Ornithine decarboxylase (ODC) catalyzes the rate-limiting step in polyamine biosynthesis and is one of the shortest-lived mammalian proteins. Its activity and proteasomal degradation are controlled by antizyme (AZ), which disrupts the active ODC homodimer and exposes proteasome-interacting surfaces. Disturbance of the polyamine biosynthesis pathway and their overproduction is associated with multiple diseases, including cancers. We employed activity assays and direct interaction analysis methods to quantify ODC-AZ interaction. Fluorometric activity assay, surface plasmon resonance, microscale thermophoresis and spectral shift assays allowed consistent determination of AZ-ODC binding with previously unavailable ... More
Ornithine decarboxylase (ODC) catalyzes the rate-limiting step in polyamine biosynthesis and is one of the shortest-lived mammalian proteins. Its activity and proteasomal degradation are controlled by antizyme (AZ), which disrupts the active ODC homodimer and exposes proteasome-interacting surfaces. Disturbance of the polyamine biosynthesis pathway and their overproduction is associated with multiple diseases, including cancers. We employed activity assays and direct interaction analysis methods to quantify ODC-AZ interaction. Fluorometric activity assay, surface plasmon resonance, microscale thermophoresis and spectral shift assays allowed consistent determination of AZ-ODC binding with previously unavailable sensitivity. Contrary to former studies of this interaction, we show that binding of AZ to ODC occurs with a single-digit nanomolar affinity. Collectively, our orthogonal assays converge on a low-nanomolar interaction (apparent KD 1-4 nM in solution), affinity substantially stronger than previous estimates in the 200-700 nM range. Our results provide new insight into the functioning of the ODC regulatory network, which affects the downstream polyamine synthesis pathway. Such sensitive tools are needed for screening compound libraries and characterizing promising candidates that could affect ODC activity and consequently, polyamine levels.
