Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.5.1.4 (deaminase)
5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The distribution of arabinosylcytosine (ara-C) and its metabolites has been measured in the liver, small intestine, spleen, and kidney of mice inoculated ip 5-6 days earlier with L1210 leukemia cells. Two major metabolites were found in the tissues--the nucleotides and the deaminated inactive product, arabinosyluracil (ara-U). The decay curve of ara-C in most of these tissues was curvilinear; the ara-C half-lives estimated from the terminal phases were 8. 11, 12, and 12 hr for spleen, kidney, intestine, and liver tissues, respectively. The ara-C half-life was not correlated with the deoxycytidine deaminase activity in the tissues. However, the deaminase activity in vitro correlated well with the amount of ara-U present in vivo. Similar analyses were made for L1210 leukemic cells and ascites fluid. A high nucleotide level was found in the cells and a significant amount of nucleotides was also identifiable in the ascites fluid. The activities of deoxycytidine kinase, but not of deoxycytidine deaminase, in host tissues of mice inoculated with L1210 leukemic cells sensitive to ara-C were greater than in those of normal mice. The phosphorylating activities in vitro correlated with the amount of nucleotide present in vivo in mice bearing L1210 leukemic cells. However, the infiltration of leukemic cells containing high kinase activities into the host tissues accounted for most, if not all, of the nucleotide level in these tissues. This is further evidenced by the fact that inoculating mice with L1210 leukemic cells resistant to ara-C did not alter the kinase activity or nucleotide levels of the host tissues; these resistant cells contain negligible amounts of ara-C phosphorylating activities.
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PMID:Correlation of mouse tissue distribution of arabinosylcytosine in vivo with enzymatic activities in vitro. 0 36

Mammalian DHOase (S-dihydroorotate amidohydrolase, EC 3.5.2.3) is part of a large multifunctional protein called CAD, which also has a carbamoyl-phosphate synthetase [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.5.5] and aspartate transcarbamoylase (carbamoyl-phosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) activities. We sequenced selected restriction fragments of a Syrian hamster CAD cDNA. The deduced amino acid sequence agreed with the sequence of tryptic peptides and the amino acid composition of the DHOase domain isolated by controlled proteolysis of CAD. Escherichia coli transformed with a recombinant plasmid containing the cDNA segment 5' to the aspartate transcarbamoylase coding region expressed a polypeptide recognized by DHOase domain-specific antibodies. Thus, the order of domains within the polypeptide is NH2-carbamoyl-phosphate synthetase-DHO-aspartate transcarbamoylase-COOH. The 334-residue DHOase domain has a molecular weight of 36,733 and a pI of 6.1. A fragment of CAD having DHOase activity that was isolated after trypsin digestion has extensions on both the NH2 (18 residues) and COOH (47-65 residues) termini of this core domain. Three of five conserved histidines are within short, highly conserved regions that may participate in zinc binding. Phylogenetic analysis clustered the monofunctional and fused DHOases separately. Although these families may have arisen by convergent evolution, we favor a model involving DHOase gene duplication and insertion into an ancestral bifunctional locus.
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PMID:Mammalian dihydroorotase: nucleotide sequence, peptide sequences, and evolution of the dihydroorotase domain of the multifunctional protein CAD. 196 94

Human APOBEC3G and several other APOBEC3 proteins have been shown to inhibit the replication of a variety of retrotransposons and retroviruses. All of these enzymes can deaminate cytosines within single-strand DNA, but the overall importance of this conserved activity in retroelement restriction has been questioned by reports of deaminase-independent mechanisms. Here, three distinct retroelements, a yeast retrotransposon, Ty1, a murine endogenous retrovirus, MusD, and a lentivirus, human immunodeficiency virus type 1 (HIV-1), were used to evaluate the relative contributions of deaminase-dependent and -independent mechanisms. Although human APOBEC3G can restrict the replication of all three of these retroelements, APOBEC3G lacking the catalytic glutamate (E259Q) was clearly defective. This phenotype was particularly clear in experiments with low levels of APOBEC3G expression. In contrast, purposeful overexpression of APOBEC3G-E259Q was able to cause modest to severe reductions in the replication of Ty1, MusD, and HIV-1(DeltaVif). The importance of these observations was highlighted by data showing that CEM-SS T-cell lines expressing near-physiologic levels of APOBEC3G-E259Q failed to inhibit the replication of HIV-1(DeltaVif), whereas similar levels of wild-type APOBEC3G fully suppressed virus infectivity. Despite the requirement for DNA deamination, uracil DNA glycosylase did not modulate APOBEC3G-dependent restriction of Ty1 or HIV-1(DeltaVif), further supporting prior studies indicating that the major uracil excision repair system of cells is not involved. In conclusion, the absolute requirement for the catalytic glutamate of APOBEC3G in Ty1, MusD, and HIV-1 restriction strongly indicates that DNA cytosine deamination is an essential part of the mechanism.
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PMID:The DNA deaminase activity of human APOBEC3G is required for Ty1, MusD, and human immunodeficiency virus type 1 restriction. 1818 15

Enzymatic deamination of bases in DNA or RNA leads to an alteration of flow of genetic information. Adenosine deaminases edit RNA (ADARs, TADs). Specialized cytidine deaminases are involved in RNA/DNA editing in lipid metabolism (APOBEC1) and in innate (APOBEC3 family) and humoral (AID) immunity. APOBEC2 is required for proper muscle development and, along with AID, was implicated in demethylation of DNA. The functions of APOBEC4, APOBEC5, and other deaminases recently discovered by bioinformatics approaches are unknown. What is the basis for the diverse biological functions of enzymes with similar enzyme structure and the same principal enzymatic reaction? AID, APOBEC1, lamprey CDA1, and APOBEC3G enzymes cause uracil DNA glycosylase-dependent induction of mutations when overproduced ectopically in bacteria or yeast. APOBEC2, on the contrary, is nonmutagenic. We studied the effects of the expression of various deaminases in yeast and bacteria. The mutagenic specificities of four deaminases, hAID, rAPOBEC1, hAPOBEC3G, and lamprey CDA1, are strikingly different. This suggests the existence of an intrinsic component of deaminase targeting. The expression of yeast CDD1 and TAD2/TAD3, human APOBEC4, Xanthomonas oryzae APOBEC5, and deaminase encoded by Micromonas sp. gene MICPUN_56782 was nonmutagenic. A lack of a mutagenic effect for Cdd1 is expected because the enzyme functions in the salvage of pyrimidine nucleotides, and it is evolutionarily distant from RNA/DNA editing enzymes. The reason for inactivity of deaminases grouped with APOBEC2 is not obvious from their structures. This can not be explained by protein insolubility and peculiarities of cellular distribution and requires further investigation.
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PMID:Mutator effects and mutation signatures of editing deaminases produced in bacteria and yeast. 2156 45

APOBEC3G is a single-stranded DNA cytosine deaminase that comprises part of the innate immune response to viruses and transposons. Although APOBEC3G is the prototype for understanding the larger mammalian polynucleotide deaminase family, no specific chemical inhibitors exist to modulate its activity. High-throughput screening identified 34 compounds that inhibit APOBEC3G catalytic activity. Twenty of 34 small molecules contained catechol moieties, which are known to be sulfhydryl reactive following oxidation to the orthoquinone. Located proximal to the active site, C321 was identified as the binding site for the inhibitors by a combination of mutational screening, structural analysis, and mass spectrometry. Bulkier substitutions C321-to-L, F, Y, or W mimicked chemical inhibition. A strong specificity for APOBEC3G was evident, as most compounds failed to inhibit the related APOBEC3A enzyme or the unrelated enzymes E. coli uracil DNA glycosylase, HIV-1 RNase H, or HIV-1 integrase. Partial, but not complete, sensitivity could be conferred to APOBEC3A by introducing the entire C321 loop from APOBEC3G. Thus, a structural model is presented in which the mechanism of inhibition is both specific and competitive, by binding a pocket adjacent to the APOBEC3G active site, reacting with C321, and blocking access to substrate DNA cytosines.
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PMID:First-in-class small molecule inhibitors of the single-strand DNA cytosine deaminase APOBEC3G. 2218 50

The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency.
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PMID:Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. 2763 11

In eukaryotes, the CRISPR-Cas9 system has now been widely used as a revolutionary genome engineering tool^{1, 2}. However, in prokaryotes, the use of nuclease-mediated genome editing tools has been limited to negative selection for the already modified cells because of its lethality^{3, 4}. Here, we report on deaminase-mediated targeted nucleotide editing (Target-AID) ⁵ adopted in Escherichia coli. Cytidine deaminase PmCDA1 fused to the nuclease-deficient CRISPR-Cas9 system achieved specific point mutagenesis at the target sites in E. coli by introducing cytosine mutations without compromising cell growth. The cytosine-to-thymine substitutions were induced mainly within an approximately five-base window of target sequences on the protospacer adjacent motif-distal side, which can be shifted depending on the length of the single guide RNA sequence. Use of a uracil DNA glycosylase inhibitor ⁶ in combination with a degradation tag (LVA tag) ⁷ resulted in a robustly high mutation efficiency, which allowed simultaneous multiplex editing of six different genes. The major multi-copy transposase genes that consist of at least 41 loci were also simultaneously edited by using four target sequences. As this system does not rely on any additional or host-dependent factors, it may be readily applicable to a wide range of bacteria.
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PMID:Deaminase-mediated multiplex genome editing in Escherichia coli. 2940 14

Base editing is an exciting new genome engineering technology. C-to-T mutations in genomic DNA have been achieved using ribonucleoprotein complexes comprised of rat APOBEC1 single-stranded DNA deaminase, Cas9 nickase (Cas9n), uracil DNA glycosylase inhibitor (UGI), and guide (g)RNA. Here, we report the first real-time reporter system for quantification of APOBEC-mediated base editing activity in living mammalian cells. The reporter expresses eGFP constitutively as a marker for transfection or transduction, and editing restores functionality of an upstream mCherry cassette through the simultaneous processing of two gRNA binding regions that each contain an APOBEC-preferred 5'TCA target site. Using this system as both an episomal and a chromosomal editing reporter, we show that human APOBEC3A-Cas9n-UGI and APOBEC3B-Cas9n-UGI base editing complexes are more efficient than the original rat APOBEC1-Cas9n-UGI construct. We also demonstrate coincident enrichment of editing events at a heterologous chromosomal locus in reporter-edited, mCherry-positive cells. The mCherry reporter also quantifies the double-stranded DNA cleavage activity of Cas9, and may therefore be adaptable for use with many different CRISPR systems. The combination of a rapid, fluorescence-based editing reporter system and more efficient, structurally defined DNA editing enzymes broadens the versatility of the rapidly expanding toolbox of genome editing and engineering technologies.
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PMID:A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC-Cas9 or cleavage by Cas9 in living cells. 2974 67

B cell-depleting therapies have been shown to ameliorate symptoms in multiple sclerosis (MS) patients; however, the mechanism of action remains unclear. Following priming with Ag, B cells undergo secondary diversification of their BCR, including BCR class-switch recombination (CSR) and somatic hypermutation (SHM), with both processes requiring the enzyme activation-induced (cytidine) deaminase. We previously reported that activation-induced (cytidine) deaminase is required for full clinical manifestation of disease in an animal model of MS (experimental autoimmune encephalomyelitis; EAE) provoked by immunization with the extracellular domain of recombinant human myelin oligodendrocyte glycoprotein (hMOG). In this study, we investigated the role of CSR versus SHM in the pathogenesis of EAE. We found that passive transfer of class-switched anti-MOG IgG1 Abs into hMOG-primed Aicda^-/- mice is sufficient to fully rescue EAE disease. In addition, we found that the nature of the Ag is an important determinant of EAE severity in Aicda^-/- mice because the lack of a diversified BCR does not affect the induction of EAE when immunized with the extracellular domain of rat MOG. To discriminate the effect of either CSR or SHM, we induced EAE in uracil DNA glycosylase-deficient mice (Ung^-/-) that exhibit a defect primarily in CSR. We observed that Ung^-/- mice exhibit milder clinical disease compared with control mice, concomitant with a reduced amount of anti-MOG IgG₁ class-switched Abs that preserved normal affinity. Collectively, these results indicate that CSR plays an important role in governing the incidence and severity of EAE induced with hMOG but not rat MOG.
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PMID:Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda^-/- and Ung^-/- Mice. 2998 Jun 12

Base editors that do not require double-stranded DNA cleavage or homology-directed repair enable higher efficiency and cleaner substitution of targeted single nucleotides in genomic DNA than conventional approaches. However, their broad applications are limited within the editing window of several base pairs from the canonical NGG protospacer adjacent motif (PAM) sequence. In this study, we fused the D10A nickase of several Streptococcus pyogenes Cas9 (SpCas9) variants with Petromyzon marinus cytidine deaminase 1 (PmCDA1) and uracil DNA glycosylase inhibitor (UGI) and developed two new effective PmCDA1-based cytosine base editors (pBEs), SpCas9 nickase (SpCas9n)-pBE and VQR nickase (VQRn)-pBE, which expanded the scope of genome targeting for cytosine-to-thymine (C-to-T) substitutions in rice. Four of six and 12 of 18 target sites selected randomly in SpCas9n-pBE and VQRn-pBE, respectively were base edited with frequencies of 4-90% in T₀ plants. The effective deaminase window typically spanned positions 1-7 within the protospacer and the single target C showed the maximum C-to-T frequency at or near position 3, counting the end distal to PAM as position 1. In addition, the modified single guide RNA (sgRNA) improved the base editing efficiencies of VQRn-pBE with 1.3- to 7.6-fold increases compared with the native sgRNA, and targets that could not be mutated using the native sgRNA were edited successfully using the modified sgRNA. These newly developed base editors can be used to realize C-to-T substitutions and may become powerful tools for both basic scientific research and crop breeding in rice.
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PMID:Increasing Cytosine Base Editing Scope and Efficiency With Engineered Cas9-PmCDA1 Fusions and the Modified sgRNA in Rice. 3113 25


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