Picking Out Protein Targets

Humans have 17 known adenosine diphosphate (ADP)-ribosyltransferases. These enzymes help regulate many cell functions by modifying target proteins by transferring ADP-ribose from their substrate, nicotinamide adenine dinucleotide (NAD⁺). The 17 enzymes have the same catalytic domain, and many of them share targets. These factors make it difficult to determine the targets of the various enzymes. Michael S. Cohen and coworkers at Oregon Health & Science University have developed a “bump-hole” strategy to identify those targets (J. Am. Chem. Soc. 2014, DOI: 10.1021/ja412897a). They create a hole by changing a single amino acid in an enzyme’s nicotinamide-binding site and create a bump by modifying the NAD⁺ substrate with an alkyl substituent in one position and with an alkyne tag in another. The mutated enzyme is the only ADP-ribosyltransferase that can accept the modified substrate (example shown). After the transfer reaction, the researchers use a biotin-azide probe to pull out the target proteins, which they identify with mass spectrometry. They used the strategy to identify the protein targets of two human ADP-ribosyltransferases in cell lysates.