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Silvestro G Conticello - One of the best experts on this subject based on the ideXlab platform.

  • Live-Cell Quantification of APOBEC1-Mediated RNA Editing: A Comparison of RNA Editing Assays.
    Methods in molecular biology (Clifton N.J.), 2020
    Co-Authors: Martina Chieca, Serena Torrini, Silvestro G Conticello
    Abstract:

    APOBEC1 is a member of the AID/APOBECs, a group of deaminases responsible for the editing of C>U in both DNA and RNA. APOBEC1 is physiologically involved in C>U RNA editing: while hundreds of targets have been discovered in mice, in humans the only well-characterized target of APOBEC1 is the apolipoprotein B (ApoB) transcript. APOBEC1 edits a CAA codon into a stop codon, which causes the translation of a truncated form of ApoB. A number of assays have been developed to investigate this process. Early assays, poisoned primer extension and Sanger sequencing, have focused on accuracy and sensitivity but rely on extraction of the RNA from tissues and cells. More recently, the need to visualize the RNA editing process directly in live cells have led to the development of fluorescence-based tools. These assays detect RNA editing through reporters whose editing causes a change in cellular localization or a change in fluorescent properties. Here we review the available assays to quantify RNA editing, and we present the protocol for cytofluorimetric analysis using a double-fluorescent reporter.

  • Dimerisation of APOBEC1 is dispensable for its RNA editing activity
    2018
    Co-Authors: Martina Chieca, Riccardo Pecori, Francesco Severi, Marco D Montini, Silvestro G Conticello
    Abstract:

    Among the AID/APOBECs - a family of DNA and RNA deaminases - APOBEC1 physiologically partakes into a complex that edits a CAA codon into UAA Stop codon in the transcript of Apolipoprotein B (ApoB), a protein crucial in the transport of lipids in the blood. Catalytically inactive mutants of APOBEC1 have a dominant negative effect on its activity, as they compete for the targeting to the ApoB mRNA. Here we show that catalytically inactive chimeras of APOBEC1 restricted to different compartments of the cell present different abilities to titrate APOBEC1-mediated RNA editing, and that the ability of APOBEC1 to interact with these mutants is the main determinant for its activity. Our results demonstrate that dimerisation, a feature common to other APOBECs targeting DNA, is not required for APOBEC1 activity on mRNA. Furthermore, APOBEC1-mediated RNA editing is a dynamic process where interplay among the components of the editing complex is regulated through the balance between availability of A1CF, one of APOBEC1 cofactors, and nuclear degradation of APOBEC1.

  • A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC-Cas9 or cleavage by Cas9 in living cells.
    Nucleic acids research, 2018
    Co-Authors: Amber St. Martin, William L. Brown, Silvestro G Conticello, Daniel J. Salamango, Artur A. Serebrenik, Nadine M. Shaban, Francesco Donati, Uday Munagala, Reuben S. Harris
    Abstract:

    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.

  • Flow-cytometric visualization of C>U mRNA editing reveals the dynamics of the process in live cells
    RNA biology, 2015
    Co-Authors: Francesco Severi, Silvestro G Conticello
    Abstract:

    APOBEC1 is the catalytic subunit of the complex that edits ApolipoproteinB (ApoB) mRNA, which specifically deaminates cytidine 6666 to uracil in the human transcript. The editing leads to the generation of a stop codon, resulting in the synthesis of a truncated form of ApoB. We have developed a method to quantitatively assay ApoB RNA editing in live cells by using a double fluorescent mCherry-EGFP chimera containing a ∼300bp fragment encompassing the region of ApoB subject to RNA editing. Coexpression of APOBEC1 together with this chimera causes specific RNA editing of the ApoB fragment. The insertion of a stop codon between the mCherry and EGFP thus induces the loss of EGFP fluorescence. Using this method we analyze the dynamics of APOBEC1-dependent RNA editing under various conditions. Namely we show the interplay of APOBEC1 with known interactors (ACF, hnRNP-C1, GRY-RBP) in cells that are RNA editing-proficient (HuH-7) or -deficient (HEK-293T), and the effects of restricted cellular localization of APO...

  • The RNA editing enzyme APOBEC1 induces somatic mutations and a compatible mutational signature is present in esophageal adenocarcinomas.
    Genome biology, 2014
    Co-Authors: Giulia Saraconi, Francesco Severi, Cesare Sala, Giorgio Mattiuz, Silvestro G Conticello
    Abstract:

    The AID/APOBECs are deaminases that act on cytosines in a diverse set of pathways and some of them have been linked to the onset of genetic alterations in cancer. Among them, APOBEC1 is the only family member to physiologically target RNA, as the catalytic subunit in the Apolipoprotein B mRNA editing complex. APOBEC1 has been linked to cancer development in mice but its oncogenic mechanisms are not yet well understood. We analyze whether expression of APOBEC1 induces a mutator phenotype in vertebrate cells, likely through direct targeting of genomic DNA. We show its ability to increase the inactivation of a stably inserted reporter gene in a chicken cell line that lacks any other AID/APOBEC proteins, and to increase the number of imatinib-resistant clones in a human cellular model for chronic myeloid leukemia through induction of mutations in the BCR-ABL1 fusion gene. Moreover, we find the presence of an AID/APOBEC mutational signature in esophageal adenocarcinomas, a type of tumor where APOBEC1 is expressed, that mimics the one preferred by APOBEC1 in vitro. Our findings suggest that the ability of APOBEC1 to trigger genetic alterations represents a major layer in its oncogenic potential. Such APOBEC1-induced mutator phenotypes could play a role in the onset of esophageal adenocarcinomas. APOBEC1 could be involved in cancer promotion at the very early stages of carcinogenesis, as it is highly expressed in Barrett's esophagus, a condition often associated with esophageal adenocarcinoma.

Nicholas O Davidson - One of the best experts on this subject based on the ideXlab platform.

  • APOBEC1 mediated C-to-U RNA editing: target sequence and trans-acting factor contribution to 177 RNA editing events in 119 murine transcripts in-vivo
    2021
    Co-Authors: Saeed Soleyman-jahi, Valerie Blanc, Nicholas O Davidson
    Abstract:

    Mammalian C-to-U RNA editing was described more than 30 years ago as a single nucleotide modification in APOB RNA in small intestine, later shown to be mediated by the RNA-specific cytidine deaminase APOBEC1. Reports of other examples of C-to-U RNA editing, coupled with the advent of genome-wide transcriptome sequencing, identified an expanded range of APOBEC1 targets. Here we analyze the cis-acting regulatory components of verified murine C-to-U RNA editing targets, including nearest neighbor as well as flanking sequence requirements and folding predictions. We summarize findings demonstrating the relative importance of trans-acting factors (A1CF, RBM47) acting in concert with APOBEC1. Using this information, we developed a multivariable linear regression model to predict APOBEC1 dependent C-to-U RNA editing efficiency, incorporating factors independently associated with editing frequencies based on 103 Sanger-confirmed editing sites, which accounted for 84% of the observed variance. Co-factor dominance was associated with editing frequency, with RNAs targeted by both RBM47 and A1CF observed to be edited at a lower frequency than RBM47 dominant targets. The model also predicted a composite score for available human C-to-U RNA targets, which again correlated with editing frequency.

  • APOBEC1 complementation factor a1cf and rbm47 interact in tissue specific regulation of c to u rna editing in mouse intestine and liver
    RNA, 2019
    Co-Authors: Valerie Blanc, Yan Xie, Susan Kennedy, Joseph H Nadeau, Jesse D Riordan, Deborah C Rubin, Blair B Madison, Jason C Mills, Nicholas O Davidson
    Abstract:

    Mammalian C to U RNA is mediated by APOBEC1, the catalytic deaminase, together with RNA binding cofactors (including A1CF and RBM47) whose relative physiological requirements are unresolved. Although A1CF complements APOBEC1 for in vitro RNA editing, A1cf -/- mice exhibited no change in apolipoproteinB (apoB) RNA editing, while Rbm47 mutant mice exhibited impaired intestinal RNA editing of apoB as well as other targets. Here we examined the role of A1CF and RBM47 in adult mouse liver and intestine, following deletion of either one or both gene products and also following forced (liver or intestinal) transgenic A1CF expression. There were minimal changes in hepatic and intestinal apoB RNA editing in A1cf -/- mice and no changes in either liver- or intestine-specific A1CF transgenic mice. Rbm47 liver-specific knockout (Rbm47 LKO ) mice demonstrated reduced editing in a subset (11 of 20) of RNA targets, including apoB. By contrast, apoB RNA editing was virtually eliminated (<6% activity) in intestine-specific (Rbm47 IKO ) mice with only five of 53 targets exhibiting C-to-U RNA editing. Double knockout of A1cf and Rbm47 in liver (AR LKO ) eliminated apoB RNA editing and reduced editing in the majority of other targets, with no changes following adenoviral APOBEC1 administration. Intestinal double knockout mice (AR IKO ) demonstrated further reduced editing (<10% activity) in four of five of the residual APOBEC1 targets identified in AR IKO mice. These data suggest that A1CF and RBM47 each function independently, yet interact in a tissue-specific manner, to regulate the activity and site selection of APOBEC1 dependent C-to-U RNA editing.

  • Reassessment of murine APOBEC1 as a retrovirus restriction factor in vivo.
    Virology, 2014
    Co-Authors: Bradley S. Barrett, Michael S. Harper, Kejun Guo, Karl J. Heilman, Nicholas O Davidson, Mario L. Santiago
    Abstract:

    APOBEC1 is a cytidine deaminase involved in cholesterol metabolism that has been linked to retrovirus restriction, analogous to the evolutionarily-related APOBEC3 proteins. In particular, murine APOBEC1 was shown to inhibit Friend retrovirus (FV) in vitro, generating high levels of C-to-T and G-to-A mutations. These observations raised the possibility that FV infection might be altered in APOBEC1-null mice. To examine this question directly, we infected wild-type and APOBEC1-null mice with FV complex and evaluated acute infection levels. Surprisingly, APOBEC1-null mice exhibited similar cellular infection levels and plasma viremia relative to wild-type mice. Moreover, next-generation sequencing analyses revealed that in contrast to APOBEC3, APOBEC1 did not enhance retroviral C-to-T and G-to-A mutational frequencies in genomic DNA. Thus, APOBEC1 neither inhibited nor significantly drove the molecular evolution of FV in vivo. Our findings reinforce that not all retrovirus restriction factors characterized as potent in vitro may be functionally relevant in vivo.

  • intestine specific expression of apobec 1 rescues apolipoprotein b rna editing and alters chylomicron production in APOBEC1 mice
    Journal of Lipid Research, 2012
    Co-Authors: Valerie Blanc, Yan Xie, Jianyang Luo, Susan Kennedy, Nicholas O Davidson
    Abstract:

    Intestinal apolipoprotein B (apoB) mRNA undergoes C-to-U editing, mediated by the catalytic deaminase apobec-1, which results in translation of apoB48. APOBEC1−/− mice produce only apoB100 and secrete larger chylomicron particles than those observed in wild-type (WT) mice. Here we show that transgenic rescue of intestinal apobec-1 expression (APOBEC1Int/O) restores C-to-U RNA editing of apoB mRNA in vivo, including the canonical site at position 6666 and also at approximately 20 other newly identified downstream sites present in WT mice. The small intestine of APOBEC1Int/O mice produces only apoB48, and the liver produces only apoB100. Serum chylomicron particles were smaller in APOBEC1Int/O mice compared with those from APOBEC1−/− mice, and the predominant fraction of serum apoB48 in APOBEC1Int/O mice migrated in lipoproteins smaller than chylomicrons, even when these mice were fed a high-fat diet. Because apoB48 arises exclusively from the intestine in APOBEC1Int/O mice and intestinal apoB48 synthesis and secretion rates were comparable to WT mice, we were able to infer the major sites of origin of serum apoB48 in WT mice. Our findings imply that less than 25% of serum apoB48 in WT mice arises from the intestine, with the majority originating from the liver.

  • Intestine-specific expression of Apobec-1 rescues apolipoprotein B RNA editing and alters chylomicron production in APOBEC1 -/- mice.
    Journal of lipid research, 2012
    Co-Authors: Valerie Blanc, Yan Xie, Jianyang Luo, Susan Kennedy, Nicholas O Davidson
    Abstract:

    Intestinal apolipoprotein B (apoB) mRNA undergoes C-to-U editing, mediated by the catalytic deaminase apobec-1, which results in translation of apoB48. APOBEC1(-/-) mice produce only apoB100 and secrete larger chylomicron particles than those observed in wild-type (WT) mice. Here we show that transgenic rescue of intestinal apobec-1 expression (APOBEC1(Int/O)) restores C-to-U RNA editing of apoB mRNA in vivo, including the canonical site at position 6666 and also at approximately 20 other newly identified downstream sites present in WT mice. The small intestine of APOBEC1(Int/O) mice produces only apoB48, and the liver produces only apoB100. Serum chylomicron particles were smaller in APOBEC1(Int/O) mice compared with those from APOBEC1(-/-) mice, and the predominant fraction of serum apoB48 in APOBEC1(Int/O) mice migrated in lipoproteins smaller than chylomicrons, even when these mice were fed a high-fat diet. Because apoB48 arises exclusively from the intestine in APOBEC1(Int/O) mice and intestinal apoB48 synthesis and secretion rates were comparable to WT mice, we were able to infer the major sites of origin of serum apoB48 in WT mice. Our findings imply that less than 25% of serum apoB48 in WT mice arises from the intestine, with the majority originating from the liver.

Michael S Neuberger - One of the best experts on this subject based on the ideXlab platform.

  • Deficiency in APOBEC2 leads to a shift in muscle fiber type, diminished body mass, and myopathy
    The Journal of biological chemistry, 2009
    Co-Authors: Yusuke Sato, Michael S Neuberger, Hans Christian Probst, Ryuichi Tatsumi, Yoshihide Ikeuchi, Cristina Rada
    Abstract:

    Abstract The apoB RNA-editing enzyme, catalytic polypeptide-like (APOBEC) family of proteins includes APOBEC1, APOBEC3, and activation-induced deaminase, all of which are zinc-dependent cytidine deaminases active on polynucleotides and involved in RNA editing or DNA mutation. In contrast, the biochemical and physiological functions of APOBEC2, a muscle-specific member of the family, are unknown, although it has been speculated, like APOBEC1, to be an RNA-editing enzyme. Here, we show that, although expressed widely in striated muscle (with levels peaking late during myoblast differentiation), APOBEC2 is preferentially associated with slow-twitch muscle, with its abundance being considerably greater in soleus compared with gastrocnemius muscle and, within soleus muscle, in slow as opposed to fast muscle fibers. Its abundance also decreases following muscle denervation. We further show that APOBEC2-deficient mice harbor a markedly increased ratio of slow to fast fibers in soleus muscle and exhibit an ∼15–20% reduction in body mass from birth onwards, with elderly mutant animals revealing clear histological evidence of a mild myopathy. Thus, APOBEC2 is essential for normal muscle development and maintenance of fiber-type ratios; although its molecular function remains to be identified, biochemical analyses do not especially argue for any role in RNA editing.

  • dna deamination in immunity aid in the context of its apobec relatives
    Advances in Immunology, 2007
    Co-Authors: Silvestro G Conticello, Marcandre Langlois, Zizhen Yang, Michael S Neuberger
    Abstract:

    Abstract The activation‐induced cytidine deaminase (AID)/apolipoprotein B RNA‐editing catalytic component (APOBEC) family is a vertebrate‐restricted subgrouping of a superfamily of zinc (Zn)‐dependent deaminases that has members distributed throughout the biological world. AID and APOBEC2 are the oldest family members with APOBEC1 and the APOBEC3s being later arrivals restricted to placental mammals. Many AID/APOBEC family members exhibit cytidine deaminase activity on polynucleotides, although in different physiological contexts. Here, we examine the AID/APOBEC proteins in the context of the entire Zn‐dependent deaminase superfamily. On the basis of secondary structure predictions, we propose that the cytosine and tRNA deaminases are likely to provide better structural paradigms for the AID/APOBEC family than do the cytidine deaminases, to which they have conventionally been compared. These comparisons yield predictions concerning likely polynucleotide‐interacting residues in AID/APOBEC3s, predictions that are supported by mutagenesis studies. We also focus on a specific comparison between AID and the APOBEC3s. Both are DNA deaminases that function in immunity and are responsible for the hypermutation of their target substrates. AID functions in the adaptive immune system to diversify antibodies with targeted DNA deamination being central to this function. APOBEC3s function as part of an innate pathway of immunity to retroviruses with targeted DNA deamination being central to their activity in retroviral hypermutation. However, the mechanism by which the APOBEC3s fulfill their function of retroviral restriction remains unresolved.

  • Evolution of the AID/APOBEC Family of Polynucleotide (Deoxy)cytidine Deaminases
    Molecular biology and evolution, 2004
    Co-Authors: Silvestro G Conticello, Cornelia Thomas, Svend K. Petersen-mahrt, Michael S Neuberger
    Abstract:

    The AID/APOBEC family (comprising AID, APOBEC1, APOBEC2, and APOBEC3 subgroups) contains members that can deaminate cytidine in RNA and/or DNA and exhibit diverse physiological functions (AID and APOBEC3 deaminating DNA to trigger pathways in adaptive and innate immunity; APOBEC1 mediating apolipoprotein B RNA editing). The founder member APOBEC1, which has been used as a paradigm, is an RNA-editing enzyme with proposed antecedents in yeast. Here, we have undertaken phylogenetic analysis to glean insight into the primary physiological function of the AID/APOBEC family. We find that although the family forms part of a larger superfamily of deaminases distributed throughout the biological world, the AID/APOBEC family itself is restricted to vertebrates with homologs of AID (a DNA deaminase that triggers antibody gene diversification) and of APOBEC2 (unknown function) identifiable in sequence databases from bony fish, birds, amphibians, and mammals. The cloning of an AID homolog from dogfish reveals that AID extends at least as far back as cartilaginous fish. Like mammalian AID, the pufferfish AID homolog can trigger deoxycytidine deamination in DNA but, consistent with its cold-blooded origin, is thermolabile. The fine specificity of its mutator activity and the biased codon usage in pufferfish IgV genes appear broadly similar to that of their mammalian counterparts, consistent with a coevolution of the antibody mutator and its substrate for the optimal targeting of somatic mutation during antibody maturation. By contrast, APOBEC1 and APOBEC3 are later evolutionary arrivals with orthologs not found in pufferfish (although synteny with mammals is maintained in respect of the flanking loci). We conclude that AID and APOBEC2 are likely to be the ancestral members of the AID/APOBEC family (going back to the beginning of vertebrate speciation) with both APOBEC1 and APOBEC3 being mammal-specific derivatives of AID and a complex set of domain shuffling underpinning the expansion and evolution of the primate APOBEC3s.

  • comparison of the differential context dependence of dna deamination by apobec enzymes correlation with mutation spectra in vivo
    Journal of Molecular Biology, 2004
    Co-Authors: Rupert Beale, Svend K Petersenmahrt, Ian N Watt, Cristina Rada, Michael S Neuberger
    Abstract:

    To investigate the extent to which in vivo mutation spectra might reflect the intrinsic specificities of active mutators, genetic and biochemical assays were used to analyse the DNA target specificities of cytidine deaminases of the APOBEC family. The results reveal the critical importance of nucleotides immediately 5′ of the targeted C for the specificity of all three enzymes studied (AID, APOBEC1 and APOBEC3G). At position −1, APOBEC1 showed a marked preference for dT, AID for dA/dG and APOBEC3G a strong preference for dC. Furthermore, AID and APOBEC3G showed distinct dependence on the nucleotide at position −2 with dA/dT being favoured by AID and dC by APOBEC3G. Most if not all activity of the recombinant deaminases on free dC could be attributed to low-level contamination by host enzymes. The target preference of APOBEC3G supports it being a major but possibly not sole contributor to HIV hypermutation without making it a dominant contribution to general HIV sequence variation. The specificity of AID as deduced from the genetic assay (which relies on inactivation of sacB of Bacillus subtilis) agrees well with that deduced by Pham et al. using an in vitro assay although we postulate that major intrinsic mutational hotspots in immunoglobulin V genes in vivo might reflect favoured sites of AID action being generated by proximal DNA targets located on opposite DNA strands. The target specificity of AID also accords with the spectrum of mutations observed in B lymphoma-associated oncogenes. The possibility of deaminase involvement in non-lymphoid human tumours is hinted at by tissue-specific differences in the spectra of dC transitions in tumour-suppressor genes. Thus, the patterns of hypermutation in antibodies and retroviruses owe much to the intrinsic sequence preferences of the AID/APOBEC family of DNA deaminases: analogous biases might also contribute to the spectra of cancer-associated mutation.

  • in vitro deamination of cytosine to uracil in single stranded dna by apolipoprotein b editing complex catalytic subunit 1 APOBEC1
    Journal of Biological Chemistry, 2003
    Co-Authors: Svend K Petersenmahrt, Michael S Neuberger
    Abstract:

    Abstract Apolipoprotein B-editing complex catalytic subunit 1 (APOBEC1) is the catalytic component of an RNA-editing complex that deaminates C6666 → U in apolipoprotein B RNA in gastrointestinal tissue, thereby generating a premature stop codon. Whereas RNA is the physiological substrate of APOBEC1, recent experiments have strongly indicated that, when expressed in bacteria, APOBEC1 and some of its homologues can deaminate cytosine in DNA. Indeed, genetic evidence demonstrates that the physiological function of activation-induced deaminase, a B lymphocyte-specific APOBEC1 homologue, is to perform targeted deamination of cytosine within the immunoglobulin locus, thereby triggering antibody gene diversification. However, biochemical evidence of in vitro DNA deamination by members of the APOBEC family is still needed. Here, we show that deamination of cytosine to uracil in DNA can be achieved in vitro using partially purified APOBEC1 from extracts of transformed Escherichia coli. Thus, APOBEC1 can deaminate cytosine in both RNA and DNA. Strikingly, its activity on DNA is specific for single-stranded DNA and exhibits dependence on local sequence context.

Reuben S. Harris - One of the best experts on this subject based on the ideXlab platform.

  • A panel of eGFP reporters for single base editing by APOBEC-Cas9 editosome complexes
    Scientific reports, 2019
    Co-Authors: A. St Martin, William L. Brown, Daniel J. Salamango, Artur A. Serebrenik, Nadine M. Shaban, Reuben S. Harris
    Abstract:

    The prospect of introducing a single C-to-T change at a specific genomic location has become feasible with APOBEC-Cas9 editing technologies. We present a panel of eGFP reporters for quantification and optimization of single base editing by APOBEC-Cas9 editosomes. Reporter utility is demonstrated by comparing activities of seven human APOBEC3 enzymes and rat APOBEC1 (BE3). APOBEC3A and RNA binding-defective variants of APOBEC3B and APOBEC3H display the highest single base editing efficiencies. APOBEC3B catalytic domain complexes also elicit the lowest frequencies of adjacent off-target events. However, unbiased deep-sequencing of edited reporters shows that all editosomes have some degree of local off-target editing. Thus, further optimization is required to generate true single base editors and the eGFP reporters described here have the potential to facilitate this process.

  • A fluorescent reporter for quantification and enrichment of DNA editing by APOBEC-Cas9 or cleavage by Cas9 in living cells.
    Nucleic acids research, 2018
    Co-Authors: Amber St. Martin, William L. Brown, Silvestro G Conticello, Daniel J. Salamango, Artur A. Serebrenik, Nadine M. Shaban, Francesco Donati, Uday Munagala, Reuben S. Harris
    Abstract:

    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.

  • Inhibiting APOBEC3 Activity with Single-Stranded DNA Containing 2′-Deoxyzebularine Analogues
    2018
    Co-Authors: Maksim V. Kvach, Reuben S. Harris, Fareeda M. Barzak, Stefan Harjes, Henry A. M. Schares, Geoffrey B. Jameson, Alex M. Ayoub, Ramkumar Moorthy, Hideki Aihara, Vyacheslav V. Filichev
    Abstract:

    APOBEC3 enzymes form part of the innate immune system by deaminating cytosine to uracil in single-stranded DNA (ssDNA) and thereby preventing the spread of pathogenic genetic information. However, APOBEC mutagenesis is also exploited by viruses and cancer cells to increase rates of evolution, escape adaptive immune responses, and resist drugs. This raises the possibility of APOBEC3 inhibition as a strategy for augmenting existing antiviral and anticancer therapies. Here we show that, upon incorporation into short ssDNAs, the cytidine nucleoside analogue 2′-deoxyzebularine (dZ) becomes capable of inhibiting the catalytic activity of selected APOBEC variants derived from APOBEC3A, APOBEC3B, and APOBEC3G, supporting a mechanism in which ssDNA delivers dZ to the active site. Multiple experimental approaches, including isothermal titration calorimetry, fluorescence polarization, protein thermal shift, and nuclear magnetic resonance spectroscopy assays, demonstrate nanomolar dissociation constants and low micromolar inhibition constants. These dZ-containing ssDNAs constitute the first substrate-like APOBEC3 inhibitors and, together, comprise a platform for developing nucleic acid-based inhibitors with cellular activity

  • APOBECs and virus restriction.
    Virology, 2015
    Co-Authors: Reuben S. Harris, Jaquelin P Dudley
    Abstract:

    The APOBEC family of single-stranded DNA cytosine deaminases comprises a formidable arm of the vertebrate innate immune system. Pre-vertebrates express a single APOBEC, whereas some mammals produce as many as 11 enzymes. The APOBEC3 subfamily displays both copy number variation and polymorphisms, consistent with ongoing pathogenic pressures. These enzymes restrict the replication of many DNA-based parasites, such as exogenous viruses and endogenous transposable elements. APOBEC1 and activation-induced cytosine deaminase (AID) have specialized functions in RNA editing and antibody gene diversification, respectively, whereas APOBEC2 and APOBEC4 appear to have different functions. Nevertheless, the APOBEC family protects against both periodic viral zoonoses as well as exogenous and endogenous parasite replication. This review highlights viral pathogens that are restricted by APOBEC enzymes, but manage to escape through unique mechanisms. The sensitivity of viruses that lack counterdefense measures highlights the need to develop APOBEC-enabling small molecules as a new class of anti-viral drugs.

  • apobec3b and aid have similar nuclear import mechanisms
    Journal of Molecular Biology, 2012
    Co-Authors: Lela Lackey, Allison M Land, Zachary L Demorest, Judd F. Hultquist, William L. Brown, Reuben S. Harris
    Abstract:

    Abstract Members of the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) protein family catalyze DNA cytosine deamination and underpin a variety of immune defenses. For instance, several family members, including APOBEC3B (A3B), elicit strong retrotransposon and retrovirus restriction activities. However, unlike the other proteins, A3B is the only family member with steady-state nuclear localization. Here, we show that A3B nuclear import is an active process requiring at least one amino acid (Val54) within an N-terminal motif analogous to the nuclear localization determinant of the antibody gene diversification enzyme AID (activation-induced cytosine deaminase). Mechanistic conservation with AID is further suggested by A3B's capacity to interact with the same subset of importin proteins. Despite these mechanistic similarities, enforced A3B expression cannot substitute for AID-dependent antibody gene diversification by class switch recombination. Regulatory differences between A3B and AID are also visible during cell cycle progression. Our studies suggest that the present-day A3B enzyme retained the nuclear import mechanism of an ancestral AID protein during the expansion of the APOBEC3 locus in primates. Our studies also highlight the likelihood that, after nuclear import, specialized mechanisms exist to guide these enzymes to their respective physiological substrates and prevent gratuitous chromosomal DNA damage.

Ba-bie Teng - One of the best experts on this subject based on the ideXlab platform.

  • Abstract 380: Vascular Pcsk9: A Mediator for Atherogenesis Independent of LDL Receptor
    Arteriosclerosis Thrombosis and Vascular Biology, 2015
    Co-Authors: Hua Sun, Michael Tan, Ba-bie Teng
    Abstract:

    PCSK9 (Proprotein convertase subtilisin/kexin type 9) increases the LDL levels by binding to hepatocyte LDL receptors (LDLR) and subjects it to degradation. We show that PCSK9 regulates apolipoprotein B (apoB) production by inhibiting its degradation process via the autophagic pathway, irrespective of the presence of LDLR. In addition to the role of PCSK9 in promoting hyperlipidemia, we hypothesized that vascular-PCSK9 in endothelial cells (EC) plays a role in initiating atherogenesis, irrespective of the presence of LDL receptor. Our laboratory has generated double knockout mice lacking both LDLR and APOBEC1 (apoB mRNA editing enzyme), named LDb, Ldlr-/-APOBEC1-/-. They have the lipoprotein phenotype mimics human with hyperlipidemia; elevated levels of VLDL and LDL with low levels of HDL. They develop atherosclerotic lesions spontaneously. To investigate the role of PCSK9 in atherogenesis, we deleted Pcsk9 gene from LDb mice to generate the triple knockout mice (named LTp, Ldlr-/-APOBEC1-/-Pcsk9-/-). In comparison to LDb mice (n=14), the LTp mice (n=8) had significantly decreased levels of cholesterol (387±10 vs. 313±14 mg/dl; p

  • Simultaneous expression of apolipoprotein B mRNA editing enzyme and scavenger receptor BI mediated by a therapeutic gene expression system.
    Atherosclerosis, 2005
    Co-Authors: Shumei Zhong, Chichi Liu, David L. Haviland, Peter A. Doris, Ba-bie Teng
    Abstract:

    Cardiovascular diseases are often accompanied by elevated LDL particles and endothelial dysfunction. We have examined the possibility of concurrently reducing LDL levels and modulating endothelial function using a single helper-dependent adenovirus vector system to simultaneously express the apolipoprotein B mRNA editing enzyme (APOBEC1) and the scavenger receptor, class B, type I (SR-BI) genes under the control of separate promoters (designated HD-C2). APOBEC1 edits apoB mRNA at nucleotide C-6666 to produce truncated apoB48 and is normally expressed in small intestine only. SR-BI is a receptor for multiple ligands with distinct tissue-specific functions. Expression of APOBEC1 in HepG2 cells resulted in apoB mRNA editing, leading to decreased apoB100 abundance (to 6% of control) and the appearance of apoB48. Editing of apoB mRNA in HepG2 cells resulted in decline in apoB mRNA levels of 50%. This was probably the result of nonsense-mediated decay of edited message, since over-expression of APOBEC1 increased neither APOBEC1 complementary factor (ACF) mRNA nor protein abundance. Over-expression of SR-BI in human endothelial cells activated endothelial nitric oxide synthase (eNOS) activity by phosphorylation of eNOS at residue Ser-1177 in the presence of HDL, leading to increased production of the anti-atherogenic molecule nitric oxide (NO). Taken together, this study demonstrates that using one vector delivery system to express two genes in two different cell types results in the cell-specific beneficial effects of decreasing apoB100 production and increasing eNOS activities. This combined gene expression approach may provide an improved therapeutic strategy by targeting multiple sites in the mechanism of cardiovascular injury.

  • normal perinatal rise in serum cholesterol is inhibited by hepatic delivery of adenoviral vector expressing apolipoprotein b mrna editing enzyme APOBEC1 in rabbits
    Journal of Surgical Research, 1999
    Co-Authors: Ba-bie Teng, Mary L. Brandt, Pedro A. Piedra, Jing Liu, Lawrence Chan
    Abstract:

    Abstract Background. Prenatal or neonatal hepatic gene delivery may result in more effective therapy for inborn errors of metabolism due to the immature immune system of the perinatal animal, and the ability to intervene prior to any significant cellular damage. Newborn New Zealand White rabbits have low serum levels of cholesterol at birth, with a significant and sustained rise of cholesterol while they are nursing. We used this physiologic hypercholesterolemia model to study the effect of adenovirus-mediated hepatic gene transfer of rat apolipoprotein B mRNA editing enzyme (APOBEC1) on modulation of plasma cholesterol levels. Methods and results. Transcutaneous injection of recombinant adenovirus expressing APOBEC1 (AvAPOBEC1) into the liver of newborn rabbits in vivo resulted in efficient APOBEC1 expression until Day 50, as detected by PCR-Southern blot analysis. By in vitro editing assay, liver extracts of AvAPOBEC1-treated rabbits were found to have apoB mRNA editing activities of ∼12, 15, and 15%, on Days 2, 10, and 20 after AvAPOBEC1 administration, compared with 0% editing activity in AvLacZ control vector-injected animals. This physiological level of APOBEC1 expression was associated with the production of apoB-48-containing lipoprotein particles from rabbit liver, with a concomitant 30% reduction in total plasma cholesterol compared to AvLacZ-treated or untreated control animals. Conclusion. Neonatal intrahepatic delivery of a first-generation adenoviral vector results in efficient gene transfer with little immune response, suggesting that repeated administration may be possible in the neonatal period.

  • Mutational analysis of apolipoprotein B mRNA editing enzyme (APOBEC1). structure-function relationships of RNA editing and dimerization.
    Journal of lipid research, 1999
    Co-Authors: Ba-bie Teng, Scott A. Ochsner, Qian Zhang, Kizhake V. Soman, Paul P. Lau, Lawrence Chan
    Abstract:

    Abstract APOBEC1 is the catalytic subunit of an enzyme complex that mediates apolipoprotein (apo) B mRNA editing. It dimerizes in vitro and requires complementation factor(s) for its editing activity. We have performed a systematic analysis of the structure–functional relationship of APOBEC1 by targeted mutagenesis of various sequence motifs within the protein. Using in vitro RNA editing assay, we found that basic amino acid clusters at the amino-terminal region R15R16R17 and R33K34, are essential for apoB mRNA editing. Mutation of R15R16R17 to K15K16K17 and mutation of R33K34 simultaneously to A33A34 almost completely abolished in vitro editing activity. The carboxy-terminal region of APOBEC1 contains a leucine-rich motif. Deletion analysis of this region indicates that residues 181 to 210 are important for in vitro apoB mRNA editing. Single amino acid substitutions demonstrate that L182, I185, and L189 are important residues required for normal editing function. Furthermore, the double mutant P190A/P191A also lost >90% of editing activity which suggests that a β turn in this region of the molecule may be essential for proper functioning of APOBEC1. It was suggested that dimerization of APOBEC1 creates an active structure for deamination of apoB mRNA. When we examined the dimerization potential of truncated APOBEC1s using both amino and carboxy termini deletion mutants, we found that amino-terminal deletions up to residue A117 did not impair dimerization activity whereas carboxy-terminal deletions showed diminished dimerization. The systematic and extensive mutagenesis experiments in this study provide information on the role of various sequence motifs identified in APOBEC1 in enzyme catalysis and dimerization.—Teng, B-B., S. Ochsner, Q. Zhang, K. V. Soman, P. P. Lau, and L. Chan. Mutational analysis of apolipoprotein B mRNA editing enzyme (APOBEC1): structure–function relationships of RNA editing and dimerization. J. Lipid Res. 1999. 40: 623–635.

  • Normal perinatal rise in serum cholesterol is inhibited by hepatic delivery of adenoviral vector expressing apolipoprotein B mRNA editing enzyme (APOBEC1) in rabbits.
    The Journal of surgical research, 1999
    Co-Authors: Ba-bie Teng, Mary L. Brandt, Pedro A. Piedra, Jing Liu, Lawrence Chan
    Abstract:

    Prenatal or neonatal hepatic gene delivery may result in more effective therapy for inborn errors of metabolism due to the immature immune system of the perinatal animal, and the ability to intervene prior to any significant cellular damage. Newborn New Zealand White rabbits have low serum levels of cholesterol at birth, with a significant and sustained rise of cholesterol while they are nursing. We used this physiologic hypercholesterolemia model to study the effect of adenovirus-mediated hepatic gene transfer of rat apolipoprotein B mRNA editing enzyme (APOBEC1) on modulation of plasma cholesterol levels. Transcutaneous injection of recombinant adenovirus expressing APOBEC1 (AvAPOBEC1) into the liver of newborn rabbits in vivo resulted in efficient APOBEC1 expression until Day 50, as detected by PCR-Southern blot analysis. By in vitro editing assay, liver extracts of AvAPOBEC1-treated rabbits were found to have apoB mRNA editing activities of approximately 12, 15, and 15%, on Days 2, 10, and 20 after AvAPOBEC1 administration, compared with 0% editing activity in AvLacZ control vector-injected animals. This physiological level of APOBEC1 expression was associated with the production of apoB-48-containing lipoprotein particles from rabbit liver, with a concomitant 30% reduction in total plasma cholesterol compared to AvLacZ-treated or untreated control animals. Neonatal intrahepatic delivery of a first-generation adenoviral vector results in efficient gene transfer with little immune response, suggesting that repeated administration may be possible in the neonatal period. Copyright 1999 Academic Press.