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Journal of Interferon & Cytokine... Jan 2011Adenosine deaminases acting on RNA (ADARs) catalyze adenosine (A) to inosine (I) editing of RNA that possesses double-stranded (ds) structure. A-to-I RNA editing results... (Review)
Review
Adenosine deaminases acting on RNA (ADARs) catalyze adenosine (A) to inosine (I) editing of RNA that possesses double-stranded (ds) structure. A-to-I RNA editing results in nucleotide substitution, because I is recognized as G instead of A both by ribosomes and by RNA polymerases. A-to-I substitution can also cause dsRNA destabilization, as I:U mismatch base pairs are less stable than A:U base pairs. Three mammalian ADAR genes are known, of which two encode active deaminases (ADAR1 and ADAR2). Alternative promoters together with alternative splicing give rise to two protein size forms of ADAR1: an interferon-inducible ADAR1-p150 deaminase that binds dsRNA and Z-DNA, and a constitutively expressed ADAR1-p110 deaminase. ADAR2, like ADAR1-p110, is constitutively expressed and binds dsRNA. A-to-I editing occurs with both viral and cellular RNAs, and affects a broad range of biological processes. These include virus growth and persistence, apoptosis and embryogenesis, neurotransmitter receptor and ion channel function, pancreatic cell function, and post-transcriptional gene regulation by microRNAs. Biochemical processes that provide a framework for understanding the physiologic changes following ADAR-catalyzed A-to-I ( = G) editing events include mRNA translation by changing codons and hence the amino acid sequence of proteins; pre-mRNA splicing by altering splice site recognition sequences; RNA stability by changing sequences involved in nuclease recognition; genetic stability in the case of RNA virus genomes by changing sequences during viral RNA replication; and RNA-structure-dependent activities such as microRNA production or targeting or protein-RNA interactions.
Topics: Adenosine Deaminase; Animals; Gene Expression Regulation, Enzymologic; Humans; Interferons; Isoenzymes; Protein Interaction Domains and Motifs; RNA Editing; RNA, Double-Stranded; RNA-Binding Proteins
PubMed: 21182352
DOI: 10.1089/jir.2010.0097 -
The Journal of Biological Chemistry Oct 1985Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of the immune system. For the purpose of studying...
Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of the immune system. For the purpose of studying the function and structure of this enzyme, we have isolated by genetic selection a mouse cell line, B-1/50, in which adenosine deaminase levels were increased 4,300-fold over the parent cell line. The enzyme was purified from these cells in large quantity and high yield by a simple two-step purification scheme. The enzyme derived from the B-1/50 cells was indistinguishable from that of the parental cells as judged by several biochemical criteria. The Km (30 microM) and Ki (4 nM) values using adenosine as substrate and 2'-deoxycoformycin as inhibitor, respectively, were identical for the enzyme derived from the parental cells as well as the adenosine deaminase gene amplification mutants. The enzyme from both cell types exhibited multiple isoelectric focusing forms which co-purified using our purification protocol. Electrophoretic analysis using sodium dodecyl sulfate-polyacrylamide gels showed that adenosine deaminase migrated with an apparent molecular weight of 41,000 or 36,000 depending on whether the enzyme was reduced or oxidized, respectively. This shift was reversible, indicating that proteolysis was not responsible for the faster migrating form. Monospecific antibodies raised against purified adenosine deaminase cross-reacted with the enzyme derived from the parental cells and precipitated 37% of the total soluble protein in the B-1/50 cells. Continued genetic selection resulted in the isolation of cells in which adenosine deaminase was overproduced by 11,400-fold and accounted for over 75% of the soluble protein.
Topics: Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Cell Line; Coformycin; Electrophoresis, Polyacrylamide Gel; Gene Amplification; Half-Life; Immunosorbent Techniques; Isoelectric Focusing; Kinetics; Mice; Molecular Weight; Mutation; Nucleoside Deaminases; Pentostatin
PubMed: 3902813
DOI: No ID Found -
BioEssays : News and Reviews in... Apr 2017Deamination of adenosine in RNA to form inosine has wide ranging consequences on RNA function including amino acid substitution to give proteins not encoded in the... (Review)
Review
Deamination of adenosine in RNA to form inosine has wide ranging consequences on RNA function including amino acid substitution to give proteins not encoded in the genome. What determines which adenosines in an mRNA are subject to this modification reaction? The answer lies in an understanding of the mechanism and substrate recognition properties of adenosine deaminases that act on RNA (ADARs). Our recent publication of X-ray crystal structures of the human ADAR2 deaminase domain bound to RNA editing substrates shed considerable light on how the catalytic domains of these enzymes bind RNA and promote adenosine deamination. Here we review in detail the deaminase domain-RNA contact surfaces and present models of how full length ADARs, bearing double stranded RNA-binding domains (dsRBDs) and deaminase domains, could process naturally occurring substrate RNAs.
Topics: Adenosine Deaminase; Catalytic Domain; Humans; Models, Molecular; Protein Conformation; RNA; RNA Editing; RNA-Binding Proteins; Substrate Specificity
PubMed: 28217931
DOI: 10.1002/bies.201600187 -
The Journal of Biological Chemistry Sep 1980Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4)-deficient patients recently were found to have abnormally high levels of dATP, a negative allosteric effector...
Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4)-deficient patients recently were found to have abnormally high levels of dATP, a negative allosteric effector of ribonucleotide reductase (ribonucleoside-diphosphate reductase, 2'-deoxyribonucleoside-diphosphate:oxidized thioredoxin 2'-oxidoreductase, EC 1.17.4.1). Therefore it was proposed that the immunodeficiency associated with adenosine deaminase deficiency is mediated through inhibition of ribonucleotide reductase and hence DNA replication. HeLa cells, treated with an adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl)adenine, and deoxyadenosine to mimic the adenosine deaminase-deficient state, were monitored to determine directly the effects on ribonucleotide reductase activity and levels. A low concentration of erythro-9-(2-hydroxy-3-nonyl)adenine, which did not inhibit cell growth, nevertheless retarded the cells in G2 + M phase of the cell cycle and increased reductase activity. Reductase activity was also elevated in cells treated with a low level of deoxyadenosine which did not affect the cell cycle or cell growth. However, ribonucleotide reductase activity was reduced to one-half of the control value in cells treated with either enough deoxyadenosine to inhibit cell growth or with a combination of erythro-9(2-hydroxy-3-nonyl)adenine and deoxyadenosine, each at concentrations which individually do not inhibit cell growth. Removal of deoxynucleotides, particularly dATP, from these extracts increased ribonucleotide reductase activity to several-fold higher than control values. The reduced activity of ribonucleotide reductase in the simulated adenosine deaminase-deficient HeLa cells provides direct evidence for the thesis that adenosine deaminase deficiency disease is mediated through elevated levels of dATP which inhibit ribonucleotide reductase. In addition, the cell cycle patterns and ribonucleotide reductase levels suggest that the regulatory substance(s) that controls the level of ribonucleotide reductase is not operative until the late S or G2 phase of the cell cycle.
Topics: Adenine; Adenosine Deaminase; Cell Division; Deoxyadenosines; Deoxyribonucleotides; HeLa Cells; Humans; Nucleoside Deaminases; Ribonucleotide Reductases
PubMed: 6997299
DOI: No ID Found -
Pediatric Rheumatology Online Journal Jul 2020Human adenosine deaminase 2 (ADA2) is an extracellular enzyme that negatively regulates adenosine-mediated cell signaling by converting adenosine to inosine. Altered...
BACKGROUND
Human adenosine deaminase 2 (ADA2) is an extracellular enzyme that negatively regulates adenosine-mediated cell signaling by converting adenosine to inosine. Altered ADA2 enzyme activity has been associated with some viral infections and rheumatic diseases. The potential utility of ADA2 as a biomarker is, however, limited by the absence of established ranges of ADA2 concentration and enzyme activity in the healthy population. It is known that ADA2 enzyme activity is lower in adults, but when (and why) this decline happens is not known. The purpose of this study was to establish normative ranges of ADA2 enzyme activity and protein concentration in the healthy pediatric population.
METHODS
We modified a commercially available ADA2 enzyme activity assay to enable higher throughput analysis of fresh, frozen and hemolyzed blood samples. With this assay and ADA2 protein immunoblotting, we analyzed ADA2 enzyme activity and protein concentration in blood plasma from a cohort of children and adolescents (n = 94) aged 5 months to 18 years. One-way ANOVA with subsequent Tukey multiple comparison test was used to analyze group differences. Reference intervals were generated using the central 95% of the population (2-97.5 percentiles).
RESULTS
ADA2 enzyme activity was consistent in fresh, frozen, and hemolyzed sera and plasma as measured by our modified assay. Analysis of plasma samples from the healthy pediatric cohort revealed that ADA2 enzyme activity is significantly lower in older children than in younger children (p < 0.0001). In contrast, there was no significant correlation between ADA2 protein concentration and either age or ADA2 enzyme activity.
CONCLUSION
We observed that ADA2 enzyme activity, but not ADA2 protein concentration, negatively correlates with age in a cohort of children and adolescents. Our findings stress the importance of appropriate age-matched controls for assessing ADA2 enzyme activity in the clinical setting.
Topics: Adenosine Deaminase; Adolescent; Age Factors; Biomarkers; Canada; Child; Correlation of Data; Enzyme Assays; Female; Humans; Infant; Intercellular Signaling Peptides and Proteins; Male; Reference Values; Signal Transduction
PubMed: 32650798
DOI: 10.1186/s12969-020-00446-5 -
Science Advances Mar 2024Chronic and aberrant nucleic acid sensing causes type I IFN-driven autoimmune diseases, designated type I interferonopathies. We found a significant reduction of...
Chronic and aberrant nucleic acid sensing causes type I IFN-driven autoimmune diseases, designated type I interferonopathies. We found a significant reduction of regulatory T cells (T) in patients with type I interferonopathies caused by mutations in or (encoding MDA5). We analyzed the underlying mechanisms using murine models and found that T-specific deletion of caused peripheral T loss and -like lethal autoimmune disorders. Similarly, knock-in mice with T-specific expression of an MDA5 gain-of-function mutant caused apoptosis of peripheral T and severe autoimmunity. Moreover, the impact of ADAR1 deficiency on T is multifaceted, involving both MDA5 and PKR sensing. Together, our results highlight the dysregulation of T homeostasis by intrinsic aberrant RNA sensing as a potential determinant for type I interferonopathies.
Topics: Humans; Mice; Animals; Autoimmunity; RNA; T-Lymphocytes, Regulatory; Autoimmune Diseases; Nucleic Acids; Adenosine Deaminase
PubMed: 38427731
DOI: 10.1126/sciadv.adk0820 -
Cell Reports Nov 2022Adenosine deaminase acting on RNA-1 (ADAR1) is a ubiquitously expressed RNA deaminase catalyzing adenosine-to-inosine editing to prevent hyperactivated cytosolic...
Adenosine deaminase acting on RNA-1 (ADAR1) is a ubiquitously expressed RNA deaminase catalyzing adenosine-to-inosine editing to prevent hyperactivated cytosolic double-stranded RNA (dsRNA) response mediated by MDA5. Here, we demonstrate that ADAR1 is essential for early B lymphopoiesis from late pro-B and large pre-B cell stages onward. ADAR1 exerts its requisite role via both MDA5-dependent and -independent pathways. Interestingly, the MDA5-dependent mechanisms regulate early pro-B to large pre-B cell transition by promoting early B cell survival. In contrast, the MDA5-independent mechanisms control large pre-B to small pre-B cell transition by regulating pre-B cell receptor (pre-BCR) expression. Moreover, we show that protein kinase R (PKR) and oligoadenylate synthetase/ribonuclease (OAS/RNase) L pathways are dispensable for ADAR1's role in early B lymphopoiesis. Importantly, we demonstrate that p150 isoform of ADAR1 exclusively accounts for ADAR1's function in early B lymphopoiesis, and its conventional dsRNA-binding, but not the Z-DNA/RNA-binding or the RNA-editing, activity is required for ADAR1's function in B cell development. Thus, our findings suggest that ADAR1 regulates early B lymphopoiesis through various mechanisms.
Topics: Adenosine Deaminase; Lymphopoiesis; RNA-Binding Proteins; RNA Editing; RNA, Double-Stranded
PubMed: 36417848
DOI: 10.1016/j.celrep.2022.111687 -
Genome Biology and Evolution Nov 2021Adenosine Deaminases that Act on RNA (ADARs) are RNA editing enzymes that play a dynamic and nuanced role in regulating transcriptome and proteome diversity. This... (Review)
Review
Adenosine Deaminases that Act on RNA (ADARs) are RNA editing enzymes that play a dynamic and nuanced role in regulating transcriptome and proteome diversity. This editing can be highly selective, affecting a specific site within a transcript, or nonselective, resulting in hyperediting. ADAR editing is important for regulating neural functions and autoimmunity, and has a key role in the innate immune response to viral infections, where editing can have a range of pro- or antiviral effects and can contribute to viral evolution. Here we examine the role of ADAR editing across a broad range of viral groups. We propose that the effect of ADAR editing on viral replication, whether pro- or antiviral, is better viewed as an axis rather than a binary, and that the specific position of a given virus on this axis is highly dependent on virus- and host-specific factors, and can change over the course of infection. However, more research needs to be devoted to understanding these dynamic factors and how they affect virus-ADAR interactions and viral evolution. Another area that warrants significant attention is the effect of virus-ADAR interactions on host-ADAR interactions, particularly in light of the crucial role of ADAR in regulating neural functions. Answering these questions will be essential to developing our understanding of the relationship between ADAR editing and viral infection. In turn, this will further our understanding of the effects of viruses such as SARS-CoV-2, as well as many others, and thereby influence our approach to treating these deadly diseases.
Topics: Adenosine Deaminase; Animals; Evolution, Molecular; Host-Pathogen Interactions; Humans; Immunity; RNA Editing; RNA Viruses; RNA, Viral; Virus Replication
PubMed: 34694399
DOI: 10.1093/gbe/evab240 -
The Journal of Biological Chemistry May 1994The double-stranded RNA (dsRNA) adenosine deaminase (DRADA) deaminates adenosine residues to inosines and creates I-U mismatched base pairs in dsRNAs. Its involvement in...
The double-stranded RNA (dsRNA) adenosine deaminase (DRADA) deaminates adenosine residues to inosines and creates I-U mismatched base pairs in dsRNAs. Its involvement in RNA editing of glutamate-gated ion channel gene transcripts in mammalian brains has been proposed as one of the biological functions for this recently identified cellular enzyme. We purified a mixture of three forms, 93, 88, and 83 kDa, of bovine DRADA proteins, all likely to be active enzymes. We determined that DRADA has a native molecular mass of approximately 100 kDa, suggesting that the enzyme exists as a monomer. The purified enzyme was not inhibited by 2'-deoxycoformycin, a transition state analog inhibitor of adenosine deaminase and AMP deaminase, suggesting that the catalytic mechanism of DRADA might be different from that of other deaminases. DRADA binds specifically to dsRNA with a dissociation constant of 0.23 nM for a synthetic dsRNA, and the Michaelis constant is 0.85 nM. These values indicate that DRADA has a much higher affinity for its substrate than other deaminases such as adenosine deaminase and AMP deaminase. DRADA may need this extremely high affinity to catalyze efficiently the modification of relatively rare substrate RNAs in the cell nucleus.
Topics: Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Cattle; Cell Nucleus; Chelating Agents; Chromatography, Gel; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Kinetics; Liver; Molecular Weight; RNA-Binding Proteins; Substrate Specificity; Zinc
PubMed: 8175781
DOI: No ID Found -
RNA (New York, N.Y.) Mar 2023Z-RNA is a higher-energy, left-handed conformation of RNA, whose function has remained elusive. A growing body of work alludes to regulatory roles for Z-RNA in the... (Review)
Review
Z-RNA is a higher-energy, left-handed conformation of RNA, whose function has remained elusive. A growing body of work alludes to regulatory roles for Z-RNA in the immune response. Here, we review how Z-RNA features present in cellular RNAs-especially containing retroelements-could be recognized by a family of winged helix proteins, with an impact on host defense. We also discuss how mutations to specific Z-contacting amino acids disrupt their ability to stabilize Z-RNA, resulting in functional losses. We end by highlighting knowledge gaps in the field, which, if addressed, would significantly advance this active area of research.
Topics: RNA; Adenosine Deaminase; Immunity, Innate; Amino Acids; Biology; DNA, Z-Form
PubMed: 36596670
DOI: 10.1261/rna.079429.122