ADP-ribosylation Cycles

National Heart, Lung, And Blood Institute

Investigators

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Abstract

Explanation (Arginine)protein ADP-ribosylation is a reversible post-translational modification in which ADP-ribose from beta-NAD+ is transferred to arginine of protein/peptide acceptors by arginine-specific ADP-ribosyltransferases (ARTs). The reaction can be reversed by ADP-ribosyl-acceptor hydrolase 1 (ARH1), which cleaves the alpha-ADP-ribosyl-arginine(protein) bond, releasing ADP-ribose and regenerating unmodified (arginine)proteins. The protein substrates that are ADP-ribosylated and then de-ADP-ribosylated form a pseudo-ADP-ribosylation cycle with the ARTs and ARHs. Our prior observation that non-enzymatic modification of the ADP-ribosylated substrate may alter its biological activity led us to investigate these pathways and their potential physiological activity. Ornithine is generated as a product of post-translational modification involving ADP-ribose-arginine when the guanidino group of ADP-ribose-arginine is non-enzymatically attacked by hydroxyl/H2O. We have previously demonstrated that ART1-catalyzed ADP-ribosylation at residues 14 and 24 of HNP-1 modulates its biological activities. Site-specific ADP-ribosylation by ART1 abolishes both the antimicrobial and cytotoxic functions of HNP-1. Notably, the conversion of ADP-ribosylated Arg14 and Arg24 in HNP-1 to ornithine eliminates its cytotoxicity toward epithelial cells while preserving its antibacterial function, indicating selective attenuation of activity via post-translational modification. HNP-(R14, 24 orn) emerges as a promising, stable peptide with reduced toxicity for epithelial cells lining the airway. Formation of ornithine from ADP-ribosylated arginine is coupled to release of “ADP-ribose-carbodiimide”, the structure of which is under investigation. Release of ornithine from attack of the hydroxyl/H2O on the guanidino group presumes that ornithine is a favored leaving group. However, an alternative leaving group is ammonium ion. We now found that the release of ammonium ion results in the formation of “des-amino-ADP-ribose-arginine” from the attack of guanidino group of water or hydroxyl ion. This compound was isolated by high-performance liquid chromatography (HPLC). The “des-amino-ADP-ribose-arginine” exhibited distinct chromatographic retention times compared to ADP-ribose-arginine and other known derivatives, confirming its unique chemical identity. Mass spectrometry (MS) analysis provided a mass consistent with the loss of an amino group from the arginine side chain, supporting the proposed displacement mechanism. Tandem mass spectrometry (MS/MS) fragmentation patterns further supported the structural assignment. Further kinetic studies showed that formation of the “des-amino-ADP-ribose-arginine” was time and temperature dependent. These studies demonstrated a slow but progressive chemical transformation of the initial ADP-ribose-arginine, suggesting that the stability of the ADP-ribose-arginine linkage is sensitive to incubation conditions. In summary, our proposed mechanism involves hydroxyl-mediated nucleophilic attack on the guanidino group, leading to either release of ammonia or ornithine. The ADP-ribose moiety by mass spectrometry is intact in “des-amino-ADP-ribose-arginine”. ARH1, an ADP-ribose-arginine hydrolase, catalyzes the cleavage of ADP-ribose-arginine, releasing ADP-ribose and generating the free guanidino group, thereby reversing the modification. We observed that ARH1 did not hydrolyze des-amino-ADP-ribose-arginine. This finding suggests that this metabolite derived from ADP-ribose-arginine may maintain prolonged signaling states, with preservation of the ADP-ribose structure, potentially playing roles in long-term cellular function and regulation. In summary, non- enzymatic ADP-ribosylation of arginine residues, catalyzed by endogenous ARTs, constitutes a post-translational modification affecting protein function and cellular signaling. Ornithine and des-amino-ADP-ribose-arginine, key metabolic products arising from alternative pathways of modification of ADP-ribosylated arginine, underscore the complexity of ADP-ribose-arginine metabolism. Current studies are directed at understanding how these alternative pathways affect biological activity.