TY - JOUR
T1 - Clickable NAD analogues for labeling substrate proteins of poly(ADP-ribose) polymerases
AU - Jiang, Hong
AU - Kim, Jun Hyun
AU - Frizzell, Kristine M.
AU - Kraus, W. Lee
AU - Lin, Hening
PY - 2010/7/14
Y1 - 2010/7/14
N2 - 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 receptorassociated 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.
AB - 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 receptorassociated 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.
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U2 - 10.1021/ja101588r
DO - 10.1021/ja101588r
M3 - Article
C2 - 20560583
AN - SCOPUS:77955810108
SN - 0002-7863
VL - 132
SP - 9363
EP - 9372
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 27
ER -