Endogenous APOBEC3A DNA Cytosine Deaminase Is Cytoplasmic and Nongenotoxic

APOBEC3A (A3A) is a myeloid lineage-specific DNA cytosine deaminase with a role in innate immunity to foreign DNA. Previous studies have shown that heterologously expressed A3A is genotoxic, suggesting that monocytes may have a mechanism to regulate this enzyme. Indeed, we observed no significant cytotoxicity when interferon was used to induce the expression of endogenous A3A in CD14⁺-enriched primary cells or the monocytic cell line THP-1. In contrast, doxycycline-induced A3A in HEK293 cells caused major cytotoxicity at protein levels lower than those observed when CD14⁺ cells were stimulated with interferon. Immunofluorescent microscopy of interferon-stimulated CD14⁺ and THP-1 cells revealed that endogenous A3A is cytoplasmic, in stark contrast to stably or transiently transfected A3A, which has a cell-wide localization. A3A constructs engineered to be cytoplasmic are also nontoxic in HEK293 cells. These data combine to suggest that monocytic cells use a cytoplasmic retention mechanism to control A3A and avert genotoxicity during innate immune responses.     

APOBEC3s: DNA‐editing human cytidine deaminases

Nucleic acid editing enzymes are essential components of the human immune system that lethally mutate viral pathogens and somatically mutate immunoglobulins. Among these enzymes are cytidine deaminases of the apolipoprotein B mRNA editing enzyme, catalytic polypeptide‐like (APOBEC) super family, each with unique target sequence specificity and subcellular localization. We focus on the DNA‐editing APOBEC3 enzymes that have recently attracted attention because of their involvement in cancer and potential in gene‐editing applications. We review and compare the crystal structures of APOBEC3 (A3) domains, binding interactions with DNA, substrate specificity, and activity. Recent crystal structures of A3A and A3G bound to ssDNA have provided insights into substrate binding and specificity determinants of these enzymes. Still many unknowns remain regarding potential cooperativity, nucleic acid interactions, and systematic quantification of substrate preference of many APOBEC3s, which are needed to better characterize the biological functions and consequences of misregulation of these gene editors.     

m6A demethylation of cytidine deaminase APOBEC3B mRNA orchestrates arsenic-induced mutagenesis

The cytidine deaminase APOBEC3B (A3B) is an endogenous inducer of somatic mutations and causes chromosomal instability by converting cytosine to uracil in single-stranded DNA. Therefore, identification of factors and mechanisms that mediate A3B expression will be helpful for developing therapeutic approaches to decrease DNA mutagenesis. Arsenic (As) is one well-known mutagen and carcinogen, but the mechanisms by which it induces mutations have not been fully elucidated. Herein, we show that A3B is upregulated and required for As-induced DNA damage and mutagenesis. We found that As treatment causes a decrease of N6-methyladenosine (m6A) modification near the stop codon of A3B, consequently increasing the stability of A3B mRNA. We further reveal that the demethylase FTO is responsible for As-reduced m6A modification of A3B, leading to increased A3B expression and DNA mutation rates in a manner dependent on the m6A reader YTHDF2. Our in vivo data also confirm that A3B is a downstream target of FTO in As-exposed lung tissues. In addition, FTO protein is highly expressed and positively correlates with the protein levels of A3B in tumor samples from human non–small cell lung cancer patients. These findings indicate a previously unrecognized role of A3B in As-triggered somatic mutation and might open new avenues to reduce DNA mutagenesis by targeting the FTO/m6A axis.     

猪APOBEC3F基因电子克隆

从EST序列着手直接寻找新基因,即利用计算机进行同源性和一致性分析、寻找感兴趣的EST、构建包含着EST的重叠群,再进行ORF判定以及蛋白质同源性分析。最终利用生物信息学技术克隆获得了猪的APOBEC3F序列。     

Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity

The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off‐target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin‐containing TCP‐1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A‐induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.     

Sub-families of alpha/beta barrel enzymes: a new adenine deaminase family

No gene coding for an adenine deaminase has been described in eukaryotes. However, physiological and genetical evidence indicates that adenine deaminases are present in the ascomycetes. We have cloned and characterised the genes coding for the adenine deaminases of Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe. The A.nidulans gene was expressed in Escherichia coli and the purified enzyme shows adenine but not adenosine deaminase activity. The open reading frames coded by the three genes are very similar and obviously related to the bacterial and eukaryotic adenosine deaminases rather than to the bacterial adenine deaminases. The latter are related to allantoinases, ureases and dihydroorotases. The fungal adenine deaminases and the homologous adenosine deaminases differ in a number of residues, some of these being clearly involved in substrate specificity. Other prokaryotic enzymes in the database, while clearly related to the above, do not fit into either sub-class, and may even have a different specificity. These results imply that adenine deaminases have appeared twice in the course of evolution, from different ancestral enzymes constructed both around the alpha/beta barrel scaffold.