Adenosine deaminase 2 regulates the activation of the toll-like receptor 9 in response to nucleic acids.

Human cells contain two types of adenosine deaminases (ADA) each with unique properties: ADA1, which is present in all cells where it modulates intracellular functions and extracellular signaling, and ADA2, which is secreted by immune cells. The exact intracellular functions of ADA2 remain undetermined and less defined than those of ADA1. ADA2 has distinct characteristics, such as low adenosine affinity, heparin-binding ability, and putative lysosomal entry. Here, we confirm that ADA2 is a lysosomal protein that binds toll-like receptor 9 (TLR9) agonists, specifically CpG oligodeoxynucleotides (CpG ODNs). We show that interferon-alpha (IFN-α) is secreted in response to TLR9 activation by CpG ODNs and natural DNA and markedly increases when ADA2 expression is downregulated in plasmacytoid dendritic cells (pDCs). Additionally, the pretreatment of pDCs with RNA further stimulates IFN-α secretion by pDCs after activation with CpG ODNs. Our findings indicate that ADA2 regulates TLR9 responses to DNA in activated pDCs. In conclusion, decreasing ADA2 expression or blocking it with specific oligonucleotides can enhance IFN-α secretion from pDCs, improving immune responses against intracellular infections and cancer.

ELISA based assays to measure adenosine deaminases concentration in serum and saliva for the diagnosis of ADA2 deficiency and cancer.

Adenosine deaminases (ADAs) are enzymes of purine metabolism converting adenosine to inosine. There are two types of ADAs in humans ADA1 and ADA2. While both ADA1 and ADA2 share the same substrate, they differ in expression, cellular localization, and catalytic properties. The genetic deficiency of ADA1 results in severe combined immunodeficiency (SCID), while lack in ADA2 (DADA2) results in multiple phenotypes ranging from systemic inflammation to vascular pathology. Clinical studies have shown that the levels of ADAs in biological fluids are altered in pathophysiological conditions, suggesting that ADA activity could be a convenient marker for the diagnosis of immune diseases and cancer. Here, we describe sensitive and straightforward ELISA assays to measure ADA1 and ADA2 concentrations in biological fluids. Analysis of the serum and saliva samples from the healthy controls and DADA2 patients revealed that ADA2 enzyme concentration is significantly lower in patients than in healthy controls. In contrast, the concentration of ADA2 increases in the serum of patients with large granular leukocyte leukemia (LGLL) and patients' saliva with head and neck cancer. Thus, this simple, non-invasive method allows for distinguishing healthy controls from the affected patient. It can be implemented in screening and diagnosis of DADA2 and follow up the treatment of LGLL and several types of head and neck cancer.

Secreted Bacterial Adenosine Deaminase Is an Evolutionary Precursor of Adenosine Deaminase Growth Factor.

Adenosine deaminases (ADAs) play a pivotal role in regulating the level of adenosine, an important signaling molecule that controls a variety of cellular responses. Two distinct ADAs, ADA1 and adenosine deaminase growth factor (ADGF aka ADA2), are known. Cytoplasmic ADA1 plays a key role in purine metabolism and is widely distributed from prokaryotes to mammals. On the other hand, secreted ADGF/ADA2 is a cell-signaling protein that was thought to be present only in multicellular organisms. Here, we discovered a bacterial homologue of ADGF/ADA2. Bacterial and eukaryotic ADGF/ADA2 possess the dimerization and PRB domains characteristic for the family, have nearly identical catalytic sites, and show similar catalytic characteristics. Most surprisingly, the bacterial enzyme has a signal sequence similar to that of eukaryotic ADGF/ADA2 and is specifically secreted into the extracellular space, where it may potentially control the level of extracellular adenosine. This finding provides the first example of evolution of an extracellular eukaryotic signaling protein from a secreted bacterial analogue with identical activity and suggests a potential role of ADGF/ADA2 in bacterial communication.

Human adenosine deaminases ADA1 and ADA2 bind to different subsets of immune cells.

At sites of inflammation and tumor growth, the local concentration of extracellular adenosine rapidly increases and plays a role in controlling the immune responses of nearby cells. Adenosine deaminases ADA1 and ADA2 (ADAs) decrease the level of adenosine by converting it to inosine, which serves as a negative feedback mechanism. Mutations in the genes encoding ADAs lead to impaired immune function, which suggests a crucial role for ADAs in immune system regulation. It is not clear why humans and other mammals possess two enzymes with adenosine deaminase activity. Here, we found that ADA2 binds to neutrophils, monocytes, NK cells and B cells that do not express CD26, a receptor for ADA1. Moreover, the analysis of CD4+ T-cell subset revealed that ADA2 specifically binds to regulatory T cells expressing CD39 and lacking the receptor for ADA1. Also, it was found that ADA1 binds to CD16- monocytes, while CD16+ monocytes preferably bind ADA2. A study of the blood samples from ADA2-deficient patients showed a dramatic reduction in the number of lymphocyte subsets and an increased concentration of TNF-α in plasma. Our results suggest the existence of a new mechanism, where the activation and survival of immune cells is regulated through the activities of ADA2 or ADA1 anchored to the cell surface.

Application of ADA1 as a new marker enzyme in sandwich ELISA to study the effect of adenosine on activated monocytes.

Enzyme-linked immunosorbent assay (ELISA) is a valuable technique to detect antigens in biological fluids. Horse radish peroxidase (HRP) is one of the most common enzymes used for signal amplification in ELISA. Despite new advances in technology, such as a large-scale production of recombinant enzymes and availability of new detection systems, limited research is devoted to finding alternative enzymes and their substrates to amplify the ELISA signals. Here, HRP-avidin was substituted with the human adenosine deaminase (hADA1)-streptavidin complex and adenosine as a detection system in commercial ELISA kits. The hADA1 ELISA was successfully used to demonstrate that adenosine, bound to A1 and A3 adenosine receptors, increases cytokine secretion by LPS activated monocytes. We show that hADA1-based ELISA has the same sensitivity, and also provides identical results, as HRP ELISA. In addition, the sensitivity of hADA1-based ELISA could be easily adjusted by changing the adenosine concentration and the incubation time. Therefore, hADA1 could be used as a detection enzyme with any commercial ELISA kit with a wide range of concentration of antigens.

Early-onset stroke and vasculopathy associated with mutations in ADA2.

BACKGROUND: We observed a syndrome of intermittent fevers, early-onset lacunar strokes and other neurovascular manifestations, livedoid rash, hepatosplenomegaly, and systemic vasculopathy in three unrelated patients. We suspected a genetic cause because the disorder presented in early childhood. METHODS: We performed whole-exome sequencing in the initial three patients and their unaffected parents and candidate-gene sequencing in three patients with a similar phenotype, as well as two young siblings with polyarteritis nodosa and one patient with small-vessel vasculitis. Enzyme assays, immunoblotting, immunohistochemical testing, flow cytometry, and cytokine profiling were performed on samples from the patients. To study protein function, we used morpholino-mediated knockdowns in zebrafish and short hairpin RNA knockdowns in U937 cells cultured with human dermal endothelial cells. RESULTS: All nine patients carried recessively inherited mutations in CECR1 (cat eye syndrome chromosome region, candidate 1), encoding adenosine deaminase 2 (ADA2), that were predicted to be deleterious; these mutations were rare or absent in healthy controls. Six patients were compound heterozygous for eight CECR1 mutations, whereas the three patients with polyarteritis nodosa or small-vessel vasculitis were homozygous for the p.Gly47Arg mutation. Patients had a marked reduction in the levels of ADA2 and ADA2-specific enzyme activity in the blood. Skin, liver, and brain biopsies revealed vasculopathic changes characterized by compromised endothelial integrity, endothelial cellular activation, and inflammation. Knockdown of a zebrafish ADA2 homologue caused intracranial hemorrhages and neutropenia - phenotypes that were prevented by coinjection with nonmutated (but not with mutated) human CECR1. Monocytes from patients induced damage in cocultured endothelial-cell layers. CONCLUSIONS: Loss-of-function mutations in CECR1 were associated with a spectrum of vascular and inflammatory phenotypes, ranging from early-onset recurrent stroke to systemic vasculopathy or vasculitis. (Funded by the National Institutes of Health Intramural Research Programs and others.).

Human adenosine deaminase 2 induces differentiation of monocytes into macrophages and stimulates proliferation of T helper cells and macrophages.

ADAs play a pivotal role in regulating the level of adenosine, a signaling molecule controlling a variety of cellular responses by binding to and activating four ADRs. Two enzymes, ADA1 and ADA2, are known to possess ADA activity in humans. Although the structure of ADA1 and its role in lymphocytic activation have been known for a long time, the structure and function of ADA2, a member of ADGF, remain enigmatic. Here, we found that ADA2 is secreted by monocytes undergoing differentiation into macrophages or DCs and that it binds to the cell surface via proteoglycans and ADRs. We demonstrate that ADA1 and ADA2 increase the rate of proliferation of monocyte-activated CD4+ T cells independently of their catalytic activity. We also show that ADA2 induces T cell-dependent differentiation of monocytes into macrophages and stimulates macrophage proliferation. Our discovery of the growth factor-like activity of ADA2 explains clinical observations and suggests that this enzyme could be used as a drug candidate to modulate the immune responses during inflammation and cancer.

Structural basis for the growth factor activity of human adenosine deaminase ADA2.

Two distinct adenosine deaminases, ADA1 and ADA2, are found in humans. ADA1 has an important role in lymphocyte function and inherited mutations in ADA1 result in severe combined immunodeficiency. The recently isolated ADA2 belongs to the novel family of adenosine deaminase growth factors (ADGFs), which play an important role in tissue development. The crystal structures of ADA2 and ADA2 bound to a transition state analogue presented here reveal the structural basis of the catalytic/signaling activity of ADGF/ADA2 proteins. In addition to the catalytic domain, the structures discovered two ADGF/ADA2-specific domains of novel folds that mediate the protein dimerization and binding to the cell surface receptors. This complex architecture is in sharp contrast with that of monomeric single domain ADA1. An extensive glycosylation and the presence of a conserved disulfide bond and a signal peptide in ADA2 strongly suggest that ADA2, in contrast to ADA1, is specifically designed to act in the extracellular environment. The comparison of catalytic sites of ADA2 and ADA1 demonstrates large differences in the arrangement of the substrate-binding pockets. These structural differences explain the substrate and inhibitor specificity of adenosine deaminases and provide the basis for a rational design of ADA2-targeting drugs to modulate the immune system responses in pathophysiological conditions.

Specific interaction between EF-G and RRF and its implication for GTP-dependent ribosome splitting into subunits.

After termination of protein synthesis, the bacterial ribosome is split into its 30S and 50S subunits by the action of ribosome recycling factor (RRF) and elongation factor G (EF-G) in a guanosine 5'-triphosphate (GTP)-hydrolysis-dependent manner. Based on a previous cryo-electron microscopy study of ribosomal complexes, we have proposed that the binding of EF-G to an RRF-containing posttermination ribosome triggers an interdomain rotation of RRF, which destabilizes two strong intersubunit bridges (B2a and B3) and, ultimately, separates the two subunits. Here, we present a 9-A (Fourier shell correlation cutoff of 0.5) cryo-electron microscopy map of a 50S x EF-G x guanosine 5'-[(betagamma)-imido]triphosphate x RRF complex and a quasi-atomic model derived from it, showing the interaction between EF-G and RRF on the 50S subunit in the presence of the noncleavable GTP analogue guanosine 5'-[(betagamma)-imido]triphosphate. The detailed information in this model and a comparative analysis of EF-G structures in various nucleotide- and ribosome-bound states show how rotation of the RRF head domain may be triggered by various domains of EF-G. For validation of our structural model, all known mutations in EF-G and RRF that relate to ribosome recycling have been taken into account. More importantly, our results indicate a substantial conformational change in the Switch I region of EF-G, suggesting that a conformational signal transduction mechanism, similar to that employed in transfer RNA translocation on the ribosome by EF-G, translates a large-scale movement of EF-G's domain IV, induced by GTP hydrolysis, into the domain rotation of RRF that eventually splits the ribosome into subunits.

Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies.

Ribosome recycling, the disassembly of the posttermination complex after each round of protein synthesis, is an essential step in mRNA translation, but its mechanism has remained obscure. In eubacteria, recycling is catalyzed by RRF (ribosome recycling factor) and EF-G (elongation factor G). By using cryo-electron microscopy, we have obtained two density maps, one of the RRF bound posttermination complex and one of the 50S subunit bound with both EF-G and RRF. Comparing the two maps, we found domain I of RRF to be in the same orientation, while domain II in the EF-G-containing 50S subunit is extensively rotated (approximately 60 degrees) compared to its orientation in the 70S complex. Mapping the 50S conformation of RRF onto the 70S posttermination complex suggests that it can disrupt the intersubunit bridges B2a and B3, and thus effect a separation of the two subunits. These observations provide the structural basis for the mechanism by which the posttermination complex is split into subunits by the joint action of RRF and EF-G.

Human ADA2 belongs to a new family of growth factors with adenosine deaminase activity.

Two distinct isoenzymes of ADA (adenosine deaminase), ADA1 and ADA2, have been found in humans. Inherited mutations in ADA1 result in SCID (severe combined immunodeficiency). This observation has led to extensive studies of the structure and function of this enzyme that have revealed an important role for it in lymphocyte activation. In contrast, the physiological role of ADA2 is unknown. ADA2 is found in negligible quantities in serum and may be produced by monocytes/macrophages. ADA2 activity in the serum is increased in various diseases in which monocyte/macrophage cells are activated. In the present study, we report that ADA2 is a heparin-binding protein. This allowed us to obtain a highly purified enzyme and to study its biochemistry. ADA2 was identified as a member of a new class of ADGFs (ADA-related growth factors), which is present in almost all organisms from flies to humans. Our results suggest that ADA2 may be active in sites of inflammation during hypoxia and in areas of tumour growth where the adenosine concentration is significantly elevated and the extracellular pH is acidic. Our finding that ADA2 co-purified and concentrated together with IgG in commercially available preparations offers an intriguing explanation for the observation that treatment with such preparations leads to non-specific immune-system stimulation.

Splitting of the posttermination ribosome into subunits by the concerted action of RRF and EF-G.

After peptide release by a class-1 release factor, the ribosomal subunits must be recycled back to initiation. We have demonstrated that the distance between a strong Shine-Dalgarno (SD) sequence and a codon in the P site is crucial for the binding stability of the deacylated tRNA in the P site of the posttermination ribosome and the in-frame maintenance of its mRNA. We show that the elongation factor EF-G and the ribosomal recycling factor RRF split the ribosome into subunits in the absence of initiation factor 3 (IF3) by a mechanism that requires both GTP and GTP hydrolysis. Taking into account that EF-G in the GTP form and RRF bind with positive cooperativity to the free 50S subunit but with negative cooperativity to the 70S ribosome, we suggest a mechanism for ribosome recycling that specifies distinct roles for EF-G, RRF, and IF3.

Guanine-nucleotide exchange on ribosome-bound elongation factor G initiates the translocation of tRNAs.

BACKGROUND: During the translation of mRNA into polypeptide, elongation factor G (EF-G) catalyzes the translocation of peptidyl-tRNA from the A site to the P site of the ribosome. According to the 'classical' model, EF-G in the GTP-bound form promotes translocation, while hydrolysis of the bound GTP promotes dissociation of the factor from the post-translocation ribosome. According to a more recent model, EF-G operates like a 'motor protein' and drives translocation of the peptidyl-tRNA after GTP hydrolysis. In both the classical and motor protein models, GDP-to-GTP exchange is assumed to occur spontaneously on 'free' EF-G even in the absence of a guanine-nucleotide exchange factor (GEF). RESULTS: We have made a number of findings that challenge both models. First, free EF-G in the cell is likely to be in the GDP-bound form. Second, the ribosome acts as the GEF for EF-G. Third, after guanine-nucleotide exchange, EF-G in the GTP-bound form moves the tRNA2-mRNA complex to an intermediate translocation state in which the mRNA is partially translocated. Fourth, subsequent accommodation of the tRNA2-mRNA complex in the post-translocation state requires GTP hydrolysis. CONCLUSION: These results, in conjunction with previously published cryo-electron microscopy reconstructions of the ribosome in various functional states, suggest a novel mechanism for translocation of tRNAs on the ribosome by EF-G. Our observations suggest that the ribosome is a universal guanosine-nucleotide exchange factor for EF-G as previously shown for the class-II peptide-release factor 3.

Peptidyl-tRNA regulates the GTPase activity of translation factors.

Rapid protein synthesis in bacteria requires the G proteins IF2, EF-Tu, EF-G, and RF3. These factors catalyze all major steps of mRNA translation in a GTP-dependent manner. Here, it is shown how the position of peptidyl-tRNA in the ribosome and presence of its peptide control the binding and GTPase activity of these translation factors. The results explain how idling GTPase activity and negative interference between different translation factors are avoided and suggest that hybrid sites for tRNA on the ribosome play essential roles in translocation of tRNAs, recycling of class 1 release factors by RF3, and recycling of ribosomes back to a new round of initiation. We also propose a model for translocation of tRNAs in two separate steps, which clarifies the roles of EF-G.GTP and GTP hydrolysis in this process.

The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site.

The Escherichia coli relBE operon encodes a toxin-antitoxin pair, RelE-RelB. RelB can reverse inhibition of protein synthesis by RelE in vivo. We have found that although RelE does not degrade free RNA, it cleaves mRNA in the ribosomal A site with high codon specificity. Among stop codons UAG is cleaved with fast, UAA intermediate and UGA slow rate, while UCG and CAG are cleaved most rapidly among sense codons. We suggest that inhibition of protein synthesis by RelE is reversed with the help of tmRNA, and that RelE plays a regulatory role in bacteria during adaptation to poor growth conditions.

A cryo-electron microscopic study of ribosome-bound termination factor RF2.

Protein synthesis takes place on the ribosome, where genetic information carried by messenger RNA is translated into a sequence of amino acids. This process is terminated when a stop codon moves into the ribosomal decoding centre (DC) and is recognized by a class-1 release factor (RF). RFs have a conserved GGQ amino-acid motif, which is crucial for peptide release and is believed to interact directly with the peptidyl-transferase centre (PTC) of the 50S ribosomal subunit. Another conserved motif of RFs (SPF in RF2) has been proposed to interact directly with stop codons in the DC of the 30S subunit. The distance between the DC and PTC is approximately 73 A. However, in the X-ray structure of RF2, SPF and GGQ are only 23 A apart, indicating that they cannot be at DC and PTC simultaneously. Here we show that RF2 is in an open conformation when bound to the ribosome, allowing GGQ to reach the PTC while still allowing SPF-stop-codon interaction. The results indicate new interpretations of accuracy in termination, and have implications for how the presence of a stop codon in the DC is signalled to PTC.

Structure of the Escherichia coli ribosomal termination complex with release factor 2.

Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.