ADP-ribosylation Cycles

Heart, Lung, And Blood Institute

Investigators

Linked publications & trials

Abstract

ADP-ribosylation, in which the ADP-ribose moiety of NAD is transferred to a target protein, is catalyzed by a family of bacterial toxins and mammalian enzymes. Some toxin ADP-ribosyltransferases (e.g., cholera toxin, diphtheria toxin) are responsible for symptoms of the diseases caused by the bacterium. Mammalian cells contain enzymes that catalyze reactions similar to the bacterial toxins. Mammalian ADP-ribosyltransferases (ARTs) can be located within the cell and on the cell surface, sometimes linked through a glycosylphosphatidylinositol anchor (ART1). Others, ART5, appear to be secreted. A family of mammalian transferases has been cloned in the laboratory; they display some structural similarities to the toxins, with amino acid identities in the catalytic site. A product of transferase-catalyzed reactions, ADP-ribose-(arginine)protein, is cleaved by a 39-kDa ADP-ribosylarginine hydrolase (ADPRH)to regenerate unmodified protein. Thus, transferases and hydrolases can catalyze opposing reactions to constitute an ADP-ribosylation cycle. An ADPRH cDNA had been cloned from human, rat, and mouse tissues and high levels of hydrolase mRNA were found in brain, spleen, and testis. To begin to understand the molecular mechanisms that regulate ADPRH gene expression, we determined the genomic structure of mouse ADPRH, and investigated promoter function. Northern analyses using different regions of the ADPRH cDNA as probes identified mRNAs of 1.7 and 3.0 kb that resulted from the use of alternative polyadenylation signals, CATAAC and ATTAAA, beginning at positions 1501 and 2885, respectively, of the nucleotide sequence (A of ATG = 1). The ADPRH gene, represented in two overlapping genomic clones, spans 9 kb with four exons and three introns. The 5'-flanking region contains features of a housekeeping gene; it has neither a TATA nor a CAAT box, but is, instead, highly GC-rich with multiple transcription initiation sites. Promoter analysis, assessed using transient transfection of PC12, NB41A3, NIH/3T3, and Hepa 1-6 cells with truncated constructs, revealed potent stimulatory (-119 to -89) and inhibitory (-161 to -119) elements, which were utilized similarly in the different cell lines. Further mutational analysis of the promoter and electrophoretic mobility-shift assays identified a positive GC-box element (-107 to -95); Sp1 and Sp3, which bound to this motif, were also detected by supershift assays. In co-transfection experiments using Drosophila SL2 cells that lack endogenous Sp1, Sp1 trans-activated the ADPRH promoter in a manner dependent on the presence of an Sp1-binding motif. The promoter activity pattern and involvement of Sp transcription factors are consistent with prior observations of widespread hydrolase expression in mammalian tissues. The lungs of patients with cystic fibrosis (CF) are colonized frequently by Pseudomonas aeruginosa, which is associated with progressive lung destruction and increased mortality. A number of virulence factors, including exotoxin A (ETA) and the type III cytotoxins (ExoS, ExoT, ExoU, and ExoY) contribute to the pathogenicity of P. aeruginosa, which contacts the plasma membrane to deliver type III cytotoxins through a channel formed by many proteins, including PopB, PopD, and PcrV. ETA enters mammalian cells via receptor-mediated endocytosis. ETA, ExoS, and ExoT are ADP-ribosyltransferases that modify different substrates in mammalian cells. ETA, like diphtheria toxin, ADP-ribosylates elongation factor 2, thereby inhibiting protein synthesis. ExoS and ExoT target different signaling pathways, but they both ADP-ribosylate an arginine residue in their substrates. The recent study characterized the appearance with time of antibodies to components of the type III system in children with CF, who were colonized early in life with P. aeruginosa expressing the type III system. Surveillance for seroconversion to type III antigens in addition to clinical symptoms and oropharyngeal cultures may facilitate early detection of P. aeruginosa infections. Cholera toxin (CT), the toxic product produced by pathogenic agent responsible for cholera, exerts its effects on cells by ADP-ribosylation of a specific arginine in the regulatory guanine nucleotide-binding protein, G alpha As. Plant polyphenols, RG-tannin, and applephenon inhibited cholera toxin CT ADP-ribosyltransferase activity and CT-induced fluid accumulation in mouse ileal loops. A high molecular weight fraction of hop bract extract (HBT) also inhibited CT ADP-ribosyltransferase activity. Binding of CT to Vero cells or to ganglioside GM1, a CT receptor, was inhibited in a concentration-dependent manner by HBT and applephenon, but not RG-tannin. Following toxin binding to cells, applephenon, HBT, and RG-tannin suppressed its internalization. HBT or applephenon precipitated CT, CT A subunit, and CT B subunit from solution, creating aggregates larger than 250 kDa. In contrast, RG-tannin precipitated CT poorly; it formed complexes with CT, CTA, or CTB, which were demonstrated with sucrose density gradient centrifugation and molecular weight exclusion filters. In agreement, CTA blocked the inhibition of CT internalization by RG-tannin. These data suggest that some plant polyphenols, similar to applephenon and HBT, bind CT, forming large aggregates in solution or, perhaps, on the cell surface and thereby suppress CT binding and internalization. In contrast, RG-tannin binding to CT did not interfere with its binding to Vero cells or GM1, but it did inhibit internalization. The natural products, or their derivatives may be useful in treating this toxin-mediated disease.