Substrate For ADP-ribosylation Research Articles

Identification and characterization of a new potent inhibitor targeting CtBP1/BARS in melanoma cells

BackgroundThe C-terminal-binding protein 1/brefeldin A ADP-ribosylation substrate (CtBP1/BARS) acts both as an oncogenic transcriptional co-repressor and as a fission inducing protein required for membrane trafficking and Golgi complex partitioning during mitosis, hence for mitotic entry. CtBP1/BARS overexpression, in multiple cancers, has pro-tumorigenic functions regulating gene networks associated with “cancer hallmarks” and malignant behavior including: increased cell survival, proliferation, migration/invasion, epithelial-mesenchymal transition (EMT).Structurally, CtBP1/BARS belongs to the hydroxyacid-dehydrogenase family and possesses a NAD(H)-binding Rossmann fold, which, depending on ligands bound, controls the oligomerization of CtBP1/BARS and, in turn, its cellular functions.Here, we proposed to target the CtBP1/BARS Rossmann fold with small molecules as selective inhibitors of mitotic entry and pro-tumoral transcriptional activities.MethodsStructured-based screening of drug databases at different development stages was applied to discover novel ligands targeting the Rossmann fold. Among these identified ligands, N-(3,4-dichlorophenyl)-4-{[(4-nitrophenyl)carbamoyl]amino}benzenesulfonamide, called Comp.11, was selected for further analysis. Fluorescence spectroscopy, isothermal calorimetry, computational modelling and site-directed mutagenesis were employed to define the binding of Comp.11 to the Rossmann fold. Effects of Comp.11 on the oligomerization state, protein partners binding and pro-tumoral activities were evaluated by size-exclusion chromatography, pull-down, membrane transport and mitotic entry assays, Flow cytometry, quantitative real-time PCR, motility/invasion, and colony assays in A375MM and B16F10 melanoma cell lines. Effects of Comp.11 on tumor growth in vivo were analyzed in mouse tumor model.ResultsWe identify Comp.11 as a new, potent and selective inhibitor of CtBP1/BARS (but not CtBP2). Comp.11 directly binds to the CtBP1/BARS Rossmann fold affecting the oligomerization state of the protein (unlike other known CtBPs inhibitors), which, in turn, hinders interactions with relevant partners, resulting in the inhibition of both CtBP1/BARS cellular functions: i) membrane fission, with block of mitotic entry and cellular secretion; and ii) transcriptional pro-tumoral effects with significantly hampered proliferation, EMT, migration/invasion, and colony-forming capabilities. The combination of these effects impairs melanoma tumor growth in mouse models. ConclusionsThis study identifies a potent and selective inhibitor of CtBP1/BARS active in cellular and melanoma animal models revealing new opportunities to study the role of CtBP1/BARS in tumor biology and to develop novel melanoma treatments.

Specificity of DNA ADP-Ribosylation Reversal by NADARs.

Recent discoveries establish DNA and RNA as bona fide substrates for ADP-ribosylation. NADAR ("NAD- and ADP-ribose"-associated) enzymes reverse guanine ADP-ribosylation and serve as antitoxins in the DarT-NADAR operon. Although NADARs are widespread across prokaryotes, eukaryotes, and viruses, their specificity and broader physiological roles remain poorly understood. Using phylogenetic and biochemical analyses, we further explore de-ADP-ribosylation activity and antitoxin functions of NADAR domains. We demonstrate that different subfamilies of NADAR proteins from representative E. coli strains and an E. coli-infecting phage retain biochemical activity while displaying specificity in providing protection from toxic guanine ADP-ribosylation in cells. Furthermore, we identify a myxobacterial enzyme within the YbiA subfamily that functions as an antitoxin for its associated DarT-unrelated ART toxin, which we termed YarT, thus presenting a hitherto uncharacterised ART-YbiA toxin-antitoxin pair. Our studies contribute to the burgeoning field of DNA ADP-ribosylation, supporting its physiological relevance within and beyond bacterial toxin-antitoxin systems. Notably, the specificity and confinement of NADARs to non-mammals infer their potential as highly specific targets for antimicrobial drugs with minimal off-target effects.

Deletion of Nmnat1 in Skeletal Muscle Leads to the Reduction of NAD+ Levels but Has No Impact on Skeletal Muscle Morphology and Fiber Types.

Nicotinamide adenine dinucleotide (NAD+) is a coenzyme that mediates many redox reactions in energy metabolism. NAD+ is also a substrate for ADP-ribosylation and deacetylation by poly (ADP-ribose) polymerase and sirtuin, respectively. Nicotinamide mononucleotide adenylyltransferase 1 (Nmnat1) is a NAD+ biosynthesizing enzyme found in the nucleus. Recent research has shown that the maintaining NAD+ levels is critical for sustaining muscle functions both in physiological and pathological conditions. However, the role of Nmnat1 in skeletal muscle remains unexplored. In this study, we generated skeletal muscle-specific Nmnat1 knockout (M-Nmnat1 KO) mice and investigated its role in skeletal muscle. We found that NAD+ levels were significantly lower in the skeletal muscle of M-Nmnat1 KO mice than in control mice. M-Nmnat1 KO mice, in contrast, had similar body weight and normal muscle histology. Furthermore, the distribution of muscle fiber size and gene expressions of muscle fiber type gene expression were comparable in M-Nmnat1 KO and control mice. Finally, we investigated the role of Nmnat1 in muscle regeneration using cardiotoxin-induced muscle injury model, but muscle regeneration appeared almost normal in M-Nmnat1 KO mice. These findings imply that Nmnat1 has a redundancy in the pathophysiology of skeletal muscle.

ADP-ribosylation of RNA and DNA: from in vitro characterization to in vivo function.

The functionality of DNA, RNA and proteins is altered dynamically in response to physiological and pathological cues, partly achieved by their modification. While the modification of proteins with ADP-ribose has been well studied, nucleic acids were only recently identified as substrates for ADP-ribosylation by mammalian enzymes. RNA and DNA can be ADP-ribosylated by specific ADP-ribosyltransferases such as PARP1–3, PARP10 and tRNA 2′-phosphotransferase (TRPT1). Evidence suggests that these enzymes display different preferences towards different oligonucleotides. These reactions are reversed by ADP-ribosylhydrolases of the macrodomain and ARH families, such as MACROD1, TARG1, PARG, ARH1 and ARH3. Most findings derive from in vitro experiments using recombinant components, leaving the relevance of this modification in cells unclear. In this Survey and Summary, we provide an overview of the enzymes that ADP-ribosylate nucleic acids, the reversing hydrolases, and the substrates’ requirements. Drawing on data available for other organisms, such as pierisin1 from cabbage butterflies and the bacterial toxin–antitoxin system DarT–DarG, we discuss possible functions for nucleic acid ADP-ribosylation in mammals. Hypothesized roles for nucleic acid ADP-ribosylation include functions in DNA damage repair, in antiviral immunity or as non-conventional RNA cap. Lastly, we assess various methods potentially suitable for future studies of nucleic acid ADP-ribosylation.

Abstract 2158: ART1 tumor expression mediates immune resistance in non-small cell lung cancer by elimination of P2 × 7R+ CD8 tissue resident memory T cells and conventional type I dendritic cells

Abstract Extracellular NAD+ (eNAD) is released from stressed and dying cells where it acts as a proinflammatory mediator. However, in the presence of mono-ADP-ribosyltransferases (ARTs) eNAD serves as a substrate for ADP-ribosylation of the P2 × 7 receptor (P2RX7), resulting in NAD-induced cell death (NICD). P2RX7 is expressed on several immune cells including CD103+ conventional type I dendritic cells (cDC1s) which are essential for initiation of tumor-specific immune responses as well as on tissue resident memory T cells (TRMs), of which high tumor infiltration has been associated with improved survival in non-small cell lung cancer (NSCLC) [1]. NICD of P2 × 7R+ TRMs was recently identified as a regulator of TRM tissue homeostasis [2]. CD38 is expressed on activated immune cells and can reduce eNAD levels by converting eNAD into ADPR. We tested the hypothesis that ART1 expression in NSCLC constitutes a mechanism of immune resistance by mediating NICD of P2 × 7R+ TRMs and P2 × 7R+ cDC1s. ART1 expression in human NSCLC samples was determined by immunohistochemistry scoring of NSCLC tumors using a tissue microarray (TMA) (n=493). In addition, matched tumor and adjacent normal lung tissue samples were analyzed for ART1 expression by qPCR (n=40) and for infiltration of TRM and cDC1s by flow cytometry. A mouse KP1 lung tumor cell line with high ART1 expression was established from KRASG12D/P53−/− mice and transduced with a doxycycline-inducible shRNA targeting ART1 (ART1KD). KP1 cells and their derivatives were intravenously injected into syngeneic C57BL/6 mice to generate orthotopic lung tumors. Lung tumor nodules were enumerated two weeks after tumor inoculation using H&E-stained FFPE lung sections and lung-derived cell suspensions were analyzed by flow cytometry for infiltration of T cell and DC subsets. Increased expression of ART1 in tumor compared to normal lung was observed in 55% of NSCLC samples by TMA analysis, and confirmed by qPCR. The frequency of P2RX7+ CD8 TRMs (p<0.05) and P2 × 7R+ cDC1s (p<0.01) were reduced in ART1+ tumors compared to matched normal lung tissue. In line with the hypothesis that CD38 protects P2 × 7R+ cells from ART1-mediated NICD, P2 × 7R+ CD8 TRMs and P2 × 7R+ cDC1s present within ART1+ tumors had elevated expression of CD38. In mice, knockdown of ART1 in KP1 tumors resulted in significantly delayed growth in immunocompetent mice (p<0.001) but not in nude or CD8-depeleted mice. In an orthotopic lung model, ART1KD resulted in a 75% decrease in lung metastatic nodules (p<0.01) and was associated with increased infiltration of CD8 T cells (P<0.0001) and cDC1s (p<0.01). In vitro experiments confirmed that CD38- P2RX7+ CD8 T cells were susceptible to ADP-ribosylation and NICD when co-cultured with ART1high lung tumor cells, while P2 × 7R+ CD8 T cells that co-expressed CD38 were resistant to NICD. In conclusion, we show that ART1 is overexpressed in NSCLC and identify ART1-mediated NICD of P2 × 7R+ TRMs and cDC1s as a possible novel immune escape mechanism in NSCLC. Furthermore, we describe a novel role of CD38 expression on immune cells in its protection against NICD. 1. Nizard, M., et al., Induction of resident memory T cells enhances the efficacy of cancer vaccine. Nat Commun, 2017. 8: p. 15221. 2. Stark, R., et al., T RM maintenance is regulated by tissue damage via P2RX7. Sci Immunol, 2018. 3(30). Citation Format: Erik Wennerberg, Clarey Hung, Amanda Valeta, Timothy McGraw, Sandra Demaria, Brendon Stiles. ART1 tumor expression mediates immune resistance in non-small cell lung cancer by elimination of P2 × 7R+ CD8 tissue resident memory T cells and conventional type I dendritic cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2158.

Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin.

Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia.

Identifying Direct Protein Targets of Poly-ADP-Ribose Polymerases (PARPs) Using Engineered PARP Variants-Orthogonal Nicotinamide Adenine Dinucleotide (NAD+) Analog Pairs.

Poly-ADP-ribose polymerases (PARPs) comprise a family of 17 distinct enzymes that catalyze the transfer of ADP-ribose from nicotinamide adenine dinucleotide (NAD+) to acceptor sites on protein targets. PARPs have been implicated in a number of essential signaling pathways regulating both normal cell function and pathophysiology. To understand the physiological role of each PARP family member in the cell we need to identify the direct targets for each unique PARP in a cellular context. PARP-family member-specific target identification is challenging because of their shared catalytic mechanism and functional redundancy. To address this challenge, we have engineered a PARP variant that efficiently uses an orthogonal NAD+ analog, an analog that endogenous PARPs cannot use, as a substrate for ADP-ribosylation. The protocols in this unit describe a general procedure for using engineered PARP variants-orthogonal NAD+ analog pairs for labeling and identifying the direct targets of the poly-subfamily of PARPs (PARPs 1-3, 5, and 6).

Molecular mechanism and functional role of brefeldin A-mediated ADP-ribosylation of CtBP1/BARS

ADP-ribosylation is a posttranslational modification that modulates the functions of many target proteins. We previously showed that the fungal toxin brefeldin A (BFA) induces the ADP-ribosylation of C-terminal-binding protein-1 short-form/BFA-ADP-ribosylation substrate (CtBP1-S/BARS), a bifunctional protein with roles in the nucleus as a transcription factor and in the cytosol as a regulator of membrane fission during intracellular trafficking and mitotic partitioning of the Golgi complex. Here, we report that ADP-ribosylation of CtBP1-S/BARS by BFA occurs via a nonconventional mechanism that comprises two steps: (i) synthesis of a BFA-ADP-ribose conjugate by the ADP-ribosyl cyclase CD38 and (ii) covalent binding of the BFA-ADP-ribose conjugate into the CtBP1-S/BARS NAD(+)-binding pocket. This results in the locking of CtBP1-S/BARS in a dimeric conformation, which prevents its binding to interactors known to be involved in membrane fission and, hence, in the inhibition of the fission machinery involved in mitotic Golgi partitioning. As this inhibition may lead to arrest of the cell cycle in G2, these findings provide a strategy for the design of pharmacological blockers of cell cycle in tumor cells that express high levels of CD38.

Inheritance of Silent rDNA Chromatin Is Mediated by PARP1 via Noncoding RNA

Faithful propagation of specific chromatin states requires re-establishment of epigenetic marks after every cell division. How the original epigenetic signature is inherited after disruption during DNA replication is still poorly understood. Here, we show that the poly(ADP-ribose)-polymerase-1 (PARP1/ARTD1) is implicated in the maintenance of silent rDNA chromatin during cell division. We demonstrate that PARP1 associates with TIP5, a subunit of the NoRC complex, via the noncoding pRNA and binds to silent rRNA genes after their replication in mid-late S phase. PARP1 represses rRNA transcription and is implicated in the formation of silent rDNA chromatin. Silent rDNA chromatin is a specific substrate for ADP-ribosylation and the enzymatic activity of PARP1 is necessary to establish rDNA silencing. The data unravel a function of PARP1 and ADP-ribosylation that serves to allow for the inheritance of silent chromatin structures, shedding light on how epigenetic marks are transmitted during each cell cycle.

Differential Regulation of P2X7 Receptor Activation by Extracellular Nicotinamide Adenine Dinucleotide and Ecto-ADP-Ribosyltransferases in Murine Macrophages and T Cells

Extracellular NAD induces the ATP-independent activation of the ionotropic P2X(7) purinergic receptor (P2X(7)R) in murine T lymphocytes via a novel covalent pathway involving ADP-ribosylation of arginine residues on the P2X(7)R ectodomain. This modification is catalyzed by ART2.2, a GPI-anchored ADP-ribosyltransferase (ART) that is constitutively expressed in murine T cells. We previously reported that ART2.1, a related ecto-ART, is up-regulated in inflammatory murine macrophages that constitutively express P2X(7)R. Thus, we tested the hypothesis that extracellular NAD acts via ART2.1 to regulate P2X(7)R function in murine macrophages. Coexpression of the cloned murine P2X(7)R with ART2.1 or ART2.2 in HEK293 cells verified that P2X(7)R is an equivalent substrate for ADP-ribosylation by either ART2.1 or ART2.2. However, in contrast with T cells, the stimulation of macrophages or HEK293 cells with NAD alone did not activate the P2X(7)R. Rather, NAD potentiated ATP-dependent P2X(7)R activation as indicated by a left shift in the ATP dose-response relationship. Thus, extracellular NAD regulates the P2X(7)R in both macrophages and T cells but via distinct mechanisms. Although ADP-ribosylation is sufficient to gate a P2X(7)R channel opening in T cells, this P2X(7)R modification in macrophages does not gate the channel but decreases the threshold for gating in response to ATP binding. These findings indicate that extracellular NAD and ATP can act synergistically to regulate P2X(7)R signaling in murine macrophages and also suggest that the cellular context in which P2X(7)R signaling occurs differs between myeloid and lymphoid leukocytes.

CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis

Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the ‘fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

Investigating Interactions Mediated by the Presynaptic Protein Bassoon in Living Cells by Foerster's Resonance Energy Transfer and Fluorescence Lifetime Imaging Microscopy

Neuronal synapses are highly specialized structures for communication between nerve cells. Knowledge about their molecular organization and dynamics is still incomplete. The large multidomain protein Bassoon plays a major role in scaffolding and organizing the cytomatrix at the active zone of neurotransmitter release in presynaptic boutons. Utilizing immunofluorescence techniques, we show that Bassoon is essential for corecruitment of its synaptic interaction partners, C-terminal binding protein 1/brefeldin A-dependent ADP-ribosylation substrate and CAZ-associated structural protein, into protein complexes upon heterologous expression in COS-7 cells. A combination of Foerster's resonance energy transfer and fluorescence lifetime imaging microscopy in the time domain was adopted to investigate the potential for the association of these proteins in the same complexes. A direct physical association between Bassoon and CtBP1 could also be observed at synapses of living hippocampal neurons. Simultaneous analysis of fluorescence decays of the donor and the acceptor probes along with their decay-associated spectra allowed a clear discrimination of energy transfer.

Pseudomonas aeruginosa ExoS ADP-ribosyltransferase inhibits ERM phosphorylation

Pseudomonas aeruginosa causes life-threatening infections in compromised and cystic fibrosis patients. Pathogenesis stems from a number of virulence factors, including four type III translocated cytotoxins: ExoS, ExoT, ExoY and ExoU. ExoS is a bifunctional toxin: the N terminus (amino acids 96-219) encodes a Rho GTPase Activating Protein (GAP) domain. The C terminus (amino acids 234-453) encodes a 14-3-3-dependent ADP-ribosyltransferase domain which transfers ADP-ribose from NAD onto substrates such as the Ras GTPases and vimentin. Ezrin/radixin/moesin (ERM) proteins have recently been identified as high-affinity substrates for ADP-ribosylation by ExoS. Expression of ExoS in HeLa cells led to a loss of phosphorylation of ERM proteins that was dependent upon the expression of ADP-ribosyltransferase activity. MALDI-MS and site-directed mutagenesis studies determined that ExoS ADP-ribosylated moesin at three C-terminal arginines (Arg553, Arg560 and Arg563), which cluster Thr558, the site of phosphorylation by protein kinase C and Rho kinase. ADP-ribosylated-moesin was a poor target for phosphorylation by protein kinase C and Rho kinase, which showed that ADP-ribosylation directly inhibited ERM phosphorylation. Expression of dominant active-moesin inhibited cell rounding elicited by ExoS, indicating that moesin is a physiological target in cultured cells. This is the first demonstration that a bacterial toxin inhibits the phosphorylation of a mammalian protein through ADP-ribosylation. These data explain how the expression of the ADP-ribosylation of ExoS modifies the actin cytoskeleton and indicate that ExoS possesses redundant enzymatic activities to depolymerize the actin cytoskeleton.

A role for BARS at the fission step of COPI vesicle formation from Golgi membrane

The core complex of Coat Protein I (COPI), known as coatomer, is sufficient to induce coated vesicular-like structures from liposomal membrane. In the context of biological Golgi membrane, both palmitoyl-coenzyme A (p-coA) and ARFGAP1, a GTPase-activating protein (GAP) for ADP-Ribosylation Factor 1, also participate in vesicle formation, but how their roles may be linked remains unknown. Moreover, whether COPI vesicle formation from Golgi membrane requires additional factors also remains unclear. We now show that Brefeldin-A ADP-Ribosylated Substrate (BARS) plays a critical role in the fission step of COPI vesicle formation from Golgi membrane. This role of BARS requires its interaction with ARFGAP1, which is in turn regulated oppositely by p-coA and nicotinamide adenine dinucleotide, which act as cofactors of BARS. Our findings not only identify a new factor needed for COPI vesicle formation from Golgi membrane but also reveal a surprising mechanism by which the roles of p-coA and GAP are linked in this process.

Crystal structure of the C3bot–RalA complex reveals a novel type of action of a bacterial exoenzyme

C3 exoenzymes from bacterial pathogens ADP-ribosylate and inactivate low-molecular-mass GTPases of the Rho subfamily. Ral, a Ras subfamily GTPase, binds the C3 exoenzymes from Clostridium botulinum and C. limosum with high affinity without being a substrate for ADP ribosylation. In the complex, the ADP-ribosyltransferase activity of C3 is blocked, while binding of NAD and NAD-glycohydrolase activity remain. Here we report the crystal structure of C3 from C. botulinum in a complex with GDP-bound RalA at 1.8 A resolution. C3 binds RalA with a helix-loop-helix motif that is adjacent to the active site. A quaternary complex with NAD suggests a mode for ADP-ribosyltransferase inhibition. Interaction of C3 with RalA occurs at a unique interface formed by the switch-II region, helix alpha3 and the P loop of the GTPase. C3-binding stabilizes the GDP-bound conformation of RalA and blocks nucleotide release. Our data indicate that C. botulinum exoenzyme C3 is a single-domain toxin with bifunctional properties targeting Rho GTPases by ADP ribosylation and Ral by a guanine nucleotide dissociation inhibitor-like effect, which blocks nucleotide exchange.

CD38 Controls ADP-Ribosyltransferase-2-Catalyzed ADP-Ribosylation of T Cell Surface Proteins

ADP-ribosyltransferase-2 (ART2), a GPI-anchored, toxin-related ADP-ribosylating ectoenzyme, is prominently expressed by murine T cells but not by B cells. Upon exposure of T cells to NAD, the substrate for ADP-ribosylation, ART2 catalyzes ADP-ribosylation of the P2X7 purinoceptor and other functionally important cell surface proteins. This in turn activates P2X7 and induces exposure of phosphatidylserine and shedding of CD62L. CD38, a potent ecto-NAD-glycohydrolase, is strongly expressed by most B cells but only weakly by T cells. Following incubation with NAD, CD38-deficient splenocytes exhibited lower NAD-glycohydrolase activity and stronger ADP-ribosylation of cell surface proteins than their wild-type counterparts. Depletion of CD38(high) cells from wild-type splenocytes resulted in stronger ADP-ribosylation on the remaining cells. Similarly, treatment of total splenocytes with the CD38 inhibitor nicotinamide 2'-deoxy-2'-fluoroarabinoside adenine dinucleotide increased the level of cell surface ADP-ribosylation. Furthermore, the majority of T cells isolated from CD38-deficient mice "spontaneously" exposed phosphatidylserine and lacked CD62L, most likely reflecting previous encounter with ecto-NAD. Our findings support the notion that ecto-NAD functions as a signaling molecule following its release from cells by lytic or nonlytic mechanisms. ART2 can sense and translate the local concentration of ecto-NAD into corresponding levels of ADP-ribosylated cell surface proteins, whereas CD38 controls the level of cell surface protein ADP-ribosylation by limiting the substrate availability for ART2.

Mutation of cysteine 214 in Gi1 alpha subunit abolishes its endogenous GTPase activity.

The functional consequences of the mutation of a conserved Cys-214 in Galpha(i1) have been investigated. We reported herein that substitutions of Cys-214 of Galpha(i1) to either alanine or tryptophan abolished the intrinsic GTPase activity. Free phosphate release from [32P]GTP-bound Galpha(i1) C214A or [32P]GTP-bound Galpha(i1) C214W was at least 30-fold lower than that of the wild-type Galpha(i1) in single-turnover GTPase assays. Consistently, tryptic proteolysis of C214A and C214W proteins showed that they were partially protected by GTP, further confirming that the GTPase activity in both mutant proteins was impaired. Expression of C214A or C214W mutants in Chinese hamster ovary K1 cells caused significant inhibition of forskolin-stimulated adenylate cyclase activity. However, the mutations did not significantly affect the GTP[S] (guanosine 5'-[gamma-[35S]thio]triphosphate)-binding activity. Both C214A and C214W mutants serve as good substrates for pertussis toxin-catalysed ADP ribosylation, indicating that they interact well with betagamma subunits. Moreover, RGS4 protein, a GTPase-activating protein for Galpha(i1), cannot interact with Cys-214 mutants even in the presence of AlF4-, which induces the transition state of Galpha. In summary, our findings suggest that C214A or C214W are GTPase-deficient mutants and can functionally serve as constitutively active forms of Galpha(i1) in cells.

Triggering of T-cell apoptosis by toxin-related ecto-ADP-ribosyltransferase ART2.

Cytotoxicity induced by protein ADP-ribosylation is a common theme of certain bacterial toxins and of the mammalian ectoenzyme ART2. Exposure of T cells to NAD, the substrate for ART2-catalyzed ADP-ribosylation, induces exposure of phosphatidylserine, uptake of propidium iodide, and fragmentation of DNA. ART2-specific antibodies raised by gene gun immunization block NAD-induced apoptosis. ART2 catalyzed ADP-ribosylation of cell membrane proteins induces formation of cytolytic membrane pores by activating the P2X7 purinoceptor. This alternative pathway to T cell apoptosis could be triggered upon the release of NAD from intracellular stores, for example, during inflammatory tissue damage.

NAD-Induced T Cell Death: ADP-Ribosylation of Cell Surface Proteins by ART2 Activates the Cytolytic P2X7 Purinoceptor

T cells express a toxin-related ADP-ribosylating ectoenzyme, ART2. Exposure of mature T cells to NAD, the substrate for ADP-ribosylation, induces cell death. ART2-catalyzed ADP-ribosylation activates the cytolytic P2X7 purinoceptor, causing calcium flux, pore formation, phosphatidylserine exposure, shedding of CD62L, cell shrinkage, and propidium iodide uptake. Interestingly, much lower NAD than ATP concentrations are required to activate P2X7. NAD-induced cell death (NICD) operates with endogenous sources of NAD released upon cell lysis. These findings identify P2X7 as a key effector of NICD and demonstrate that P2X7 can be activated by an endogenous ligand other than ATP. Our results delineate an alternative mechanism for inducing T cell death and set an interesting precedent for immunoregulation via crosstalk between NAD-dependent ADP-ribosyltransferases and purinoceptors.

Thiol-modifying phenylarsine oxide inhibits guanine nucleotide binding of Rho but not of Rac GTPases.

Phenylarsine oxide (PAO) is a phosphotyrosine phosphatase inhibitor that cross-links vicinal thiol groups, thereby inactivating phosphatases possessing XCysXXCysX motifs. The RhoA-GTPase, but not the Rac1-GTPase, also possesses vicinal cysteines within the guanine nucleotide-binding region (aa 13-20) and the phosphohydrolase activity site. Treatment of Caco-2 cells with PAO showed a dose-dependent reorganization of the actin cytoskeleton, indicating involvement of Rho GTPases. As tested by pull-down experiments, RhoA, but not Rac1, from cell lysates was inactivated by PAO in a concentration-dependent manner. Modification of RhoA by PAO resulted in altered mobility on SDS-polyacrylamide gel electrophoresis, and PAO-modified RhoA was no longer substrate for C3-catalyzed ADP-ribosylation. Furthermore, RhoA treated with PAO, but not Rac1 treated with PAO, lost its property to bind to guanine nucleotides. Matrix-assisted laser desorption ionization-mass analysis of PAO-modified RhoA showed a mass shift according to an adduction of a single PAO molecule per molecule RhoA. Further analysis of Glu-C-generated RhoA peptides confirmed binding of PAO to a peptide harboring the guanine nucleotide binding region. Thus, PAO does not exclusively inhibit phosphotyrosine phosphatases but also inactivates RhoA by alteration of nucleotide binding.