APOBEC-Induced Mutagenesis in Cancer.

The initiation, progression, and relapse of cancers often result from mutations occurring within somatic cells. Consequently, processes that elevate mutation rates accelerate carcinogenesis and hinder the development of long-lasting therapeutics. Recent sequencing of human cancer genomes has identified patterns of mutations, termed mutation signatures, many of which correspond to specific environmentally induced and endogenous mutation processes. Some of the most frequently observed mutation signatures are caused by dysregulated activity of APOBECs, which deaminate cytidines in single-stranded DNA at specific sequence motifs causing C-to-T and C-to-G substitutions. In humans, APOBEC-generated genetic heterogeneity in tumor cells contributes to carcinogenesis, metastasis, and resistance to therapeutics. Here, we review the current understanding of APOBECs' role in cancer mutagenesis and impact on disease and the biological processes that influence APOBEC mutagenic capacity.

Evolution of Inosine-Specific Endonuclease V from Bacterial DNase to Eukaryotic RNase.

Endonuclease V (EndoV) cleaves the second phosphodiester bond 3' to a deaminated adenosine (inosine). Although highly conserved, EndoV homologs change substrate preference from DNA in bacteria to RNA in eukaryotes. We have characterized EndoV from six different species and determined crystal structures of human EndoV and three EndoV homologs from bacteria to mouse in complex with inosine-containing DNA/RNA hybrid or double-stranded RNA (dsRNA). Inosine recognition is conserved, but changes in several connecting loops in eukaryotic EndoV confer recognition of 3 ribonucleotides upstream and 7 or 8 bp of dsRNA downstream of the cleavage site, and bacterial EndoV binds only 2 or 3 nt flanking the scissile phosphate. In addition to the two canonical metal ions in the active site, a third Mn2+ that coordinates the nucleophilic water appears necessary for product formation. Comparison of EndoV with its homologs RNase H1 and Argonaute reveals the principles by which these enzymes recognize RNA versus DNA.

Poly(ADP-Ribose) Polymerase 2 Recruits Replication Protein A to Sites of LINE-1 Integration to Facilitate Retrotransposition.

Long interspersed element-1 (LINE-1 or L1) retrotransposition poses a threat to genome integrity, and cells have evolved mechanisms to restrict retrotransposition. However, how cellular proteins facilitate L1 retrotransposition requires elucidation. Here, we demonstrate that single-strand DNA breaks induced by the L1 endonuclease trigger the recruitment of poly(ADP-ribose) polymerase 2 (PARP2) to L1 integration sites and that PARP2 activation leads to the subsequent recruitment of the replication protein A (RPA) complex to facilitate retrotransposition. We further demonstrate that RPA directly binds activated PARP2 through poly(ADP-ribosyl)ation and can protect single-strand L1 integration intermediates from APOBEC3-mediated cytidine deamination in vitro. Paradoxically, we provide evidence that RPA can guide APOBEC3A, and perhaps other APOBEC3 proteins, to sites of L1 integration. Thus, the interplay of L1-encoded and evolutionarily conserved cellular proteins is required for efficient retrotransposition; however, these interactions also may be exploited to restrict L1 retrotransposition in the human genome.

Serine deamination by human serine racemase synergizes with antibiotics to curtail the replication of Chlamydia trachomatis.

The obligate intracellular bacterium, Chlamydia trachomatis, has evolved to depend on its human host for many metabolites, including most amino acids and three of the four nucleotides. Given this, it is not surprising that depletion of a single amino acid in the host cell growth medium blocks chlamydial replication. Paradoxically, supra-normal levels of some amino acids also block productive replication of Chlamydia. Here, we have determined how elevated serine levels, generated by exogenous supplementation, impede chlamydial inclusion development and reduce the generation of infectious progeny. Our findings reveal that human serine racemase, which is broadly expressed in multiple tissues, potentiates the anti-chlamydial effect of elevated serine concentrations. In addition to reversibly converting l-serine to d-serine, serine racemase also deaminates serine via ß-elimination. We have determined that d-serine does not directly impact Chlamydia; rather, ammonia generated by serine deamination limits the productive chlamydial replication. Our findings imply that ammonia produced within host cells can traverse the chlamydial inclusion membrane. Further, this property of serine deaminase can be exploited to sensitize Chlamydia to concentrations of doxycycline that are otherwise not bactericidal. Because exogenously elevated levels of serine can be tolerated over extended periods, the broad expression pattern of serine racemase indicates it to be a host enzyme whose activity can be directed against multiple intracellular bacterial pathogens. From a therapeutic perspective, demonstrating host metabolism can be skewed to generate an anti-bacterial metabolite that synergizes with antibiotics, we believe our results provide a new approach to target intracellular pathogens.

Germline MBD4 deficiency causes a multi-tumor predisposition syndrome.

We report an autosomal recessive, multi-organ tumor predisposition syndrome, caused by bi-allelic loss-of-function germline variants in the base excision repair (BER) gene MBD4. We identified five individuals with bi-allelic MBD4 variants within four families and these individuals had a personal and/or family history of adenomatous colorectal polyposis, acute myeloid leukemia, and uveal melanoma. MBD4 encodes a glycosylase involved in repair of G:T mismatches resulting from deamination of 5'-methylcytosine. The colorectal adenomas from MBD4-deficient individuals showed a mutator phenotype attributable to mutational signature SBS1, consistent with the function of MBD4. MBD4-deficient polyps harbored somatic mutations in similar driver genes to sporadic colorectal tumors, although AMER1 mutations were more common and KRAS mutations less frequent. Our findings expand the role of BER deficiencies in tumor predisposition. Inclusion of MBD4 in genetic testing for polyposis and multi-tumor phenotypes is warranted to improve disease management.

Site-directed A → I RNA editing as a therapeutic tool: moving beyond genetic mutations.

Adenosine deamination by the ADAR family of enzymes is a natural process that edits genetic information as it passes through messenger RNA. Adenosine is converted to inosine in mRNAs, and this base is interpreted as guanosine during translation. Realizing the potential of this activity for therapeutics, a number of researchers have developed systems that redirect ADAR activity to new targets, ones that are not normally edited. These site-directed RNA editing (SDRE) systems can be broadly classified into two categories: ones that deliver an antisense RNA oligonucleotide to bind opposite a target adenosine, creating an editable structure that endogenously expressed ADARs recognize, and ones that tether the catalytic domain of recombinant ADAR to an antisense RNA oligonucleotide that serves as a targeting mechanism, much like with CRISPR-Cas or RNAi. To date, SDRE has been used mostly to try and correct genetic mutations. Here we argue that these applications are not ideal SDRE, mostly because RNA edits are transient and genetic mutations are not. Instead, we suggest that SDRE could be used to tune cell physiology to achieve temporary outcomes that are therapeutically advantageous, particularly in the nervous system. These include manipulating excitability in nociceptive neural circuits, abolishing specific phosphorylation events to reduce protein aggregation related to neurodegeneration or reduce the glial scarring that inhibits nerve regeneration, or enhancing G protein-coupled receptor signaling to increase nerve proliferation for the treatment of sensory disorders like blindness and deafness.

Impact of ADAR-induced editing of minor viral RNA populations on replication and transmission of SARS-CoV-2.

Adenosine deaminases acting on RNA (ADAR) are RNA-editing enzymes that may restrict viral infection. We have utilized deep sequencing to determine adenosine to guanine (A→G) mutations, signifying ADAR activity, in clinical samples retrieved from 93 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected patients in the early phase of the COVID-19 pandemic. A→G mutations were detected in 0.035% (median) of RNA residues and were predominantly nonsynonymous. These mutations were rarely detected in the major viral population but were abundant in minor viral populations in which A→G was more prevalent than any other mutation (P < 0.001). The A→G substitutions accumulated in the spike protein gene at positions corresponding to amino acids 505 to 510 in the receptor binding motif and at amino acids 650 to 655. The frequency of A→G mutations in minor viral populations was significantly associated with low viral load (P < 0.001). We additionally analyzed A→G mutations in 288,247 SARS-CoV-2 major (consensus) sequences representing the dominant viral population. The A→G mutations observed in minor viral populations in the initial patient cohort were increasingly detected in European consensus sequences between March and June 2020 (P < 0.001) followed by a decline of these mutations in autumn and early winter (P < 0.001). We propose that ADAR-induced deamination of RNA is a significant source of mutated SARS-CoV-2 and hypothesize that the degree of RNA deamination may determine or reflect viral fitness and infectivity.

ADAR Family Proteins: A Structural Review.

This review aims to highlight the structures of ADAR proteins that have been crucial in the discernment of their functions and are relevant to future therapeutic development. ADAR proteins can correct or diversify genetic information, underscoring their pivotal contribution to protein diversity and the sophistication of neuronal networks. ADAR proteins have numerous functions in RNA editing independent roles and through the mechanisms of A-I RNA editing that continue to be revealed. Provided is a detailed examination of the ADAR family members-ADAR1, ADAR2, and ADAR3-each characterized by distinct isoforms that offer both structural diversity and functional variability, significantly affecting RNA editing mechanisms and exhibiting tissue-specific regulatory patterns, highlighting their shared features, such as double-stranded RNA binding domains (dsRBD) and a catalytic deaminase domain (CDD). Moreover, it explores ADARs' extensive roles in immunity, RNA interference, and disease modulation, demonstrating their ambivalent nature in both the advancement and inhibition of diseases. Through this comprehensive analysis, the review seeks to underline the potential of targeting ADAR proteins in therapeutic strategies, urging continued investigation into their biological mechanisms and health implications.

Rampant C-to-U deamination accounts for the intrinsically high mutation rate in SARS-CoV-2 spike gene.

The high mutation rate of SARS-CoV-2 largely complicates our control of the pandemic. In particular, it is currently unclear why the spike (S) gene has an extraordinarily high mutation rate among all SARS-CoV-2 genes. By analyzing the occurrence of fixed synonymous mutations between SARS-CoV-2 and RaTG13, and profiling the DAF (derived allele frequency) of polymorphic synonymous sites among millions of worldwide SARS-CoV-2 strains, we found that both fixed and polymorphic mutations show higher mutation rates in the S gene than other genes. The majority of mutations are C-to-T, representing the APOBEC-mediated C-to-U deamination instead of the previously proposed A-to-I deamination. Both in silico and in vivo evidence indicated that the S gene is more likely to be single-stranded compared to other SARS-CoV-2 genes, agreeing with the APOBEC preference of ssRNA. We conclude that the single-stranded property of the S gene makes it a favorable target for C-to-U deamination, leading to its excessively high mutation rate compared to other non-S genes. In conclusion, APOBEC, rather than ADAR, is the "editor-in-chief" of SARS-CoV-2 RNAs. This work helps us to understand the molecular mechanism underlying the mutation and evolution of SARS-CoV-2, and we believe it will contribute to the control of the pandemic.

A Novel Approach of Electrocatalytic Deamination From Aromatic Amide to Diarylimide on Ni-PTFE Modified Electrode.

A hydrophobic Ni-PTFE modified electrode has been prepared by constant current and cathodic electroplating with a nickel sheet as substrate in a PTFE suspension. Then the Ni-PTFE modified electrode was used for electroreduction from aromatic amide to diarylimide. The electrochemical characterizations such as cyclic voltammogram, EIS, polarization curves, and electrode stability have been carried out by electrochemical workstation. The structure of the electroreduction product diarylimide was characterized by 1H NMR, FT-IR, MS(Mass Spectrum), and EA(Elemental Analyzer). Based on the hydrophobicity of the electrode, an approach suggested that the phenyl ketone radical may be formed by electroreductive deamination at the cathode. With the construction of C-N bond by the radical coupling, the electrocatalytic reduction may be comprised of a one-electron process including an ECC (Electrochemical-Chemical-Chemical) process. The electroreduction of aromatic amide to diarylimide may be controlled by both charge migration and concentration polarization. Electrocatalytic reduction of aromatic amides on Ni-PTFE modified electrodes is all well conversion ratio.

Metal-Free Visible-Light Induced Oxidative Cleavage of C(sp3 )-C, and C(sp3 )-N Bonds of Nitriles, Alcohols, and Amines.

Selective cleavage of unstrained (sp3 ) C-C/ C-N bonds under mild conditions is highly challenging due to the higher bond dissociation energy. A visible light mediated metal-free oxidative dehomologation of aryl acetonitriles, primary alcohols and diols to carboxylic acids via organophotocatalyzed C(sp3 )-CN, C(sp3 )-C(OH) bond cleavage is reported. Notably, this methodology was further extended towards selective synthesis of aldehydes via deamination of both primary as well as secondary amines. This mild protocol features wide array of substrate variation with excellent functional group tolerance, preparative-scale synthesis, and operational simplicity. Possible mechanisms for these transformations were demonstrated through a series of control experiments.

Development of growth selection system and pocket engineering of d-amino acid oxidase to enhance selective deamination activity toward d-phosphinothricin.

D-amino acid oxidase (DAAO)-catalyzed selective oxidative deamination is a very promising process for synthesizing l-amino acids including l-phosphinothricin ( l-PPT, a high-efficiency and broad-spectrum herbicide). However, the wild-type DAAO's low activity toward unnatural substrates like d-phosphinothricin ( d-PPT) hampers its application. Herein, a DAAO from Caenorhabditis elegans (CeDAAO) was screened and engineered to improve the catalytic potential on d-PPT. First, we designed a novel growth selection system, taking into account the intricate relationship between the growth of Escherichia coli (E. coli) and the catalytic mechanism of DAAO. The developed system was used for high-throughput screening of gene libraries, resulting in the discovery of a variant (M6) with significantly increased catalytic activity against d-PPT. The variant displays different catalytic properties on substrates with varying hydrophobicity and hydrophilicity. Analysis using Alphafold2 modeling and molecular dynamic simulations showed that the reason for the enhanced activity was the substrate-binding pocket with enlarged size and suitable charge distribution. Further QM/MM calculations revealed that the crucial factor for enhancing activity lies in reducing the initial energy barrier of the reductive half reaction. Finally, a comprehensive binding-model index to predict the enhanced activity of DAAO toward d-PPT, and an enzymatic deracemization approach was developed, enabling the efficient synthesis of l-PPT with remarkable efficiency.

Structural basis for recognition of distinct deaminated DNA lesions by endonuclease Q.

Spontaneous deamination of DNA cytosine and adenine into uracil and hypoxanthine, respectively, causes C to T and A to G transition mutations if left unrepaired. Endonuclease Q (EndoQ) initiates the repair of these premutagenic DNA lesions in prokaryotes by cleaving the phosphodiester backbone 5' of either uracil or hypoxanthine bases or an apurinic/apyrimidinic (AP) lesion generated by the excision of these damaged bases. To understand how EndoQ achieves selectivity toward these structurally diverse substrates without cleaving undamaged DNA, we determined the crystal structures of Pyrococcus furiosus EndoQ bound to DNA substrates containing uracil, hypoxanthine, or an AP lesion. The structures show that substrate engagement by EndoQ depends both on a highly distorted conformation of the DNA backbone, in which the target nucleotide is extruded out of the helix, and direct hydrogen bonds with the deaminated bases. A concerted swing motion of the zinc-binding and C-terminal helical domains of EndoQ toward its catalytic domain allows the enzyme to clamp down on a sharply bent DNA substrate, shaping a deep active-site pocket that accommodates the extruded deaminated base. Within this pocket, uracil and hypoxanthine bases interact with distinct sets of amino acid residues, with positioning mediated by an essential magnesium ion. The EndoQ-DNA complex structures reveal a unique mode of damaged DNA recognition and provide mechanistic insights into the initial step of DNA damage repair by the alternative excision repair pathway. Furthermore, we demonstrate that the unique activity of EndoQ is useful for studying DNA deamination and repair in mammalian systems.

Addition of α-ketoglutaric acid (AKG) reduces deamination in Chinese perch (Siniperca chuatsi) fed with fermented soybean meal as a substitute for fishmeal.

To alleviate amino acid imbalances in fermented soybean meal as a replacement for fishmeal feeds, this study evaluated the effects of adding lysine (Lys), methionine (Met), and α-ketoglutaric acid (AKG) to fermented soybean meals for Chinese perch. Chinese perch (34 ± 3 g) were fed five diets for 66 days (fishmeal as the protein source of the basal diet [FM]; fermented soybean meal as a substitute for 30% fishmeal in the soybean meal diet [FSM]; addition of crystalline Lys and Met [AA]; addition of α-ketoglutaric acid [AKG]; and simultaneous addition of crystalline Lys, Met, and AKG [BA] to the soybean meal diet). At the end of the feeding trial, the FSM group had the highest feeding rate and the lowest weight gain rate among all the groups. The FM group had the highest protein retention and the lowest feed efficiency among the groups. The mRNA transcription level of genes related to the AMP-activating protein (AMPK) signaling pathway and amino acid response (AAR) signaling pathway (lkb1, atf4, and gcn2) were highest in the AA group (P < 0.05) but lower in the AKG and BA groups. In the AKG group, the mRNA transcription level of the gluconeogenesis pathway-related gene (pepck and g6pase) was significantly higher than that in the other four groups, but the mRNA transcription level of genes related to amino acid catabolism (gdh and ampd) was lower. Among all the groups, the FSM group had the lowest mRNA transcription level of genes associated with the mammalian target of rapamycin (mTOR) signaling pathway (mtor and s6k). These findings imply that the feeding rate of Chinese perch in the fermented soybean meal group was the highest, but the protein retention was the lowest, while the addition of Lys, Met, and AKG improved protein retention. In conclusion, the addition of AKG to fermented soybean meal as a fishmeal substitute reduced amino acid deamination, enhanced gluconeogenesis, and increased protein deposition, which contributed to the growth of Chinese perch, alleviated amino acid imbalances, and improved the feed utilization of Chinese perch.

Unified Approach to Deamination and Deoxygenation Through Isonitrile Hydrodecyanation: A Combined Experimental and Computational Investigation.

Herein, we describe a general hydrodefunctionalization protocol of alcohols and amines through a common isonitrile intermediate. To cleave the relatively inert C-NC bond, we leveraged dual hydrogen atom transfer (HAT) and photoredox catalysis to generate a nucleophilic boryl radical, which readily forms an imidoyl radical intermediate from the isonitrile. Rapid ß-scission then accomplishes defunctionalization. This method has been applied to the hydrodefunctionalization of both amine and alcohol-containing pharmaceuticals, natural products, and biomolecules. We extended this approach to the reduction of carbonyls and olefins to their saturated counterparts, as well as the hydrodecyanation of alkyl nitriles. Both experimental and computational studies demonstrate a facile ß-scission of the imidoyl radical, and reconcile differences in reactivity between nitriles and isonitriles within our protocol.

Site-Specific Deaminative Trifluoromethylation of Aliphatic Primary Amines.

The introduction of trifluoromethyl groups into organic molecules is of paramount importance in modern synthetic chemistry and medicinal chemistry. While methods for constructing C(sp2 )-CF3 bonds have been well established, the advancement of practical and comprehensive approaches for forming C(sp3 )-CF3 bonds remains considerably restricted. In this work, we describe an efficient and site-specific deaminative trifluoromethylation reaction of aliphatic primary amines to afford the corresponding alkyl trifluoromethyl compounds. The reaction proceeds at room temperature with readily accessible N-anomeric amide (Levin's reagent) and bench-stable bpyCu(CF3 )3 (Grushin's reagent, bpy=2,2'-bipyridine) under blue light. The protocol features mild reaction conditions, good functional group tolerance, and moderate to good yields. Remarkably, the method can be applied to the direct, late-stage trifluoromethylation of natural products and bioactive molecules. Experimental mechanistic studies were conducted, and a radical mechanism is proposed, wherein the dual roles of Grushin's reagent have been elucidated.

Natural polyphenols convert proteins into histone-binding ligands.

Antioxidants are sensitive to oxidation and are immediately converted into their oxidized forms that can react with proteins. We have recently found that proteins incubated with oxidized vitamin C (dehydroascorbate) gain a new function as a histone-binding ligand. This finding led us to predict that antioxidants, through conversion to their oxidized forms, may generally have similar functions. In the present study, we identified several natural polyphenols as a source of histone ligands and characterized the mechanism for the interaction of protein-bound polyphenols with histone. Through screening of 25 plant-derived polyphenols by assessing their ability to convert bovine serum albumin into histone ligands, we identified seven polyphenols, including (-)-epigallocatechin-3-O-gallate (EGCG). Additionally, we found that the histone tail domain, which is a highly charged and conformationally flexible region, is involved in the interaction with the polyphenol-modified proteins. Further mechanistic studies showed the involvement of a complex heterogeneous group of the polyphenol-derived compounds bound to proteins as histone-binding elements. We also determined that the interaction of polyphenol-modified proteins with histones formed aggregates and exerted a protective effect against histone-mediated cytotoxicity toward endothelial cells. These findings demonstrated that histones are one of the major targets of polyphenol-modified proteins and provide important insights into the chemoprotective functions of dietary polyphenols.

Measurement of deaminated cytosine adducts in DNA using a novel hybrid thymine DNA glycosylase.

The hydrolytic deamination of cytosine and 5-methylcytosine drives many of the transition mutations observed in human cancer. The deamination-induced mutagenic intermediates include either uracil or thymine adducts mispaired with guanine. While a substantial array of methods exist to measure other types of DNA adducts, the cytosine deamination adducts pose unusual analytical problems, and adequate methods to measure them have not yet been developed. We describe here a novel hybrid thymine DNA glycosylase (TDG) that is comprised of a 29-amino acid sequence from human TDG linked to the catalytic domain of a thymine glycosylase found in an archaeal thermophilic bacterium. Using defined-sequence oligonucleotides, we show that hybrid TDG has robust mispair-selective activity against deaminated U:G and T:G mispairs. We have further developed a method for separating glycosylase-released free bases from oligonucleotides and DNA followed by GC-MS/MS quantification. Using this approach, we have measured for the first time the levels of total uracil, U:G, and T:G pairs in calf thymus DNA. The method presented here will allow the measurement of the formation, persistence, and repair of a biologically important class of deaminated cytosine adducts.

Acceleration of the Deamination of Cytosine through Photo-Crosslinking.

Herein, we report the major factor for deamination reaction rate acceleration, i.e., hydrophilicity, by using various 5-substituted target cytosines and by carrying out deamination at high temperatures. Through substitution of the groups at the 5'-position of the cytosine, the effect of hydrophilicity was understood. It was then used to compare the various modifications of the photo-cross-linkable moiety as well as the effect of the counter base of the cytosine to edit both DNA and RNA. Furthermore, we were able to achieve cytosine deamination at 37 °C with a half-life in the order of a few hours.

Non-random Codon Usage of Synonymous and Non-synonymous Mutations in the Human HLA-A Gene.

The structure and function of human leucocyte antigen (HLA-A) is well known and is an extremely variable protein. From the public HLA-A database, we chose 26 high frequency HLA-A alleles (45% of sequenced alleles). Using five arbitrary references from these alleles, we analyzed synonymous mutations at the third codon position (sSNP3) and non-synonymous mutations (NSM). Both mutation types showed non-random locations of 29 sSNP3 codons and 71 NSM codons in the five reference lists. Most sSNP3 codons show identical mutation types with many mutations resulting from cytosine deamination. We proposed 23 ancestral parents of sSNP3 in five reference sequences using conserved parents in five unidirectional codons and 18 majority parents in reciprocal codons. These 23 proposed ancestral parents show exclusive codon usage of G3 or C3 parents located on both DNA strands that mutate to A3 or T3 variants mostly (76%) by cytosine deamination The sSNP3 and NSM show clear separation of the two variant types with most sSNP3 located in conserved areas in exons 2, 3 and 4, compared to most NSM appearing in two Variable Areas with no sSNP3 in the latter parts of exons 2 (α1) and 3 (α2). The Variable Areas contain NSM (polymorphic) residues at the center of the groove that bind the foreign peptide. We find distinctly different mutation patterns in NSM codons from those of sSNP3. Namely, G-C to A-T mutation frequency was much smaller, suggesting that evolutional pressures of deamination and other mechanisms applied to the two areas are significantly different.