Clickable NAD analogues for labeling substrate proteins of poly(ADP-ribose) polymerases.
Poly(ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple adenine diphosphate ribose (ADP-ribose) units from nicotinamide adenine dinucleotide (NAD) to substrate proteins. There are 17 PARPs in humans. Several PARPs, such as PARP-1 and Tankyrase-1, are known to play important roles in DNA repair, transcription, mitosis, and telomere length maintenance. To better understand the functions of PARPs at a molecular level, it is necessary to know what substrate proteins PARPs modify. Here we report clickable NAD analogues that can be used to label PARP substrate proteins. The clickable NAD analogues have a terminal alkyne group which allows the conjugation of fluorescent or affinity tags to the substrate proteins. Using this method, PARP-1 and tankyrase-1 substrate proteins were labeled by a fluorescent tag and visualized on SDS-PAGE gel. Using a biotin affinity tag, we were able to isolate and identify a total of 79 proteins as potential PARP-1 substrates. These include known PARP-1 substrate proteins, including histones and heterogeneous nuclear ribonucleoproteins. About 40% of the proteins were also identified in recent proteomic studies as potential PARP-1 substrates. Among the identified potential substrates, we further demonstrated that tubulin and three mitochondrial proteins, TRAP1 (TNF receptor-associated protein 1), citrate synthase, and GDH (glutamate dehydrogenase 1), are substrates of PARP-1 in vitro. These results demonstrate that the clickable NAD analogue is useful for labeling, in-gel detection, isolation, and identification of the substrate proteins of PARPs and will help to understand the biological functions of PARPs.
Jiang
Hong
H
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14850, USA.
Kim
Jun Hyun
JH
Frizzell
Kristine M
KM
Kraus
W Lee
WL
Lin
Hening
H
D019921:Biotinylation; D005456:Fluorescent Dyes; D006801:Humans; D009243:NAD / Q000031:analogs & derivatives* / Q000737:chemistry; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry*; D013379:Substrate Specificity; D038501:Tankyrases
D005456:Fluorescent Dyes; D009243:NAD; D011065:Poly(ADP-ribose) Polymerases; D011506:Proteins; D038501:Tankyrases; C418109:TNKS protein, human