Characterization of TCDD-inducible poly-ADP-ribose polymerase (TIPARP/ARTD14) catalytic activity

Abstract

Here, we report the biochemical characterization of the mono-ADP-ribosyltransferase 2,3,7,8-tetrachlorodibenzo-p-dioxin poly-ADP-ribose polymerase (TIPARP/ARTD14/PARP7), which is known to repress aryl hydrocarbon receptor (AHR)-dependent transcription. We found that the nuclear localization of TIPARP was dependent on a short N-terminal sequence and its zinc finger domain. Deletion and in vitro ADP-ribosylation studies identified amino acids 400–657 as the minimum catalytically active region, which retained its ability to mono-ADP-ribosylate AHR. However, the ability of TIPARP to ADP-ribosylate and repress AHR in cells was dependent on both its catalytic activity and zinc finger domain. The catalytic activity of TIPARP was resistant to meta-iodobenzylguanidine but sensitive to iodoacetamide and hydroxylamine, implicating cysteines and acidic side chains as ADP-ribosylated target residues. Mass spectrometry identified multiple ADP-ribosylated peptides in TIPARP and AHR. Electron transfer dissociation analysis of the TIPARP peptide 33ITPLKTCFK41 revealed cysteine 39 as a site for mono-ADP-ribosylation. Mutation of cysteine 39 to alanine resulted in a small, but significant, reduction in TIPARP autoribosylation activity, suggesting that additional amino acid residues are modified, but loss of cysteine 39 did not prevent its ability to repress AHR. Our findings characterize the subcellular localization and mono-ADP-ribosyltransferase activity of TIPARP, identify cysteine as a mono-ADP-ribosylated residue targeted by this enzyme, and confirm the TIPARP-dependent mono-ADP-ribosylation of other protein targets, such as AHR.

Does NLRP3 Inflammasome and Aryl Hydrocarbon Receptor Play an Interlinked Role in Bowel Inflammation and Colitis-Associated Colorectal Cancer?

“…This indicates AhRR may also be involved with other signalling pathways and have other cellular functions, since the Ahrr genes contain binding sites that are recognised by the NF-κB and zinc-finger transcription factors of the Sp1 family [132]. Lastly, the mono-ADP-ribosyltransferase, TCDD-inducible Poly ADP-Ribose Polymerase (TiPARP), has been shown in several studies to negatively regulate AhR transactivation; comparably with AhRR but utilising different mechanisms, through a negative feedback loop in the AhR signalling pathway and modulating its expression [133][134][135][136].…”

A Quantitative HILIC–MS/MS Assay of the Metabolic Response of Huh-7 Cells Exposed to 2,3,7,8-Tetrachlorodibenzo-p-Dioxin

“…In this study, we aimed to find changes in the levels of polar metabolites using TCDD-exposed Huh-7 cells, which is a common in vitro toxicology model. Further, Huh-7 cells are a suitable model, as AHR is expressed and active [19,44,45].…”

Progress and outlook in studying the substrate specificities of PARPs and related enzymes

“…With thousands of sites in the human cell, serine ADP-ribosylation appears to be a major contributor to the proteome complexity. Further promising candidates include cysteine [23,57,60] and tyrosine [23,51,58], which might be the targets of specific PARP family members; several other amino acids are also possible as acceptors. Moreover, it has emerged that PARPs and related ARTs can modify not only proteins, but also nucleic acids.…”

“…However, since it is not clear whether these originate from automodification or are synthesised by some other ARTs present in the cell, it is too early to draw conclusions about the specificity of these enzymes. A specific cysteine automodification site has been identified on PARP7 in vitro [60]. Generally speaking, however, glutamate/aspartate specificity might be the most common for PARPs, with best-supported examples includingin addition to PARP1 and PARP2also PARP3 [57,61] and tankyrases 1 and 2 (PARP5a and PARP5b) [62,63].…”