ADP-ribosylation Cycles

National Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

Explanation 1.Prior studies demonstrated that cholera toxin, the product of the bacteria Vibrio cholerae, is an ADP-ribosyltransferase that utilizes NAD as a substrate and catalyzes the hydrolysis to ADP-ribose and nicotinamide, as well as transfer of the ADP-ribose to free arginine and arginine residues in proteins. Others showed that the key modified protein was a guanine nucleotide-binding known as G(alpha)s. Based on our findings, we showed that mammalian tissues possessed NAD:arginine ADP-ribosyltransferases. We then asked why the ADP-ribosylation was not toxic, as is the case with cholera toxin, and proposed that mammalian cells had enzymes that hydrolyzed ADP-ribose-arginine(protein). Thus, arginine-specific mono-ADP-ribosylation is a reversible post-translational modification; arginine-specific, cholera toxin-like mono-ADP-ribosyltransferases (ARTC1s) transfer ADP-ribose from NAD+ to arginine, followed by cleavage of ADP-ribose-(arginine)protein bond by ADP-ribosylarginine hydrolase 1 (ARH1), generating unmodified (arginine)protein. ARTC1 has been shown to increase tumorigenicity that is also increased by Arh1 deficiency. In this study, Artc1-KO and Artc1/Arh1-double-KO mice were generated and their properties compared. Artc1-deficient mice showed decreased spontaneous tumorigenesis and increased age-dependent, multi-organ inflammation with upregulation of pro-inflammatory cytokine TNF(alpha). In a xenograft model using tumorigenic Arh1-KO mouse embryonic fibroblasts (MEFs), tumorigenicity was decreased in Artc1-KO and heterozygous recipient mice, with tumor infiltration by CD8+ T cells and macrophages, leading to necroptosis, suggesting that ARTC1 promotes the tumor microenvironment. Furthermore, Artc1/Arh1-double-KO MEFs showed decreased tumorigenesis in nude mice, thus both tumor cells as well as tumor microenvironment require ARTC1. By echocardiography and MRI, Artc1-KO and heterozygous mice showed male-specific, reduced myocardial contractility. Furthermore, Artc1-KO male hearts exhibited enhanced susceptibility to myocardial ischemia-reperfusion-induced injury with increased receptor-interacting protein kinase 3 (RIP3) protein levels compared to WT mice, suggesting that ARTC1 suppresses necroptosis. Overall survival rate of Artc1-KO was less than their Artc1-WT counterparts, primarily due to enhanced immune response and inflammation. Thus, anti-ARTC1 agents may reduce tumorigenesis but may increase multi-organ inflammation and decrease cardiac contractility. 2.COVID 19-related studies: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for a global pandemic that resulted in more than 6-million deaths worldwide. The virus encodes several non-structural proteins (Nsps) that contain elements capable of disrupting cellular processes. Among these Nsp proteins, Nsp3 contains macrodomains, e.g., Mac1, Mac2, Mac3, with potential effects on host cells. Mac1 has been shown to increase SARS-CoV-2 virulence and disrupt ADP-ribosylation pathways in mammalian cells. Viral macrodomains have been shown to cleave the ADP-ribose-acceptor bond, generating free ADP-ribose. By this reaction, the macrodomain-containing proteins interfere with ADP-ribose homeostasis in host cells. Here, we examined potential hydrolytic activities of SARS-CoV-2 Mac1, 2, and 3 on substrates containing ADP-ribose. Mac1 cleaved -NAD + , but not -NAD + , consistent with stereospecificity at the C-1 bond. This reaction is similar to those catalyzed by ARH1 and ARH3 as well as other macrodomains. In contrast to ARH1 and ARH3, Mac1 did not require Mg 2+ for optimal activity. Mac1 also hydrolyzed O-acetyl-ADP-ribose and ADP-ribose-1-phosphate, but Mac2 and Mac3 were inactive in these reactions. In addition, Mac1 did not cleave -ADP-ribose-(arginine) and ADP-ribose-(serine)-histone H3 peptide, suggesting that Mac1 hydrolyzes ADP-ribose attached to O- and N-linked functional groups, with specificity at the catalytic site in the ADP-ribose moiety. We conclude that SARS-CoV-2 Mac1 may exert anti-viral activity by reversing host-mediated ADP-ribosylation. New insights on Nsp3 Macrodomain activities may shed light on potential SARS-CoV-2 therapeutic targets.