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Archives of Microbiology Dec 1980Adenosine deaminase (EC 3.5.4.4) was found to occur in the extract of Azotobacter vinelandii, strain 0, and purified by heating at 65 degrees C, fractionation with...
Adenosine deaminase (EC 3.5.4.4) was found to occur in the extract of Azotobacter vinelandii, strain 0, and purified by heating at 65 degrees C, fractionation with ammonium sulfate, DEAE-cellulose chromatography and gel filtration on Sephadex G-150. Purified adenosine deaminase was effectively stabilized by the addition of ethylene glycol. The molecular weight of the enzyme was estimated to be 66,000 by gel filtration on Sephadex G-150. The enzyme specifically attacked adenosine and 2'-deoxyadenosine to the same extent, and formycin A to a lesser extent. The pH optimum of the enzyme was observed at pH 7.2. Double reciprocal plot of initial velocity versus adenosine concentration was concave upward, and Hill interaction coefficient was calculated to be 1.5, suggesting the allosteric binding of the substrate. ATP inhibited adenosine deaminase in an allosteric manner, whereas other nucleotides were without effect. The physiological significance of the enzyme was discussed in relation to salvage pathway of purine nucleotides.
Topics: Adenosine Deaminase; Allosteric Regulation; Azotobacter; Hydrogen-Ion Concentration; Kinetics; Molecular Weight; Nucleoside Deaminases; Substrate Specificity
PubMed: 7212927
DOI: 10.1007/BF00406163 -
Current Gene Therapy 2019Members of the adenosine deaminase acting on RNA (ADAR) family of enzymes consist of double-stranded RNA-binding domains (dsRBDs) and a deaminase domain (DD) that... (Comparative Study)
Comparative Study
INTRODUCTION
Members of the adenosine deaminase acting on RNA (ADAR) family of enzymes consist of double-stranded RNA-binding domains (dsRBDs) and a deaminase domain (DD) that converts adenosine (A) into inosine (I), which acts as guanosine (G) during translation. Using the MS2 system, we engineered the DD of ADAR1 to direct it to a specific target. The aim of this work was to compare the deaminase activities of ADAR1-DD and various isoforms of ADAR2-DD.
MATERIALS AND METHODS
We measured the binding affinity of the artificial enzyme system on a Biacore ™ X100. ADARs usually target dsRNA, so we designed a guide RNA complementary to the target RNA, and then fused the guide sequence to the MS2 stem-loop. A mutated amber (TAG) stop codon at 58 amino acid (TGG) of EGFP was targeted. After transfection of these three factors into HEK 293 cells, we observed fluorescence signals of various intensities.
RESULTS
ADAR2-long without the Alu-cassette yielded a much higher fluorescence signal than ADAR2-long with the Alu-cassette. With another isoform, ADAR2-short, which is 81 bp shorter at the C-terminus, the fluorescence signal was undetectable. A single amino acid substitution of ADAR2-long-DD (E488Q) rendered the enzyme more active than the wild type. The results of fluorescence microscopy suggested that ADAR1-DD is more active than ADAR2-long-DD. Western blots and sequencing confirmed that ADAR1-DD was more active than any other DD.
CONCLUSION
This study provides information that should facilitate the rational use of ADAR variants for genetic restoration and treatment of genetic diseases.
Topics: Adenosine Deaminase; Genetic Code; Genetic Therapy; HEK293 Cells; Humans; Protein Isoforms; RNA; RNA Editing; RNA-Binding Proteins
PubMed: 30426900
DOI: 10.2174/1566523218666181114122116 -
Nature Biotechnology Sep 2019Current tools for targeted RNA editing rely on the delivery of exogenous proteins or chemically modified guide RNAs, which may lead to aberrant effector activity,...
Current tools for targeted RNA editing rely on the delivery of exogenous proteins or chemically modified guide RNAs, which may lead to aberrant effector activity, delivery barrier or immunogenicity. Here, we present an approach, called leveraging endogenous ADAR for programmable editing of RNA (LEAPER), that employs short engineered ADAR-recruiting RNAs (arRNAs) to recruit native ADAR1 or ADAR2 enzymes to change a specific adenosine to inosine. We show that arRNA, delivered by a plasmid or viral vector or as a synthetic oligonucleotide, achieves editing efficiencies of up to 80%. LEAPER is highly specific, with rare global off-targets and limited editing of non-target adenosines in the target region. It is active in a broad spectrum of cell types, including multiple human primary cell types, and can restore α-L-iduronidase catalytic activity in Hurler syndrome patient-derived primary fibroblasts without evoking innate immune responses. As a single-molecule system, LEAPER enables precise, efficient RNA editing with broad applicability for therapy and basic research.
Topics: Adenosine Deaminase; Animals; Cell Line; Genetic Engineering; Humans; RNA; RNA Editing; RNA-Binding Proteins
PubMed: 31308540
DOI: 10.1038/s41587-019-0178-z -
Nature Biotechnology Sep 2019Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted...
Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BE-PACE) to improve their editing efficiency and target sequence compatibility. We used BE-PACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.
Topics: Adenosine Deaminase; Animals; Base Sequence; CRISPR-Cas Systems; Cell Line; Directed Molecular Evolution; Gene Editing; Gene Expression Regulation, Enzymologic; Gene Targeting; Humans; INDEL Mutation; Mice
PubMed: 31332326
DOI: 10.1038/s41587-019-0193-0 -
Progress in Nucleic Acid Research and... 2006
Review
Topics: Adenosine Deaminase; Animals; Communicable Diseases; Humans; Interferons; RNA, Double-Stranded; RNA-Binding Proteins; eIF-2 Kinase
PubMed: 16891177
DOI: 10.1016/S0079-6603(06)81010-X -
Journal of Cellular Biochemistry Mar 1989Adenosine deaminase (ADA) deficiency in humans is one cause of severe combined immunodeficiency. When ADA fails to catalyze the deamination of adenosine and... (Review)
Review
Adenosine deaminase (ADA) deficiency in humans is one cause of severe combined immunodeficiency. When ADA fails to catalyze the deamination of adenosine and deoxyadenosine, the levels of deoxyadenosine that accumulate are toxic to lymphoid cells. Patients with complete ADA deficiency (e.g., with less than 5% normal ADA catalytic activity) lack both B- and T-lymphocyte function. B-lymphoblast cell lines derived from patients with ADA deficiency have been analyzed at multiple levels. Blot hybridization and S1 nuclease analysis of ADA messenger RNA (mRNA) indicates that the majority of ADA-deficient cell lines have ADA mRNA in the same abundance and size as in normal cell lines. Sequence analysis of ADA cDNAs derived from these mRNAs shows that the majority of mutations are single base changes that alter the amino acid sequence. Expression analysis proves that these point mutations lead to deficiency of ADA catalytic activity. Several cell lines have mutations that alter mRNA transcription or processing. These include a point mutation in one allele of an ADA-deficient cell line that leads to deletion of exon 4 during mRNA splicing. In addition, two cell lines are homozygous for large deletions of the gene that are the result of homologous recombination. Subjects with partial ADA deficiency have undetectable ADA activity in their erythrocytes, variable activity in their lymphoid cells, and normal immunological function. Analysis of the ADA catalytic activity of partially deficient cell lines indicates that the mutations involved affect protein stability. However, the mutations causing partial ADA deficiency are as yet undefined.
Topics: Adenosine Deaminase; Animals; Blotting, Northern; Cell Line; Mice; Mutation; Nucleoside Deaminases; RNA, Messenger; Transcription, Genetic
PubMed: 2651461
DOI: 10.1002/jcb.240390302 -
Hospital Practice (Office Ed.) Jun 1987
Topics: Adenosine Deaminase; Bone Marrow Transplantation; Chromosome Deletion; Chromosomes, Human, Pair 20; Female; Humans; Male; Nucleoside Deaminases
PubMed: 3108284
DOI: 10.1080/21548331.1987.11703253 -
Cell Mar 2023Adenosine-to-inosine RNA editing has been proposed to be involved in a bacterial anti-phage defense system called RADAR. RADAR contains an adenosine triphosphatase...
Adenosine-to-inosine RNA editing has been proposed to be involved in a bacterial anti-phage defense system called RADAR. RADAR contains an adenosine triphosphatase (RdrA) and an adenosine deaminase (RdrB). Here, we report cryo-EM structures of RdrA, RdrB, and currently identified RdrA-RdrB complexes in the presence or absence of RNA and ATP. RdrB assembles into a dodecameric cage with catalytic pockets facing outward, while RdrA adopts both autoinhibited tetradecameric and activation-competent heptameric rings. Structural and functional data suggest a model in which RNA is loaded through the bottom section of the RdrA ring and translocated along its inner channel, a process likely coupled with ATP-binding status. Intriguingly, up to twelve RdrA rings can dock one RdrB cage with precise alignments between deaminase catalytic pockets and RNA-translocation channels, indicative of enzymatic coupling of RNA translocation and deamination. Our data uncover an interesting mechanism of enzymatic coupling and anti-phage defense through supramolecular assemblies.
Topics: RNA; Adenosine Triphosphate; Adenosine Deaminase
PubMed: 36764292
DOI: 10.1016/j.cell.2023.01.026 -
The Journal of Sexual Medicine Sep 2010Priapism featured with painful prolonged penile erection is dangerous and commonly seen in sickle cell disease (SCD). The preventive approaches or effective treatment...
INTRODUCTION
Priapism featured with painful prolonged penile erection is dangerous and commonly seen in sickle cell disease (SCD). The preventive approaches or effective treatment options for the disorder are limited because of poor understanding of its pathogenesis. Recent studies have revealed a novel role of excess adenosine in priapism caused by heightened cavernosal relaxation, and therefore present an intriguing mechanism-based therapeutic possibility.
AIM
The aim of this study was to determine the therapeutic effects of adenosine deaminase (ADA) enzyme therapy to lower adenosine in priapism.
METHODS
Both ADA-deficient mice and SCD transgenic (Tg) mice display priapism caused by excessive adenosine. Thus, we used these two distinct lines of mouse models of priapism as our investigative tools. Specifically, we treated both of these mice with different dosages of polyethylene glycol-modified ADA (PEG-ADA) to reduce adenosine levels in vivo. At the end points of the experiments, we evaluated the therapeutic effects of PEG-ADA treatment by measuring adenosine levels and monitoring the cavernosal relaxation.
MAIN OUTCOME MEASURES
Adenosine levels in penile tissues were measured by high-performance liquid chromatography, and cavernosal relaxation was quantified by electrical field stimulation (EFS)-induced corporal cavernosal strip (CCS) assays.
RESULTS
We found that lowering adenosine levels in penile tissues by PEG-ADA treatment from birth in ADA-deficient mice prevented the increased EFS-induced CCS relaxation associated with priapism. Intriguingly, in both ADA-deficient mice and SCD Tg mice with established priapism, we found that normalization of adenosine levels in penile tissues by PEG-ADA treatment relieved the heightened EFS-induced cavernosal relaxation in priapism.
CONCLUSIONS
Our studies have identified that PEG-ADA is a novel, safe, and mechanism-based drug to prevent and correct excess adenosine-mediated increased cavernosal relaxation seen in two independent priapic animal models, and suggested its therapeutic possibility in men suffering from priapism.
Topics: Adenosine Deaminase; Animals; Chromatography, High Pressure Liquid; Disease Models, Animal; Dose-Response Relationship, Drug; Electric Stimulation; Male; Mice; Mice, Knockout; Muscle Relaxation; Muscle, Smooth; Penis; Polyethylene Glycols; Priapism
PubMed: 19845544
DOI: 10.1111/j.1743-6109.2009.01552.x -
Reumatismo Dec 2023Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited autoinflammatory disease characterized by systemic inflammation and immunodeficiency. Infliximab...
Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited autoinflammatory disease characterized by systemic inflammation and immunodeficiency. Infliximab proved to be favorable in the treatment of this condition. This case report is concerned with a DADA2 deficient patient treated with infliximab. This is a rare case of DADA2 in a 32-year-old female patient. The patient was admitted with a clinical presentation of erythema, ulcers, and pruritus on both legs and ankles, accompanied by red ulcerative oral lesions, fatigue, malaise, and dizziness. The patient's genetic analysis was positive for DADA2. Treatment based on TNF-α inhibition was highly effective for this patient. We used laboratory testing and punch biopsy as differential diagnostic tools, where antinuclear antibody positivity, high prolactin levels, and high serum C-reactive protein were observed. The punch biopsy revealed both orthohyperkeratosis and parahyperkeratosis of the dermis, diffuse core fragments, plasma in the stratum corneum, and hypergranulous acanthosis. DADA2 treatment is centered on tumor necrosis factor α suppression. Although high-dose systemic glucocorticoids can reduce inflammation in the initial stages of the disease, most patients have a resistant or relapsing response to tapering attempts. The prevalence of undiagnosed cases of autoinflammatory diseases is anticipated to diminish with the growing awareness of them.
Topics: Female; Humans; Adult; Adenosine Deaminase; Infliximab; Intercellular Signaling Peptides and Proteins; Inflammation; Mutation
PubMed: 38115782
DOI: 10.4081/reumatismo.2023.1543