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At least 19 recordsLinked to original sources
PubMed1996

Gene transfer of cytidine deaminase apoBEC-1 lowers lipoprotein(a) in transgenic mice and induces apolipoprotein B editing in rabbits.

Apolipoprotein (apo) B100 is an essential component of low-density lipoproteins (LDL) and lipoprotein(a) [Lp(a)]. In mammals, apoB can be edited post-transcriptionally to encode a truncated form of apoB (apoB48) that is unable to form either of these atherogenic lipoproteins. To study the effect of increasing hepatic apoB editing activity on formation of Lp(a), a recombinant adenovirus encoding rat apoBEC-1, the cytidine deaminase component of the apoB mRNA editing complex, was administered to human apoB/apo(a) transgenic mice. This resulted in expression of apoBEC-1 in hepatocytes of these mice, increased hepatic editing of human apoB mRNA, and decreased plasma levels of human apoB100 and Lp(a). The apoBEC-1 recombinant adenovirus was also administered to rabbits, an animal which, like humans, naturally lacks hepatic apoB editing. Expression of the exogenous apoBEC-1 in rabbit liver resulted in editing of up to 10% of apoB mRNA. Hepatic apoB editing was associated with lower LDL levels in these rabbits relative to those treated with a control adenovirus. However, LDL levels were elevated significantly in both animals as a result of adenovirus injection. These studies demonstrate that introduction of the cytidine deaminase apoBEC-1 is sufficient to induce hepatic apoB editing in an animal lacking this activity, and that induction of editing could serve as a novel approach for lowering plasma concentrations of the atherogenic lipoproteins Lp(a) and LDL.

APOBEC-1 Deaminase↗
PubMed2006

APOBEC deaminases as cellular antiviral factors: a novel natural host defense mechanism.

The APOBEC (acronym for apolipoprotein B editing catalytic polypeptide) family of cytidine deaminases are widely distributed in the biological world and play a central role in diverse enzymatic pathways. Members of this family (APOBEC3G and APOBEC3F) have been recently shown to be able to restrict HIV-1 replication in physiologically relevant target cells (macrophages, lymphocytes), presumably by triggering extensive deamination of the viral RNA/DNA replication intermediates. This natural antiretroviral host defense mechanism is counteracted by the HIV-1 protein Vif, which is able to target APOBECs to degrade. The so-called "Vif/APOBEC3G paradigm" has been confirmed by a growing literature. However, evidence arising from recent studies has expanded this view, showing that the replication of other viruses is also restricted by APOBEC family members and suggesting antiviral mechanism(s) of action unrelated to the catalytic activity of these proteins. Furthermore, evolutionary investigations on primates have shown that APOBEC3 gene expansion might be related to an ancient adaptive selection to prevent endogenous genetic instability, indicating an additional ancient protective role of APOBECs. This article is aimed at broadening the current knowledge about the antiviral activity of the APOBEC members and to highlight the notion that their role(s) might be more general than previously anticipated.

APOBEC-1 Deaminase↗
PubMed2003

RNA-editing cytidine deaminase Apobec-1 is unable to induce somatic hypermutation in mammalian cells.

Antibody diversification by somatic hypermutation, gene conversion, and class switch recombination is completely dependent on activation-induced cytidine deaminase (AID). A recent report showing induction of DNA mutations in Escherichia coli by overexpression of AID, Apobec-1, and related members of the RNA-editing cytidine deaminase family suggested that they may directly modify deoxycytidine in DNA in mammalian cells (DNA-editing model). We therefore examined whether Apobec-1 bona fide RNA-editing enzyme could show somatic hypermutation and class switching activities in murine B lymphocytes and fibroblasts. Unlike AID, Apobec-1 was unable to induce somatic hypermutation or class switching. The results force a reevaluation of the physiological significance of the DNA deaminase activities of AID and Apobec-1 in E. coli and in vitro.

3T3 Cells↗
PubMed2024

An in vitro cytidine deaminase assay to monitor APOBEC activity on DNA.

APOBEC enzymes promote the deamination of cytosine (C) to uracil (U) in DNA to defend cells against viruses but also serve as a predominant source of mutations in cancer genomes. This protocol describes an assay to monitor APOBEC deaminase activity in vitro on a synthetic DNA oligonucleotide. The method described here focuses specifically on APOBEC3B to illustrate the different steps of the assay. However, the protocol can be applied to monitor the DNA deaminase activity of any other member of the APOBEC family, such as APOBEC3A. This assay involves preparing APOBEC3B-expressing cell extract or purifying APOBEC3B by immunoprecipitation, followed by incubation with a single-stranded DNA containing a TpC motif. The deaminated cytosine is then removed by recombinant Uracil DNA Glycosylase present in the reaction to form an abasic site. The abasic site creates a weakness in the DNA's backbone, causing the DNA to be cleaved under high temperatures and alkaline conditions. Denaturing gel electrophoresis is used to separate cleaved DNA from full-length DNA, enabling the quantification of the percentage of deamination induced by APOBEC3B. This protocol can be used to determine the presence of APOBEC and the regulation of APOBEC activity in specific cell lines, to study substrate preference targeted by different members of the APOBEC family and different APOBEC mutants, or to determine the efficiency and specificity of inhibitor compounds against APOBEC enzymes.

Cytidine Deaminase↗
PubMed2004

The structure of a yeast RNA-editing deaminase provides insight into the fold and function of activation-induced deaminase and APOBEC-1.

Activation-induced deaminase (AID) uses base deamination for class-switch recombination and somatic hypermutation and is related to the mammalian RNA-editing enzyme apolipoprotein B editing catalytic subunit 1 (APOBEC-1). CDD1 is a yeast ortholog of APOBEC-1 that exhibits cytidine deaminase and RNA-editing activity. Here, we present the crystal structure of CDD1 at 2.0-A resolution and its use in comparative modeling of APOBEC-1 and AID. The models explain dimerization and the need for trans-acting loops that contribute to active site formation. Substrate selectivity appears to be regulated by a central active site "flap" whose size and flexibility accommodate large substrates in contrast to deaminases of pyrimidine metabolism that bind only small nucleosides or free bases. Most importantly, the results suggested both AID and APOBEC-1 are equally likely to bind single-stranded DNA or RNA, which has implications for the identification of natural AID targets.

APOBEC-1 Deaminase↗
PubMed2006

Restriction of foamy viruses by APOBEC cytidine deaminases.

Foamy viruses (FVs) are nonpathogenic retroviruses infecting many species of mammals, notably primates, cattle, and cats. We have examined whether members of the apolipoprotein B-editing catalytic polypeptide-like subunit (APOBEC) family of antiviral cytidine deaminases restrict replication of simian FV. We show that human APOBEC3G is a potent inhibitor of FV infectivity in cell culture experiments. This antiviral activity is associated with cytidine editing of the viral genome. Both molecular FV clones and primary uncloned viruses were susceptible to APOBEC3G, and viral infectivity was also inhibited by murine and simian APOBEC3G homologues, as well as by human APOBEC3F. Wild-type and bet-deleted viruses were similarly sensitive to this antiviral activity, suggesting that Bet does not significantly counteract APOBEC proteins. Moreover, we did not detect FV sequences that may have been targeted by APOBEC in naturally infected macaques, but we observed a few G-to-A substitutions in humans that have been accidentally contaminated by simian FV. In infected hosts, the persistence strategy employed by FV might be based on low levels of replication, as well as avoidance of cells expressing large amounts of active cytidine deaminases.

APOBEC-3G Deaminase↗
PubMed1996

Apolipoprotein B RNA editing enzyme-deficient mice are viable despite alterations in lipoprotein metabolism.

RNA editing in the nucleus of higher eukaryotes results in subtle changes to the RNA sequence, with the ability to effect dramatic changes in biological function. The first example to be described and among the best characterized, is the cytidine-to-uridine editing of apolipoprotein B (apo-B) RNA. The editing of apo-B RNA is mediated by a novel cytidine deaminase, apobec-1, which has acquired the ability to bind RNA. The stop translation codon generated by the editing of apo-B RNA truncates the full-length apo-B100 to form apo-B48. The recent observations of tumor formation in Apobec-1 transgenic animals, together with the fact that Apobec-1 is expressed in numerous tissues lacking apo-B, raises the issue of whether this enzyme is essential for a variety of posttranscriptional editing events. To directly test this, mice were created with a null mutation in Apobec-1 using homologous recombination in embryonic stem cells. Mice, homozygous for this mutation, were viable and made apo-B100 but not apo-B48. The null animals were fertile, and a variety of histological, behavioral, and morphological analyses revealed no phenotype other than abnormalities in lipoprotein metabolism, which included an increased low density lipoprotein fraction and a reduction in high density lipoprotein cholesterol. These studies demonstrate that neither apobec-1 nor apo-B48 is essential for viability and suggest that the major role of apobec-1 may be confined to the modulation of lipid transport.

APOBEC-1 Deaminase↗
PubMed2004

Ancient adaptive evolution of the primate antiviral DNA-editing enzyme APOBEC3G.

Host genomes have adopted several strategies to curb the proliferation of transposable elements and viruses. A recently discovered novel primate defense against retroviral infection involves a single-stranded DNA-editing enzyme, APOBEC3G, that causes hypermutation of HIV. The HIV-encoded virion infectivity factor (Vif) protein targets APOBEC3G for destruction, setting up a genetic conflict between the APOBEC3G and Vif genes. This kind of conflict leads to rapid fixation of mutations that alter amino acids at the protein-protein interface, referred to as positive selection. We show that the APOBEC3G gene has been subject to strong positive selection throughout the history of primate evolution. Unexpectedly, this selection appears more ancient than, and is likely only partially caused by, modern lentiviruses. Furthermore, five additional APOBEC genes in the human genome appear to be engaged in similar genetic conflicts, displaying some of the highest signals for positive selection in the human genome. Despite being only recently discovered, editing of RNA and DNA may thus represent an ancient form of host defense in primate genomes.

APOBEC Deaminases↗
PubMed1996

Hyperediting of multiple cytidines of apolipoprotein B mRNA by APOBEC-1 requires auxiliary protein(s) but not a mooring sequence motif.

An RNA-binding cytidine deaminase (APOBEC-1) and unidentified auxiliary protein(s) are required for apolipoprotein (apo) B mRNA editing. A sequence motif on apoB mRNA ("mooring sequence," nucleotides 6671-6681) is obligatory for the editing of cytidine 6666 (C6666), the only cytidine on apoB mRNA converted to uridine in normal animals. Transgenic animals with hepatic overexpression of APOBEC-1 develop liver tumors, and other non-apoB mRNAs are edited, suggesting a loss of the normally precise specificity. In this study, we examined apoB mRNA from these transgenic animals to determine if cytidines aside from C6666 are edited. Multiple cytidines downstream from C6666 in apoB mRNA were edited extensively by the overexpressed APOBEC-1. This pathophysiological "hyperediting" could be mimicked in vitro by incubating a synthetic apoB RNA substrate with the transgenic mouse liver extracts. Multiple cytidines in the synthetic apoB RNA were edited by recombinant APOBEC-1 but only with supplementation of the auxiliary protein(s). Mutations in the mooring sequence markedly decreased the normal editing of C6666 but, surprisingly, increased the hyperediting of downstream cytidines. Furthermore, cytidines in an apoB RNA substrate lacking the mooring sequence were also edited in vitro. These results indicate that the hyperediting of apoB mRNA by overexpressed APOBEC-1 depends upon auxiliary protein(s) but is independent of the mooring sequence motif. These results suggest that hyperediting may represent the first step in a two-step recognition model for normal apoB mRNA editing.

APOBEC-1 Deaminase↗
PubMed2000

APOBEC-1 dependent cytidine to uridine editing of apolipoprotein B RNA in yeast.

Cytidine to uridine editing of apolipoprotein B (apoB) mRNA requires the cytidine deaminase APOBEC-1 as well as a tripartite sequence motif flanking a target cytidine in apoB mRNA and an undefined number of auxiliary proteins that mediate RNA recognition and determine site-specific editing. Yeast engineered to express APOBEC-1 and apoB mRNA supported editing under conditions of late log phase growth and stationary phase. The cis -acting sequence requirements and the intracellular distribution of APOBEC-1 in yeast were similar to those described in mammalian cells. These findings suggest that auxiliary protein functions necessary for the assembly of editing complexes, or 'editosomes', are expressed in yeast and that the distribution of editing activity is to the cell nucleus.

APOBEC-1 Deaminase↗
PubMed2006

The APOBEC-2 crystal structure and functional implications for the deaminase AID.

APOBEC-2 (APO2) belongs to the family of apolipoprotein B messenger RNA-editing enzyme catalytic (APOBEC) polypeptides, which deaminates mRNA and single-stranded DNA. Different APOBEC members use the same deamination activity to achieve diverse human biological functions. Deamination by an APOBEC protein called activation-induced cytidine deaminase (AID) is critical for generating high-affinity antibodies, and deamination by APOBEC-3 proteins can inhibit retrotransposons and the replication of retroviruses such as human immunodeficiency virus and hepatitis B virus. Here we report the crystal structure of APO2. APO2 forms a rod-shaped tetramer that differs markedly from the square-shaped tetramer of the free nucleotide cytidine deaminase, with which APOBEC proteins share considerable sequence homology. In APO2, two long alpha-helices of a monomer structure prevent the formation of a square-shaped tetramer and facilitate formation of the rod-shaped tetramer via head-to-head interactions of two APO2 dimers. Extensive sequence homology among APOBEC family members allows us to test APO2 structure-based predictions using AID. We show that AID deamination activity is impaired by mutations predicted to interfere with oligomerization and substrate access. The structure suggests how mutations in patients with hyper-IgM-2 syndrome inactivate AID, resulting in defective antibody maturation.

APOBEC Deaminases↗
PubMed1999

Disproportionate relationship between APOBEC-1 expression and apolipoprotein B mRNA editing activity.

Apolipoprotein B (apoB) mRNA editing is a site-specific (nucleotide 6666) cytidine to uridine transition catalyzed by a cytidine deaminase, APOBEC-1, in the context of a multiprotein complex referred to as the C/U editosome. This report quantifies for the first time the effect of altering APOBEC-1 protein abundance on the proportion of edited apoB mRNAs using transfected McArdle rat hepatoma cells which had been sorted by flow cytometry into populations expressing different levels of green fluorescent protein-APOBEC-1 chimera, GFP-APOBEC. A correlation was observed in which increased expression of GFP-APOBEC protein resulted in a higher proportion of edited apoB mRNA. The number of enzyme molecules required to increase the proportion of edited apoB RNAs was disproportionately high relative to that which might have been predicted from a typical catalytic relationship. Moreover, editing of apoB mRNA at inappropriate sites (promiscuous editing) occurred in response to overexpressing GFP-APOBEC. The data suggest that experimental manipulation of APOBEC-1 abundance in the absence of other regulatory considerations will always result in some level of promiscuous editing. Coordinate expression of APOBEC-1 and the auxiliary proteins and/or regulation of their interactions may be required to increase editing activity without losing editing-site fidelity.

APOBEC-1 Deaminase↗
PubMed2003

Intestinal lipoprotein assembly in apobec-1-/- mice reveals subtle alterations in triglyceride secretion coupled with a shift to larger lipoproteins.

Mammalian enterocytes express apolipoprotein (apo)B-48, which is produced after posttranscriptional RNA editing of the nuclear apoB-100 transcript by the catalytic deaminase apobec-1. Earlier studies in apobec-1-/- mice revealed an apoB-100-only lipoprotein profile but no gross defects in triglyceride absorption. However, subtle defects may have been obscured by the mixed genetic background. In addition, the intrinsic susceptibility to proteolytic degradation of intestinal apoB-100 and apoB-48 has been questioned. Accordingly, we examined triglyceride absorption, intestinal apoB expression, and lipoprotein secretion in apobec-1-/- mice backcrossed into a C57BL/6 background. Inbred apobec-1-/- mice absorb triglyceride normally, yet secrete triglyceride-rich lipoproteins more slowly than wild-type congenic controls. There was comparable induction of apoB synthesis in response to fat feeding in both genotypes, but apoB-100 was preferentially retained and more extensively degraded than apoB-48. By contrast, synthesis, secretion, and content of apo A-IV were indistinguishable in apobec-1-/- and wild-type mice with 100% recovery, suggesting no degradation of this apoprotein in either genotype. Newly secreted lipoproteins from isolated enterocytes of wild-type mice revealed apoB-48 in both high-density lipoproteins and very low-density lipoproteins. By contrast, apobec-1-/- mice secreted apoB-100-containing particles that were almost exclusively in the low and very low-density lipoproteins range with no apoB-100-containing high-density lipoproteins. These studies establish the existence of preferential degradation of intestinal apoB-100 and subtle defects in triglyceride secretion in apobec-1-/- mice, coupled with a shift to the production of larger particles, findings that suggest an important divergence in intestinal lipoprotein assembly pathways with the different isoforms of apoB.

APOBEC-1 Deaminase↗
PubMed1996

In vitro reconstitution of apolipoprotein B RNA editing activity from recombinant APOBEC-1 and McArdle cell extracts.

Apolipoprotein B (apoB) RNA editing activity involves a site-specific cytidine to uridine transition catalyzed by a cytidine deaminase, APOBEC-1, in the context of a multi-protein-containing editosome. In the absence of yet to be characterized "auxiliary" proteins, APOBEC-1 lacks RNA editing capacity. Recombinant APOBEC-1 has been engineered to bind nickel resin and used in affinity chromatography of the auxiliary proteins from McArdle rat hepatoma cell extracts. We demonstrate activation of APOBEC-1 RNA editing activity under these conditions through the association of a subset of extract proteins having approximate molecular masses of 145, 87, 75, 66, 61, and 50 kDa and a heterogeneous grouping of 45- to 35-kDa proteins. These data suggest that the components of the editosome can be partially purified from extracts through APOBEC-1 affinity chromatography.

Animals↗
PubMed2003

A novel nuclear localization signal in the auxiliary domain of apobec-1 complementation factor regulates nucleocytoplasmic import and shuttling.

C to U editing of the nuclear apolipoprotein B (apoB) transcript is mediated by a core enzyme containing a catalytic deaminase, apobec-1, and an RNA binding subunit, apobec-1 complementation factor (ACF). ACF expression is predominantly nuclear, including mutant proteins with deletions of a putative nuclear localization signal. We have now identified a novel 41-residue motif (ANS) in the auxiliary domain of ACF that functions as an authentic nuclear localization signal. ANS-green fluorescence protein and ANS-beta-galactosidase chimeras were both expressed exclusively in the nucleus, whereas wild-type chimeras or an ACF deletion mutant lacking the ANS were cytoplasmic. Nuclear accumulation of ACF is transcription-dependent, temperature-sensitive, and reversible, features reminiscent of a shuttling protein. ACF relocates to the cytoplasm after actinomycin D treatment, an effect blocked by the CRM1 inhibitor leptomycin B. Heterokaryon assays confirmed directly that ACF shuttles in vivo. ACF binds to the protein carrier, transportin 2 in vivo, and colocalizes to the nucleus as determined by confocal microscopy. Co-immunoprecipitation experiments revealed that transportin 2 binds directly to the ANS motif. These data suggest that directed nuclear localization and compartmentalization of the core complex of the apoB RNA editing enzyme is regulated through a dominant targeting sequence (ANS) contained within ACF.

Active Transport, Cell Nucleus↗
PubMed2004

Gene structure and expression of the mouse APOBEC-1 complementation factor: multiple transcriptional initiation sites and a spliced variant with a premature stop translation codon.

Editing of apolipoprotein (apo) B mRNA is mediated by an enzyme-complex that consists of the catalytic cytidine deaminase APOBEC-1 and the mRNA binding protein APOBEC-1 complementation factor or APOBEC-1 stimulating protein (ACF/ASP). Here we describe the detailed characterization of the structure, expression and splicing pattern of the mouse ACF/ASP gene. ACF/ASP mRNA is mainly expressed in mouse liver, small intestine and kidney. The deduced protein sequences of ACF/ASP from mouse and man share an identity of 93%. The mouse ACF/ASP gene consists of 12 exons and gives rise predominantly to full-length transcripts. To a minor extent (<10%) ACF/ASP mRNA with unspliced exon 8 is generated in liver, kidney and small intestine that encodes a truncated protein with a predicted molecular weight of 43 kDa. The promoter of the mouse ACF/ASP gene lacks a canonical TATA-box, but contains a cluster of Sp1 binding sites and uses multiple transcriptional initiation sites. Transfection studies demonstrated a preference of this promoter for cell lines derived from the gastrointestinal tract and proved the location of the promoter core region. The high sequence identity between man and mouse-much higher as observed for APOBEC-1-indicates a strong evolutionary constraint on the structure-function relationship of ACF/ASP, most probably due to a central role in editing and processing of apo B mRNA.

Alternative Splicing↗
PubMed1998

Ethanol increases apolipoprotein B mRNA editing in rat primary hepatocytes and McArdle cells.

Apolipoprotein B (apoB) mRNA editing involves a site-specific cytidine to uridine transition catalyzed by the cytidine deaminase, APOBEC-1, in the context of and regulated by a multi-protein-containing editosome. ApoB mRNA editing in vivo is subject to tissue specific, developmental and metabolic regulation. We demonstrate for the first time that the amount of edited apoB mRNA in rat primary hepatocytes is markedly increased subsequent to transient treatment with ethanol in vitro. The apparent change in editing efficiency was dose-dependent (from 0.1%-2.4% initial ethanol dose) and occurred with rapid onset. The proportion of edited apoB mRNA was also markedly enhanced in a rat hepatoma cell line, McArdle RH7777 cells and in a stable McArdle cell line over-expressing APOBEC-1 by transient treatment with 2.5 % ethanol. In contrast, the apoB mRNA editing in a human hepatoma cell line, HepG2 cells and a stable HepG2 cell line over-expressing APOBEC-1 did not respond to ethanol treatment. The data support the possibility that editing activity is ethanol-responsive but suggest that this change is cell type-specific.

Animals↗
PubMed1999

Tissue-specific differences in the role of RNA 3' of the apolipoprotein B mRNA mooring sequence in editosome assembly.

Site-specific editing of apolipoprotein B (apoB) mRNA by the cytidine deaminase, APOBEC-1 is proposed to require interactions of auxiliary protein(s) with an eleven nucleotide element, the mooring sequence, located 3' of the C --> U editing site. An analysis of the RNA sequence dependence for protein-RNA interactions and editosome assembly in rat liver and the small intestine demonstrated that the mooring sequence was a minimal requirement for these interactions. Sequences 3' of the mooring sequence either interacted with 66 kDa and 44 kDa proteins or enhanced the interactions of these proteins with the mooring sequence. The data also suggested tissue-specific differences in the relative importance of the 3' cis-acting 'enhancer' elements in the efficiency or stability of editosome assembly. We propose that the previously demonstrated differences in apoB mRNA editing efficiency and its regulation in liver and intestine may in part be due to differences in auxiliary protein interactions with apoB mRNA 3' of the mooring sequence.

Animals↗