ADP-ribosyltransferases: plastic tools for inactivating protein and small molecular weight targets.

ADP-ribosyltransferases (ADPRTs) form an interesting class of enzymes with well-established roles as potent bacterial toxins and metabolic regulators. ADPRTs catalyze the transfer of the ADP-ribose moiety from NAD(+) onto specific substrates including proteins. ADP-ribosylation usually inactivates the function of the target. ADPRTs have become adapted to function in extra- and intracellular settings. Regulation of ADPRT activity can be mediated by ligand binding to associated regulatory domains, proteolytic cleavage, disulphide bond reduction, and association with other proteins. Crystallisation has revealed a conserved core set of elements that define an unusual minimal scaffold of the catalytic domain with remarkably plastic sequence requirements--only a single glutamic acid residue critical to catalytic activity is invariant. These inherent properties of ADPRTs suggest that the ADPRT catalytic fold is an attractive, malleable subject for protein design.     

The best defense is a good offense--Salmonella deploys an ADP-ribosylating toxin

The dramatic clinical manifestations of toxigenic infections such as cholera and diphtheria occur without substantial bacterial invasion. Disease is mediated by the secretion of potent toxins that use ADP-ribosylation as the catalytic mechanism underlying their action. ADP-ribosylating toxins comprise a large family, including the cholera, diphtheria, pertussis and Escherichia coli heat-labile (LT) toxins, and all produce disease by altering key metabolic processes after transfer of an ADP-ribose moiety from NAD to specific host-cell target proteins. A new paradigm implicating ADP-ribosylation during intracellular pathogenesis is beginning to emerge from recent research in Salmonella.     

Bacterial ADP-ribosylating toxins: molecular structures and signal transducing functions

Mono-ADP-ribosylation is a posttranslational modification of proteins employed by a variety of bacterial ADP-ribosylating toxins to modify the metabolism of target cells. The ADP-ribosyltransferases of bacterial toxins, in general, use NAD as a substrate for covalent modification by ADP-ribose to certain GTP-binding proteins (G proteins) as signal transducers resulting in altered enzymatic activity of the membrane enzymes as effectors. Such a mechanism has the potential of being of importance in the physiological regulation of cellular metabolism, particularly if the process is reversible. These ADP-ribosylating toxins are characterized in Table 1.     

Structural and biochemical characterization of NarE, an iron-containing ADP-ribosyltransferase from Neisseria meningitidis

NarE is a 16 kDa protein identified from Neisseria meningitidis, one of the bacterial pathogens responsible for meningitis. NarE belongs to the family of ADP-ribosyltransferases (ADPRT) and catalyzes the transfer of ADP-ribose moieties to arginine residues in target protein acceptors. Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and alter essential functions of eukaryotic cells. NarE is further the first ADPRT which could be shown to bind iron through a Fe-S center, which is crucial for the catalytic activity. Here we present the NMR solution structure of NarE, which shows structural homology to other ADPRTs. Using NMR titration experiments we could identify from Chemical Shift Perturbation data both the NAD binding site, which is in perfect agreement with a consensus sequence analysis between different ADPRTs, as well as the iron coordination site, which consists of 2 cysteines and 2 histidines. This atypical iron coordination is also capable to bind zinc. These results could be fortified by site-directed mutagenesis of the catalytic region, which identified two functionally crucial residues. We could further identify a main interaction region of NarE with antibodies using two complementary methods based on antibody immobilization, proteolytic digestion, and mass spectrometry. This study combines structural and functional features of NarE providing for the first time a characterization of an iron-dependent ADPRT.     

Functional aspects of protein mono-ADP-ribosylation

Mono-ADP-ribosylation is the enzymatic transfer of ADP-ribose from NAD(+) to acceptor proteins. It is catalysed by cellular ADP-ribosyltransferases and certain bacterial toxins. There are two subclasses of cellular enzymes: the ectoenzymes that modify targets such as integrins, defensin and other cell surface molecules; and the intracellular enzymes that act on proteins involved in cell signalling and metabolism, such as the beta-subunit of heterotrimeric G proteins, GRP78/BiP and elongation factor 2. The genes that encode the ectoenzymes have been cloned and their protein products are well characterized, yet little is known about the intracellular ADP-ribosyltransferases, which may be part of a novel protein family with an important role in regulating cell function. ADP-ribosylation usually leads to protein inactivation, providing a mechanism to inhibit protein functions in both physiological and pathological conditions.     

Pathophysiological aspects of cellular pyridine nucleotide metabolism: focus on the vascular endothelium. Review

In recent years, pyridine nucleotides NAD(H) and NADP(H) have been established as an important molecules in physiological and pathophysiological signaling and cell injury pathways. Protein modification is catalyzed by ADP-ribosyl transferases that attach the ADP-ribose moiety of NAD+ to specific aminoacid residues of the acceptor proteins, with significant changes in the function of these acceptors. Mono(ADP-ribosyl)ation reactions have been implicated to play a role both in physiological responses and in cellular responses to bacterial toxins. Cyclic ADP-ribose formation also utilizes NAD+ and primarily serves as physiological, signal transduction mechanisms regulating intracellular calcium homeostasis. In pathophysiological conditions associated with oxidative stress (such as various forms of inflammation and reperfusion injury), activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) occurs, with subsequent, substantial fall in cellular NAD+ and ATP levels, which can determine the viability and function of the affected cells. In addition, NADPH oxidases can significantly affect the balance and fate of NAD+ and NADP in oxidatively stressed cells and can facilitate the generation of various positive feedback cycles of injury. Under severe oxidant conditions, direct oxidative damage to NAD+ has also been reported. The current review focuses on PARP and on NADPH oxidases, as pathophysiologically relevant factors in creating disturbances in the cellular pyridine nucleotide balance. A separate section describes how these mechanisms apply to the pathogenesis of endothelial cell injury in selected cardiovascular pathophysiological conditions.     

Immunochemical detection of guanine nucleotide binding proteins mono-ADP-ribosylated by bacterial toxins

Rabbits immunized with ADP-ribose chemically conjugated to carrier proteins developed antibodies reactive against guanine nucleotide binding proteins (G proteins) that had been mono-ADP-ribosylated by bacterial toxins. Antibody reactivity on immunoblots was strictly dependent on incubation of substrate proteins with both toxin and NAD and was quantitatively related to the extent of ADP-ribosylation. Gi, Go, and transducin (ADP-ribosylated by pertussis toxin) and elongation factor II (EF-II) (ADP-ribosylated by pseudomonas exotoxin) all reacted with ADP-ribose antibodies. ADP-ribose antibodies detected the ADP-ribosylation of an approximately 40-kilodalton (kDa) membrane protein related to Gi in intact human neutrophils incubated with pertussis toxin and the ADP-ribosylation of an approximately 90-kDa cytosolic protein, presumably EF-II, in intact HUT-102 cells incubated with pseudomonas exotoxin. ADP-ribose antibodies represent a novel tool for the identification and study of G proteins and other substrates for bacterial toxin ADP-ribosylation.     

Crystal structure and novel recognition motif of rho ADP-ribosylating C3 exoenzyme from Clostridium botulinum: structural insights for recognition specificity and catalysis

Clostridium botulinum C3 exoenzyme inactivates the small GTP-binding protein family Rho by ADP-ribosylating asparagine 41, which depolymerizes the actin cytoskeleton. C3 thus represents a major family of the bacterial toxins that transfer the ADP-ribose moiety of NAD to specific amino acids in acceptor proteins to modify key biological activities in eukaryotic cells, including protein synthesis, differentiation, transformation, and intracellular signaling. The 1.7 A resolution C3 exoenzyme structure establishes the conserved features of the core NAD-binding beta-sandwich fold with other ADP-ribosylating toxins despite little sequence conservation. Importantly, the central core of the C3 exoenzyme structure is distinguished by the absence of an active site loop observed in many other ADP-ribosylating toxins. Unlike the ADP-ribosylating toxins that possess the active site loop near the central core, the C3 exoenzyme replaces the active site loop with an alpha-helix, alpha3. Moreover, structural and sequence similarities with the catalytic domain of vegetative insecticidal protein 2 (VIP2), an actin ADP-ribosyltransferase, unexpectedly implicates two adjacent, protruding turns, which join beta5 and beta6 of the toxin core fold, as a novel recognition specificity motif for this newly defined toxin family. Turn 1 evidently positions the solvent-exposed, aromatic side-chain of Phe209 to interact with the hydrophobic region of Rho adjacent to its GTP-binding site. Turn 2 evidently both places the Gln212 side-chain for hydrogen bonding to recognize Rho Asn41 for nucleophilic attack on the anomeric carbon of NAD ribose and holds the key Glu214 catalytic side-chain in the adjacent catalytic pocket. This proposed bipartite ADP-ribosylating toxin turn-turn (ARTT) motif places the VIP2 and C3 toxin classes into a single ARTT family characterized by analogous target protein recognition via turn 1 aromatic and turn 2 hydrogen-bonding side-chain moieties. Turn 2 centrally anchors the catalytic Glu214 within the ARTT motif, and furthermore distinguishes the C3 toxin class by a conserved turn 2 Gln and the VIP2 binary toxin class by a conserved turn 2 Glu for appropriate target side-chain hydrogen-bonding recognition. Taken together, these structural results provide a molecular basis for understanding the coupled activity and recognition specificity for C3 and for the newly defined ARTT toxin family, which acts in the depolymerization of the actin cytoskeleton. This beta5 to beta6 region of the toxin fold represents an experimentally testable and potentially general recognition motif region for other ADP-ribosylating toxins that have a similar beta-structure framework.     

New insights into the mode of action of the actin ADP-ribosylating virulence factors Salmonella enterica SpvB and Clostridium botulinum C2 toxin

The C2 toxin from Clostridium botulinum represents the prototype of the family of binary actin-ADP-ribosylating toxins. These toxins covalently transfer ADP-ribose from nicotinamide adenine dinucleotide (NAD(+)) onto arginine-177 of actin in the cytosol of eukaryotic cells resulting in depolymerization of actin filaments and cell rounding. The C2 toxin consists of two non-linked proteins, the enzyme component C2I and the binding and translocation component C2II, which delivers C2I into host cells. The ADP-ribosyltransferase SpvB from Salmonella enterica also modifies actin, but is delivered into the host cell cytosol from intracellular growing Salmonella, most likely via type-III-secretion. We characterized the mode of action of SpvB in comparison to C2 toxin in vitro and in intact cells. We identified arginine-177 as the target for SpvB-catalyzed mono-ADP-ribosylation of actin. To compare the cellular responses following modification of actin by SpvB or by the binary toxins without the influence of other Salmonella virulence factors, we constructed a cell-permeable fusion toxin to deliver the catalytic domain of SpvB (C/SpvB) into the cytosol of target cells. This review summarizes recent findings of research on the actin ADP-ribosylating toxins regarding their cellular uptake, molecular mode of action and the cellular consequences following ADP-ribosylation of actin.     

Common structure of the catalytic sites of mammalian and bacterial toxin ADP-ribosyltransferases

The amino acid sequences of several bacterial toxin ADP-ribosyltransferases, rabbit skeletal muscle transferases, and RT6.2, a rat T-cell NAD glycohydrolase, contain three separate regions of similarity, which can be aligned. Region I contains a critical histidine or arginine residue, region II, a group of closely spaced aromatic amino acids, and region III, an active-site glutamate which is at times seen as part of an acidic amino acid-rich sequence. In some of the bacterial ADP-ribosyltransferases, the nicotinamide moiety of NAD has been photo-crosslinked to this glutamate, consistent with its position in the active site. The similarities within these three regions, despite an absence of overall sequence similarity among the several transferases, are consistent with a common structure involved in NAD binding and ADP-ribose transfer.     

Cholera- and anthrax-like toxins are among several new ADP-ribosyltransferases

Chelt, a cholera-like toxin from Vibrio cholerae, and Certhrax, an anthrax-like toxin from Bacillus cereus, are among six new bacterial protein toxins we identified and characterized using in silico and cell-based techniques. We also uncovered medically relevant toxins from Mycobacterium avium and Enterococcus faecalis. We found agriculturally relevant toxins in Photorhabdus luminescens and Vibrio splendidus. These toxins belong to the ADP-ribosyltransferase family that has conserved structure despite low sequence identity. Therefore, our search for new toxins combined fold recognition with rules for filtering sequences--including a primary sequence pattern--to reduce reliance on sequence identity and identify toxins using structure. We used computers to build models and analyzed each new toxin to understand features including: structure, secretion, cell entry, activation, NAD+ substrate binding, intracellular target binding and the reaction mechanism. We confirmed activity using a yeast growth test. In this era where an expanding protein structure library complements abundant protein sequence data--and we need high-throughput validation--our approach provides insight into the newest toxin ADP-ribosyltransferases.     

Demonstration and partial characterization of ADP-ribosylation in Pseudomonas maltophilia.

ADP-ribosylation of proteins occurs in many eukaryotes, and it is also the mechanism of action of a growing number of important bacterial toxins. To date, however, there is only one well-characterized ADP-ribosylation system where the ADP-ribosyltransferase and the substrate protein are both bacterial in origin, namely within the nitrogen-fixing bacterium Rhodospirillum rubrum. The present paper demonstrates the endogenous ADP-ribosylation of two proteins of Mr 32,000 and 20,000 within Pseudomonas maltophilia, a Gram-negative aerobe. The proteins have been partially purified: two apparently separate species of modified protein can be separated by ion-exchange chromatography and gel filtration (V0 and Mr 158,000 - Vi). The substrate protein(s) either has, or is co-eluted with, NAD+ glycohydrolase activity. The modification is mono-ADP-ribosyl in nature. The linkage between the acceptor amino acid and the ADP-ribose moiety is alkali-labile and stable to hydroxylamine, possibly indicating an S-glycosidic bond. The activity appears to be a true ADP-ribosylation reaction and not an NAD+ glycohydrolase activity followed by non-enzymic addition of ADP-ribose to protein. The results presented here indicate that ADP-ribosylation may have a wider significance within prokaryotic systems than previously thought.     

Crystal structure of the RNA 2'-phosphotransferase from Aeropyrum pernix K1

In the final step of tRNA splicing, the 2'-phosphotransferase catalyzes the transfer of the extra 2'-phosphate from the precursor-ligated tRNA to NAD. We have determined the crystal structure of the 2'-phosphotransferase protein from Aeropyrum pernix K1 at 2.8 Angstroms resolution. The structure of the 2'-phosphotransferase contains two globular domains (N and C-domains), which form a cleft in the center. The N-domain has the winged helix motif, a subfamily of the helix-turn-helix family, which is shared by many DNA-binding proteins. The C-domain of the 2'-phosphotransferase superimposes well on the NAD-binding fold of bacterial (diphtheria) toxins, which catalyze the transfer of ADP ribose from NAD to target proteins, indicating that the mode of NAD binding by the 2'-phosphotransferase could be similar to that of the bacterial toxins. The conserved basic residues are assembled at the periphery of the cleft and could participate in the enzyme contact with the sugar-phosphate backbones of tRNA. The modes by which the two functional domains recognize the two different substrates are clarified by the present crystal structure of the 2'-phosphotransferase.     

The Neisseria meningitidis ADP-Ribosyltransferase NarE Enters Human Epithelial Cells and Disrupts Epithelial Monolayer Integrity

Many pathogenic bacteria utilize ADP-ribosylating toxins to modify and impair essential functions of eukaryotic cells. It has been previously reported that Neisseria meningitidis possesses an ADP-ribosyltransferase enzyme, NarE, retaining the capacity to hydrolyse NAD and to transfer ADP-ribose moiety to arginine residues in target acceptor proteins. Here we show that upon internalization into human epithelial cells, NarE gains access to the cytoplasm and, through its ADP-ribosylating activity, targets host cell proteins. Notably, we observed that these events trigger the disruption of the epithelial monolayer integrity and the activation of the apoptotic pathway. Overall, our findings provide, for the first time, evidence for a biological activity of NarE on host cells, suggesting its possible involvement in Neisseria pathogenesis.     

Bacterial glycosyltransferase toxins

Mono‐glycosylation of host proteins is a common mechanism by which bacterial protein toxins manipulate cellular functions of eukaryotic target host cells. Prototypic for this group of glycosyltransferase toxins are Clostridium difficile toxins A and B, which modify guanine nucleotide‐binding proteins of the Rho family. However, toxin‐induced glycosylation is not restricted to the Clostridia. Various types of bacterial pathogens including Escherichia coli, Yersinia, Photorhabdus and Legionella species produce glycosyltransferase toxins. Recent studies discovered novel unexpected variations in host protein targets and amino acid acceptors of toxin‐catalysed glycosylation. These findings open new perspectives in toxin as well as in carbohydrate research.     

Mechanism of nicotinamide inhibition and transglycosidation by Sir2 histone/protein deacetylases

Silent information regulator 2 (Sir2) enzymes catalyze NAD+-dependent protein/histone deacetylation, where the acetyl group from the lysine epsilon-amino group is transferred to the ADP-ribose moiety of NAD+, producing nicotinamide and the novel metabolite O-acetyl-ADP-ribose. Sir2 proteins have been shown to regulate gene silencing, metabolic enzymes, and life span. Recently, nicotinamide has been implicated as a direct negative regulator of cellular Sir2 function; however, the mechanism of nicotinamide inhibition was not established. Sir2 enzymes are multifunctional in that the deacetylase reaction involves the cleavage of the nicotinamide-ribosyl, cleavage of an amide bond, and transfer of the acetyl group ultimately to the 2'-ribose hydroxyl of ADP-ribose. Here we demonstrate that nicotinamide inhibition is the result of nicotinamide intercepting an ADP-ribosyl-enzyme-acetyl peptide intermediate with regeneration of NAD+ (transglycosidation). The cellular implications are discussed. A variety of 3-substituted pyridines was found to be substrates for enzyme-catalyzed transglycosidation. A Br?nsted plot of the data yielded a slope of +0.98, consistent with the development of a nearly full positive charge in the transition state, and with basicity of the attacking nucleophile as a strong predictor of reactivity. NAD+ analogues including beta-2'-deoxy-2'-fluororibo-NAD+ and a His-to-Ala mutant were used to probe the mechanism of nicotinamide-ribosyl cleavage and acetyl group transfer. We demonstrate that nicotinamide-ribosyl cleavage is distinct from acetyl group transfer to the 2'-OH ribose. The observed enzyme-catalyzed formation of a labile 1'-acetylated-ADP-fluororibose intermediate using beta-2'-deoxy-2'-fluororibo-NAD+ supports a mechanism where, after nicotinamide-ribosyl cleavage, the carbonyl oxygen of acetylated substrate attacks the C-1' ribose to form an initial iminium adduct.     

Cytolethal distending toxins

The cytolethal distending toxins (CDTs) constitute the most recently discovered family of bacterial protein toxins. CDTs are unique among bacterial toxins as they have the ability to induce DNA double strand breaks (DSBs) in both proliferating and nonproliferating cells, thereby causing irreversible cell cycle arrest or death of the target cells. CDTs are encoded by three linked genes (cdtA, cdtB and cdtC) which have been identified among a variety of Gram-negative pathogenic bacteria. All three of these gene products are required to constitute the fully active holotoxin, and this is in agreement with the recently determined crystal structure of CDT. The CdtB component has functional homology with mammalian deoxyribonuclease I (DNase I). Mutation of the conserved sites necessary for this catalytic activity prevents the induction of DSBs as well as all subsequent intoxication responses of target cells. CDT is endocytosed via clathrin-coated pits and requires an intact Golgi complex to exert the cytotoxic activity. Several issues remain to be elucidated regarding CDT biology, such as the detailed function(s) of the CdtA and CdtC subunits, the identity of the cell surface receptor(s) for CDT, the final steps in the cellular internalization pathway, and a molecular understanding of how CDT interacts with DNA. Moreover, the role of CDTs in the pathogenesis of diseases still remains unclear.     

Functional comparison of the NAD binding cleft of ADP-ribosylating toxins

Although a common core structure forms the active site of ADP-ribosylating (ADPRT) toxins, the limited-sequence homology within this region suggests that different mechanisms are being used by toxins to perform their shared function. To explain differences in their mechanisms of NAD binding and hydrolysis, the functional interrelationship of residues predicted to perform similar functions in the beta3-strand of the NAD binding cleft of different ADPRT toxins was compared. Replacing Tyr54 in the A-subunit of diphtheria toxin (DTA) with a serine, its functional homologue in cholera toxin (CT), resulted in the loss of catalytic function but not NAD binding. The catalytic role of the aromatic portion of Tyr54 in the ADPRT reaction was confirmed by the ability of a Tyr54-to-phenylalanine DTA mutant to retain ADPRT activity. In reciprocal studies, positioning a tyrosine in the beta3-strand of the A1-subunit of CT (CTA1) caused both loss of function and altered structure. The restricted flexibility of the CTA1 active site relative to function became evident upon the loss of ADPRT activity when a conservative Val60-to-leucine mutation was performed. We conclude from our studies that DT and CT maintain a similar mechanism of NAD binding but differ in their mechanisms of NAD hydrolysis. The aromatic moiety at position 54 in DT is integral to NAD hydrolysis, while NAD hydrolysis in CT appears highly dependent on the precise positioning of specific residues within the beta3-strand of the active-site cleft.     

Characterization of NAD:arginine ADP-ribosyltransferases

NAD:arginine mono-ADP-ribosyltransferases catalyze the transfer of ADP-ribose from NAD to the guanidino group of arginine on a target protein. Deduced amino acid sequences of one family (ART1) of mammalian ADP-ribosyltransferases, cloned from muscle and lymphocytes, show hydrophobic amino and carboxyl termini consistent with glycosylphosphatidylinositol (GPI)-anchored proteins. The proteins, overexpressed in mammalian cells transfected with the transferase cDNAs, are released from the cell surface with phosphatidylinositol-specific phospholipase C (PI-PLC), and display immunological and biochemical characteristics consistent with a cell surface, GPI-anchored protein. In contrast, the deduced amino acid sequence of a second family (ART5) of transferases, cloned from murine lymphoma cells and expressed in high abundance in testis, displays a hydrophobic amino terminus, consistent with a signal sequence, but lacks a hydrophobic signal sequence at its carboxyl terminus, suggesting that the protein is destined for export. Consistent with the surface localization of the GPI-linked transferases, multiple surface substrates have been identified in myotubes and activated lymphocytes, and, notably, include integrin subunits. Similar to the bacterial toxin ADP-ribosyltransferases, the mammalian transferases contain the characteristic domains involved in NAD binding and ADP-ribose transfer, including a highly acidic region near the carboxy terminus, which, when disrupted by in vitro mutagenesis, results in a loss of enzymatic activity. The carboxyl half of the protein, synthesized as a fusion protein in E. coli, possessed NADase, but not ADP-ribosyltransferase activity. These findings are consistent with the existence at the carboxyl terminus of ART1 of a catalytically active domain, capable of hydrolyzing NAD, but not of transferring ADP-ribose to a guanidino acceptor.