Engineering the Substrate Specificity of ADP-Ribosyltransferases for Identifying Direct Protein Targets

Adenosine
diphosphate ribosyltransferases (ARTDs; ARTD1–17 in humans)
are emerging as critical regulators of cell function in both normal
physiology and disease. These enzymes transfer the ADP-ribose moiety
from its substrate, nicotinamide adenine dinucleotide (NAD+), to amino acids of target proteins. The functional redundancy and
overlapping target specificities among the 17 ARTDs in humans make
the identification of direct targets of individual ARTD family members
in a cellular context a formidable challenge. Here we describe the
rational design of orthogonal NAD+ analogue-engineered
ARTD pairs for the identification of direct protein targets of individual
ARTDs. Guided by initial inhibitor studies with nicotinamide analogues
containing substituents at the C-5 position, we synthesized an orthogonal
NAD+ variant and found that it is used as a substrate for
several engineered ARTDs (ARTD1, -2, and -6) but not their wild-type
counterparts. Comparing the target profiles of ARTD1 (PARP1) and ARTD2
(PARP2) in nuclear extracts highlighted the semi-complementary, yet
distinct, protein targeting. Using affinity purification followed
by tandem mass spectrometry, we identified 42 direct ARTD1 targets
and 301 direct ARTD2 targets. This represents a powerful new technique
for identifying direct protein targets of individual ARTD family members,
which will facilitate studies delineating the pathway from ARTD activation
to a given cellular response.