HEK293T cells were transfected with control, PCBP1, PCBP2, or PCBP1 and -2 siRNA and then treated with 20 μM Dfo and [³H]spermidine. Hpu and Dhp were measured as in B, and the percentage of Hpu in the total modified lysine (Hpu + Dhp) is shown. Assays were replicated three times; error bars represent SEM.