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Proceedings of the National Academy of... May 2015Activation-induced cytidine deaminase (AID) is essential for antibody class switch recombination (CSR) and somatic hypermutation (SHM). AID originally was postulated to...
Activation-induced cytidine deaminase (AID) is essential for antibody class switch recombination (CSR) and somatic hypermutation (SHM). AID originally was postulated to function as an RNA-editing enzyme, based on its strong homology with apolipoprotein B mRNA-editing enzyme, catalytic polypeptide 1 (APOBEC1), the enzyme that edits apolipoprotein B-100 mRNA in the presence of the APOBEC cofactor APOBEC1 complementation factor/APOBEC complementation factor (A1CF/ACF). Because A1CF is structurally similar to heterogeneous nuclear ribonucleoproteins (hnRNPs), we investigated the involvement of several well-known hnRNPs in AID function by using siRNA knockdown and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated disruption. We found that hnRNP K deficiency inhibited DNA cleavage and thereby induced both CSR and SHM, whereas hnRNP L deficiency inhibited only CSR and somewhat enhanced SHM. Interestingly, both hnRNPs exhibited RNA-dependent interactions with AID, and mutant forms of these proteins containing deletions in the RNA-recognition motif failed to rescue CSR. Thus, our study suggests that hnRNP K and hnRNP L may serve as A1CF-like cofactors in AID-mediated CSR and SHM.
Topics: APOBEC-1 Deaminase; Cell Line, Tumor; Cell Separation; Cytidine Deaminase; DNA; Flow Cytometry; Genetic Complementation Test; HEK293 Cells; Heterogeneous-Nuclear Ribonucleoprotein K; Heterogeneous-Nuclear Ribonucleoprotein L; Heterogeneous-Nuclear Ribonucleoproteins; Humans; Immunoglobulin A; Immunoglobulin Class Switching; Immunoglobulin Heavy Chains; Mutation; Protein Binding; Protein Structure, Tertiary; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; RNA-Binding Proteins; Recombination, Genetic; Somatic Hypermutation, Immunoglobulin
PubMed: 25902538
DOI: 10.1073/pnas.1506167112 -
International Journal of Molecular... May 2022The radiosensitization of tumor cells is one of the promising approaches for enhancing radiation damage to cancer cells and limiting radiation effects on normal tissue....
The radiosensitization of tumor cells is one of the promising approaches for enhancing radiation damage to cancer cells and limiting radiation effects on normal tissue. In this study, we performed a comprehensive screening of radiosensitization targets in human lung cancer cell line A549 using an shRNA library and identified () as a candidate target. is an innate restriction factor that inhibits HIV-1 infection as a cytidine deaminase. knockdown with siRNA showed an increased radiosensitivity in several cancer cell lines, including pancreatic cancer MIAPaCa2 cells and lung cancer A549 cells. Cell cycle analysis revealed that knockdown increased S-phase arrest in MIAPaCa2 and G2/M arrest in A549 cells after γ-irradiation. DNA double-strand break marker γH2AX level was increased in -knocked-down MIAPaCa2 cells after γ-irradiation. Using a xenograft model of A549 in mice, enhanced radiosensitivity by a combination of X-ray irradiation and knockdown was observed. These results suggest that the functional inhibition of sensitizes cancer cells to radiation by attenuating the activation of the DNA repair pathway, suggesting that could be useful as a target for the radiosensitization of cancer therapy.
Topics: APOBEC-3G Deaminase; Animals; Apoptosis; Cell Line, Tumor; Cytidine Deaminase; G2 Phase Cell Cycle Checkpoints; Gamma Rays; Humans; Lung Neoplasms; Mice; Radiation Tolerance
PubMed: 35563460
DOI: 10.3390/ijms23095069 -
Molecular Cell Aug 2017Activation-induced cytidine deaminase (AID) initiates both class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. Mechanisms of...
Activation-induced cytidine deaminase (AID) initiates both class switch recombination (CSR) and somatic hypermutation (SHM) in antibody diversification. Mechanisms of AID targeting and catalysis remain elusive despite its critical immunological roles and off-target effects in tumorigenesis. Here, we produced active human AID and revealed its preferred recognition and deamination of structured substrates. G-quadruplex (G4)-containing substrates mimicking the mammalian immunoglobulin switch regions are particularly good AID substrates in vitro. By solving crystal structures of maltose binding protein (MBP)-fused AID alone and in complex with deoxycytidine monophosphate, we surprisingly identify a bifurcated substrate-binding surface that explains structured substrate recognition by capturing two adjacent single-stranded overhangs simultaneously. Moreover, G4 substrates induce cooperative AID oligomerization. Structure-based mutations that disrupt bifurcated substrate recognition or oligomerization both compromise CSR in splenic B cells. Collectively, our data implicate intrinsic preference of AID for structured substrates and uncover the importance of G4 recognition and oligomerization of AID in CSR.
Topics: APOBEC Deaminases; Animals; Antibody Diversity; B-Lymphocytes; Cytidine Deaminase; DNA; Humans; Immunoglobulin Class Switching; Immunoglobulin Switch Region; Mice; Models, Molecular; Mutation; Nucleic Acid Conformation; Protein Binding; Protein Conformation; Recombination, Genetic; Spleen; Structure-Activity Relationship; Substrate Specificity
PubMed: 28757211
DOI: 10.1016/j.molcel.2017.06.034 -
Philosophical Transactions of the Royal... Mar 2009
Topics: Antibodies; Cytidine Deaminase; Cytosine; DNA; DNA Repair; Deamination; Gene Rearrangement; Homeodomain Proteins; Immunity, Active
PubMed: 19008190
DOI: 10.1098/rstb.2008.0236 -
Scientific Reports Oct 2021The cytidine deaminase, APOBEC3A (A3A), is a prominent source of mutations in multiple cancer types. These APOBEC-signature mutations are non-uniformly distributed...
The cytidine deaminase, APOBEC3A (A3A), is a prominent source of mutations in multiple cancer types. These APOBEC-signature mutations are non-uniformly distributed across cancer genomes, associating with single-stranded (ss) DNA formed during DNA replication and hairpin-forming sequences. The biochemical and cellular factors that influence these specificities are unclear. We measured A3A's cytidine deaminase activity in vitro on substrates that model potential sources of ssDNA in the cell and found that A3A is more active on hairpins containing 4 nt ssDNA loops compared to hairpins with larger loops, bubble structures, replication fork mimics, ssDNA gaps, or linear DNA. Despite pre-bent ssDNAs being expected to fit better in the A3A active site, we determined A3A favors a 4 nt hairpin substrate only 2- to fivefold over linear ssDNA substrates. Addition of whole cell lysates or purified RPA to cytidine deaminase assays more severely reduced A3A activity on linear ssDNA (45 nt) compared to hairpin substrates. These results indicate that the large enrichment of A3A-driven mutations in hairpin-forming sequences in tumor genomes is likely driven in part by other proteins that preferentially bind longer ssDNA regions, which limit A3A's access. Furthermore, A3A activity is reduced at ssDNA associated with a stalled T7 RNA polymerase, suggesting that potential protein occlusion by RNA polymerase also limits A3A activity. These results help explain the small transcriptional strand bias for APOBEC mutation signatures in cancer genomes and the general targeting of hairpin-forming sequences in the lagging strand template during DNA replication.
Topics: Cytidine Deaminase; DNA Replication; DNA, Single-Stranded; DNA-Binding Proteins; Enzyme Activation; Gene Expression; Humans; Nucleic Acid Conformation; Protein Binding; Proteins; Substrate Specificity; Transcription, Genetic
PubMed: 34697369
DOI: 10.1038/s41598-021-00435-y -
MSphere Dec 2022Anelloviruses are the most common viruses infecting humans. Every human carries a nonpathogenic personal anellovirus virome (anellome), yet it is unknown which...
Anelloviruses are the most common viruses infecting humans. Every human carries a nonpathogenic personal anellovirus virome (anellome), yet it is unknown which mechanisms contribute to its stability. Here, we assessed the dynamics and impact of a host antiviral defense mechanism-cytidine deaminase activity leading to C to U editing in anelloviruses-on the stability of the anellome. We investigated anellome sequence data obtained from serum samples collected every 6 months from two healthy subjects followed for more than 30 years. The subjects were infected by a total of 64 anellovirus lineages. Minus-stranded C to U editing was observed in lineages belonging to the , , and genera. The edited genomes were present within virus particles, therefore editing must have occurred at the late stages of the virus life cycle. Editing was favored by 5'-T contexts in the virus genome, indicating that apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like, catalytic subunit 3 or A3 (APOBEC3) proteins are involved. Within a lineage, mutational dynamics varied over time and few fixations of mutations were detected, indicating that C to U editing is a dead end for a virus genome. We detected an editing coldspot in the GC-rich regions, suggesting that the GC-rich region is crucial for genome packaging, since only packaged virus particles were included in the analysis. Finally, we noticed a lineage-specific reduced concentration after an editing event, yet no clearance. In conclusion, cytidine deaminase activity does not clear anelloviruses, nor does it play a major role in virus evolution, but it does contribute to the stability of the anellome. Despite significant attention on anellovirus research, the interaction between the anellovirus virome and the human host remains unknown. We show the dynamics of APOBEC3-mediated cytidine deaminase activity on anelloviruses during a 30-year period of chronic infection and postulate that this antiviral mechanism controls anelloviruses. These results expand our knowledge of anellovirus-host interactions, which may be important for the design of gene therapies.
Topics: Humans; Anelloviridae; Gene Editing; Cytosine; Antiviral Agents; Cytidine Deaminase
PubMed: 36374042
DOI: 10.1128/msphere.00506-22 -
Protein Science : a Publication of the... Feb 2020Human immunodeficiency virus (HIV) is a retroviral pathogen that targets human immune cells such as CD4 T cells, macrophages, and dendritic cells. The human... (Review)
Review
Human immunodeficiency virus (HIV) is a retroviral pathogen that targets human immune cells such as CD4 T cells, macrophages, and dendritic cells. The human apolipoprotein B mRNA- editing catalytic polypeptide 3 (APOBEC3 or A3) cytidine deaminases are a key class of intrinsic restriction factors that inhibit replication of HIV. When HIV-1 enters the cell, the immune system responds by inducing the activation of the A3 family proteins, which convert cytosines to uracils in single-stranded DNA replication intermediates, neutralizing the virus. HIV counteracts this intrinsic immune response by encoding a protein termed viral infectivity factor (Vif). Vif targets A3 to an E3 ubiquitin ligase complex for poly-ubiquitination and proteasomal degradation. Vif is unique in that it can recognize and counteract multiple A3 restriction factor substrates. Structural biology studies have provided significant insights into the overall architectures and functions of Vif and A3 proteins; however, a structure of the Vif-A3 complex has remained elusive. In this review, we summarize and reanalyze experimental data from recent structural, biochemical, and functional studies to provide key perspectives on the residues involved in Vif-A3 protein-protein interactions.
Topics: APOBEC Deaminases; Crystallography, X-Ray; Cytidine Deaminase; Humans; Models, Molecular; Protein Binding; vif Gene Products, Human Immunodeficiency Virus
PubMed: 31518043
DOI: 10.1002/pro.3729 -
MAbs 2022Affinity maturation, an essential component of antibody engineering, is crucial for developing therapeutic antibodies. Cell display system coupled with somatic...
Affinity maturation, an essential component of antibody engineering, is crucial for developing therapeutic antibodies. Cell display system coupled with somatic hypermutation (SHM) initiated by activation-induced cytidine deaminase (AID) is a commonly used technique for affinity maturation. AID introduces targeted DNA lesions into hotspots of immunoglobulin (Ig) gene loci followed by erroneous DNA repair, leading to biased mutations in the complementary determining regions. However, systems that use an mimicking mechanism often require several rounds of selection to enrich clones possessing accumulated mutations. We previously described the human ADLib® system, which features autonomous, AID-mediated diversification in Ig gene loci of a chicken B cell line DT40 and streamlines human antibody generation and optimization in one integrated platform. In this study, we further engineered DT40 capable of receiving exogenous antibody genes and examined whether the antibody could be affinity matured. The Ig genes of three representative anti-hVEGF-A antibodies originating from the human ADLib® were introduced; the resulting human IgG1 antibodies had up to 76.4-fold improvement in binding affinities (sub-picomolar ) within just one round of optimization, owing to efficient accumulation of functional mutations. Moreover, we successfully improved the affinity of a mouse hybridoma-derived anti-hCDCP1 antibody using the engineered DT40, and the observed mutations remained effective in the post-humanized antibody as exhibited by an 8.2-fold increase of cytotoxicity without compromised physical stability. These results demonstrated the versatility of the novel B cell-based affinity maturation system as an easy-to-use antibody optimization tool regardless of the species of origin. ADLib®: Autonomously diversifying library, ADLib® KI-AMP: ADLib® knock-in affinity maturation platform, AID: activation-induced cytidine deaminase, CDRs: complementary-determining regions, DIVAC: diversification activator, ECD: extracellular domain, FACS: fluorescence-activated cell sorting, FCM: flow cytometry, HC: heavy chainIg: immunoglobulin, LC: light chain, NGS: next-generation sequencing, PBD: pyrrolobenzodiazepine, SHM: somatic hypermutation, SPR: surface plasmon resonance.
Topics: Animals; Humans; Mice; B-Lymphocytes; Cytidine Deaminase; DNA; Immunoglobulin G; Somatic Hypermutation, Immunoglobulin
PubMed: 36202784
DOI: 10.1080/19420862.2022.2122275 -
Seminars in Immunology Aug 2012The creation of a highly diverse antibody repertoire requires the synergistic activity of a DNA mutator, known as activation-induced deaminase (AID), coupled with an... (Review)
Review
The creation of a highly diverse antibody repertoire requires the synergistic activity of a DNA mutator, known as activation-induced deaminase (AID), coupled with an error-prone repair process that recognizes the DNA mismatch catalyzed by AID. Instead of facilitating the canonical error-free response, which generally occurs throughout the genome, DNA mismatch repair (MMR) participates in an error-prone repair mode that promotes A:T mutagenesis and double-strand breaks at the immunoglobulin (Ig) genes. As such, MMR is capable of compounding the mutation frequency of AID activity as well as broadening the spectrum of base mutations; thereby increasing the efficiency of antibody maturation. We here review the current understanding of this MMR-mediated process and describe how the MMR signaling cascade downstream of AID diverges in a locus dependent manner and even within the Ig locus itself to differentially promote somatic hypermutation (SHM) and class switch recombination (CSR) in B cells.
Topics: Animals; Antibody Diversity; Cytidine Deaminase; DNA Mismatch Repair; Deamination; Humans; Mutation; Ubiquitination
PubMed: 22703640
DOI: 10.1016/j.smim.2012.05.005 -
Molecular & Cellular Proteomics : MCP May 2024Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements....
Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination-independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence are not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and mapped a diverse set of protein-protein and protein-RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein-folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein-protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology. Data are available via ProteomeXchange with the identifier PXD044275.
Topics: Humans; Cytidine Deaminase; Protein Interaction Maps; Deamination; APOBEC Deaminases; Aminohydrolases; HEK293 Cells; Cytosine Deaminase; APOBEC-3G Deaminase; Spliceosomes; Protein Binding; Mass Spectrometry; RNA; Minor Histocompatibility Antigens
PubMed: 38548018
DOI: 10.1016/j.mcpro.2024.100755