Functions and regulation of the APOBEC family of proteins

Abstract

APOBEC1 is a cytidine deaminase that edits messenger RNAs and was the first enzyme in the APOBEC family to be functionally characterized. Under appropriate conditions APOBEC1 also deaminates deoxycytidine in single-stranded DNA (ssDNA). The other ten members of the APOBEC family have not been fully characterized however several have deoxycytidine deaminase activity on ssDNAs. Despite the nucleic acid substrate preferences of different APOBEC proteins, a common feature appears to be their intrinsic ability to bind to RNA as well as to ssDNA. RNA binding to APOBEC proteins together with protein-protein interactions, post-translation modifications and subcellular localization serve as biological modulators controlling the DNA mutagenic activity of these potentially genotoxic proteins.

Figure 1. Models for A1 and A3G complexes with nucleic acids

A. The tripartite apoB mRNA editing motif consisting of the mooring sequence, and spacer 3’ of the C to be edited and the enhancer element 5’ of the edited C is shown with a cartoon of an editosome assembled upon it. An A1 C-terminal dimer is positioned for site-selective C to U editing by virtue of its association with ACF dimers that are shown bound to the mooring sequence. For the purposes of presentation only one A1 dimer is shown bound to one ACF through C-terminal to N-terminal (respectively) interactions (although the precise stoichiometry is unknown). Most of the three RNA recognition motifs comprising greater than the N-terminal half of ACF are required for optimal A1 binding. ACF dimerization requires only the N-terminal half of ACF. B. a. An A3G monomer is shown containing an N- and C-terminal ZDD (label as ‘N’ and ‘C’). Nucleic acid deficient A3G forms a heterogeneous mixture of oligomers consisting mostly of dimers in solution and an A3G concentration-dependent small population of monomers and tetramers. b. A3G dimers to bind ssDNA substrates (black line with the CCA editing site) and must form at minimum a tetramer for enzymatic activity. c. RNA (red line) competes for ssDNA binding by displacing ssDNA and binding at the same site or d. RNA competes for ssDNA binding by binding at a distal site and causing an allosteric change in A3G conformation, preventing ssDNA binding.

Figure 2. APOBEC family localization and activity

A. Bar diagrams of human APOBEC proteins and their relative alignments according to exon junctions. The ZDD motifs (black) and number of amino acids for each protein are indicated. The (*) next to the ZDD for APOBEC4 indicates that it is divergent from the consensus ZDD. The relative subcellular distribution is on the left with N for nuclear and C for cytoplasmic, and ‘?’ if localization is unknown. The size of N or C indicates relative distribution. The deaminase activity is indicated by a + or − and the +/− for APOBEC2 indicates mixed results depending on the system (see text). B. A chart of A3 proteins’ activity on different mobile genetic elements, with + for active and − for inactive and ND when activity has been not determined. The (monocytes) under the A3A HIV activity indicates that it has only been shown to be active in monocyte derived cells. The information in A and B was compiled from references within the text.

Figure 3. Localization of the APOBEC proteins by in situ fluorescence

A. APOBEC-1 (A1) with a C-terminal HA tag was expressed by transfection in McArdle rat hepatoma cells. Immunocytochemical localization of the HA tag in fixed cells shows that A1 is distributed throughout the cytoplasm and nucleus (DAPI) but is not localized in the nucleolus. A1 shuttles between the cytoplasm and nucleus. B. Activation Induced Deaminase (AID) as a C-terminal GFP chimeric protein was expressed in transfected human embryonic kidney cells 293T and visualized in live cells. AID appears predominantly in the cytoplasm (left panel, four transfected cells shown) but its rapid shuttling activity can be demonstrated by inhibiting its CRM1-dependent nuclear export with Leptomycin B (LMB) (right panel, two transfected cells shown). C. A3G as an N-terminal mCherry chimeric protein was expressed in transfected HEK293T cells and visualized in live cells. A3G is restricted to the cytoplasm because it has no nuclear localization signal but does have a cytoplasmic retention signal. D. Natively expressed A3G in synchronized H9 cells after fixing and staining with anti-A3G polyclonal antibody. DAPI stained mitotic chromosomes (left top, anaphase; left bottom, telophase) are segregated in the mitotic cells from cytoplasmic A3G. Original magnification was 40X.