Search Results (30)

Mesothelioma is a rare and aggressive cancer linked to asbestos exposure and characterized by rapid metastasis and poor prognosis. Inhibition of adenosine deaminase acting on dsRNA 2 (ADAR2) RNA binding but not ADAR2 editing has shown antitumor effects in mesothelioma. Natural compounds from the Traditional Chinese Medicine (TCM) database were docked to the RNA-binding interface of ADAR2’s second dsRNA binding domain (dsRBD2), and their drug-likeness and predicted safety were assessed. Eight ligands (ZINC000085597263, ZINC000085633079, ZINC000014649947, ZINC000034512861, ZINC000070454124, ZINC000085594944, ZINC000085633008, and ZINC000095909822) showed high binding affinity to dsRBD2 from molecular mechanics Poisson–Boltzmann surface area (MM/PBSA) calculations. Protein–ligand interactions were analyzed to identify key residues contributing to these binding affinities. Molecular dynamics (MD) simulations of dsRBD–ligand–RNA complexes revealed that four compounds (ZINC000085597263, ZINC000085633079, ZINC000014649947, and ZINC000034512861) had negative binding affinities to dsRBD2 in the presence of the RNA substrate GluR-2. Key residues, including Val164, Met165, Lys209, and Lys212, were crucial for ligand binding, even with RNA present, suggesting these compounds could inhibit dsRBD2’s RNA-binding function. The predicted biological activities of these compounds indicate potential anticancer properties, particularly for the treatment of mesothelioma. These compounds are structurally similar to known anti-mesothelioma agents or anticancer drugs, highlighting their therapeutic potential. Current mesothelioma treatments are limited. Optimization of these compounds, alone or in combination with current therapeutics, has potential for mesothelioma treatment. Additionally, five high-quality full-length ADAR2 models were developed. These models provide insights into ADAR2 function, mutation impacts, and potential areas for protein engineering to enhance stability, RNA-binding specificity, or protein interactions, particularly concerning dimerization or complex formation with other proteins and RNAs. Full article

The Ascomycete genus Cordyceps affects plant crops significantly, filling an important ecological niche. Cordyceps militaris (L.) Fr. presents many health benefits for humans, but its population history has not been reported. The objective of this research was to report the collection, population structure, demographic history, diversity, and cytosine deaminases of 43 wild strains of C. militaris in China through resequencing using an Illumina HiseqTM platform. All strains were assigned to the warm, subtropical, and middle temperate zone populations, confirmed by ADMIXTURE-1.3.0, PCA, and phylogenic analysis. Their population sizes declined historically, suggesting that this species suffered from bottlenecks in the wild. LD decays (r²) revealed a north-to-south migration pattern of wild C. militaris, consistent with the MSMC2-v2.1.4 analysis. The regions of high Pi were aggregating at the chromosomes CP023325.1 (51) and CP023323.1 (9), playing a key role in adaptation, especially for the sites on cytosine deaminase. Within the species, genetic differentiation was relatively high among the three populations (F_st = 0.083, 0.092, and even 0.109). According to the artificial intelligence-assisted (RoseTTAFold) predicted structures of the cytosine deaminases, they were classified into eight clades with unique, distinct, and structurally conserved domains, offering a potential suite of single- and double-stranded deaminases of great promise as tunable base editors for therapeutic and agricultural breeding applications. These provided new insights for mining novel proteins from macrofungi, structurally and functionally. Full article

SNAP-tag and Halo-tag have been employed to achieve targeted RNA editing by directing the deaminase domain of human ADAR to specific sites in the transcriptome. This targeting is facilitated by short guide RNAs (gRNAs) complementary to the target transcript, which are chemically modified with benzylguanine or chloroalkane moieties to enable covalent binding to the respective self-labeling enzymes. However, broad application of this approach has been limited by challenges such as low scalability, the requirement for specialized chemical expertise and equipment, and labor-intensive protocols. In this study, we introduce streamlined, efficient protocols for the synthesis and purification of these linkers, suitable for SNAP-tag and Halo-tag applications, without the need for advanced chemical equipment. Our methods enable linker coupling in a kit-like manner and support the high-yield production of modified gRNAs. We demonstrate that the newly synthesized linkers and gRNA designs perform similarly to previously published constructs with regard to RNA editing efficiency. Moreover, large-scale production of modified gRNAs facilitates their use in studies involving cellular uptake and in vivo experiments. Full article

RNA editing is a significant mechanism underlying genetic variation and protein molecule alteration; C-to-U RNA editing, specifically, is important in the regulation of mammalian genetic diversity. The ability to define and limit accesses of enzymatic machinery to avoid the modification of unintended targets is key to the success of RNA editing. Identification of the core component of the apoB RNA editing holoenzyme, APOBEC, and investigation into new candidate genes encoding other elements of the complex could reveal further details regarding APOBEC-mediated mRNA editing. Menkes disease is a recessive X-chromosome-linked hereditary syndrome in humans, caused by defective copper metabolism due to mutations in the ATP7A gene, which encodes a copper transport protein. Here, we generated plasmids encoding the MS2 system and the APOBEC1 deaminase domain and used a guide RNA with flanking MS2 sites to restore mutated Atp7a in fibroblasts from a macular mouse model of Menkes disease withs T>C mutation. Around 35% of the mutated C nucleotide (nt) was restored to U, demonstrating that our RNA editing system is reliable and has potential for therapeutic clinical application. RNA base editing via human RNA-guided cytidine deaminases is a potentially attractive approach for in vivo therapeutic application and provides opportunities for new developments in this field. Full article

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. Full article

Background: Hyperuricaemia (HUA) is a disorder of purine metabolism in the body. We previously synthesized a hesperitin (Hsp)-Cu(II) complex and found that the complex possessed strong uric acid (UA)-reducing activity in vitro. In this study we further explored the complex’s UA-lowering and nephroprotective effects in vivo. Methods: A mouse with HUA was used to investigate the complex’s hypouricemic and nephroprotective effects via biochemical analysis, RT-PCR, and Western blot. Results: Hsp-Cu(II) complex markedly decreased the serum UA level and restored kidney tissue damage to normal in HUA mice. Meanwhile, the complex inhibited liver adenosine deaminase (ADA) and xanthine oxidase (XO) activities to reduce UA synthesis and modulated the protein expression of urate transporters to promote UA excretion. Hsp-Cu(II) treatment significantly suppressed oxidative stress and inflammatory in the kidney, reduced the contents of cytokines and inhibited the activation of the nucleotide-binding oligomerization domain (NOD)-like receptor thermal protein domain associated protein 3 (NLRP3) inflammatory pathway. Conclusions: Hsp-Cu(II) complex reduced serum UA and protected kidneys from renal inflammatory damage and oxidative stress by modulating the NLRP3 pathway. Hsp-Cu(II) complex may be a promising dietary supplement or nutraceutical for the therapy of hyperuricemia. Full article

Adenosine deaminases acting on RNA (ADARs) have double-stranded RNA binding domains and a deaminase domain (DD). We used the MS2 system and specific guide RNAs to direct ADAR1-DD to target adenosines in the mRNA encoding-enhanced green fluorescence protein. Using this system in transfected HEK-293 cells, we evaluated the effects of changing the length and position of the guide RNA on the efficiency of conversion of amber (TAG) and ochre (TAA) stop codons to tryptophan (TGG) in the target. Guide RNAs of 19, 21 and 23 nt were positioned upstream and downstream of the MS2-RNA, providing a total of six guide RNAs. The upstream guide RNAs were more functionally effective than the downstream guide RNAs, with the following hierarchy of efficiency: 21 nt > 23 nt > 19 nt. The highest editing efficiency was 16.6%. Off-target editing was not detected in the guide RNA complementary region but was detected 50 nt downstream of the target. The editing efficiency was proportional to the amount of transfected deaminase but inversely proportional to the amount of the transfected guide RNA. Our results suggest that specific RNA editing requires precise optimization of the ratio of enzyme, guide RNA, and target RNA. Full article

The dynamic processes operating on genomic DNA, such as gene expression and cellular division, lead inexorably to topological challenges in the form of entanglements, catenanes, knots, “bubbles”, R-loops, and other outcomes of supercoiling and helical disruption. The resolution of toxic topological stress is the function attributed to DNA topoisomerases. A prominent example is the negative supercoiling (nsc) trailing processive enzymes such as DNA and RNA polymerases. The multiple equilibrium states that nscDNA can adopt by redistribution of helical twist and writhe include the left-handed double-helical conformation known as Z-DNA. Thirty years ago, one of our labs isolated a protein from Drosophila cells and embryos with a 100-fold greater affinity for Z-DNA than for B-DNA, and identified it as topoisomerase II (gene Top2, orthologous to the human UniProt proteins TOP2A and TOP2B). GTP increased the affinity and selectivity for Z-DNA even further and also led to inhibition of the isomerase enzymatic activity. An allosteric mechanism was proposed, in which topoII acts as a Z-DNA-binding protein (ZBP) to stabilize given states of topological (sub)domains and associated multiprotein complexes. We have now explored this possibility by comprehensive bioinformatic analyses of the available protein sequences of topoII representing organisms covering the whole tree of life. Multiple alignment of these sequences revealed an extremely high level of evolutionary conservation, including a winged-helix protein segment, here denoted as Zτ, constituting the putative structural homolog of Zα, the canonical Z-DNA/Z-RNA binding domain previously identified in the interferon-inducible RNA Adenosine-to-Inosine-editing deaminase, ADAR1p150. In contrast to Zα, which is separate from the protein segment responsible for catalysis, Zτ encompasses the active site tyrosine of topoII; a GTP-binding site and a GxxG sequence motif are in close proximity. Quantitative Zτ-Zα similarity comparisons and molecular docking with interaction scoring further supported the “B-Z-topoII hypothesis” and has led to an expanded mechanism for topoII function incorporating the recognition of Z-DNA segments (“Z-flipons”) as an inherent and essential element. We further propose that the two Zτ domains of the topoII homodimer exhibit a single-turnover “conformase” activity on given G(ate) B-DNA segments (“Z-flipins”), inducing their transition to the left-handed Z-conformation. Inasmuch as the topoII-Z-DNA complexes are isomerase inactive, we infer that they fulfill important structural roles in key processes such as mitosis. Topoisomerases are preeminent targets of anti-cancer drug discovery, and we anticipate that detailed elucidation of their structural–functional interactions with Z-DNA and GTP will facilitate the design of novel, more potent and selective anti-cancer chemotherapeutic agents. Full article

The NPR1 (nonexpressor of pathogenesis-related genes 1) gene is an activator of the systemic acquisition of resistance (SAR) in plants and is one of the central factors in their response to pathogenic bacterial infestation, playing an important role in plant disease resistance. Potato (Solanum tuberosum) is a crucial non-grain crop that has been extensively studied. However, the identification and analysis of the NPR1-like gene within potato have not been understood well. In this study, a total of six NPR1-like proteins were identified in potato, and phylogenetic analysis showed that the six NPR1-like proteins in Solanum tuberosum could be divided into three major groups with NPR1-related proteins from Arabidopsis thaliana and other plants. Analysis of the exon–intron patterns and protein domains of the six NPR1-like genes from potato showed that the exon–intron patterns and protein domains of the NPR1-like genes belonging to the same Arabidopsis thaliana subfamily were similar. By performing quantitative real-time PCR (qRT-PCR) analysis, we found that six NPR1-like proteins have different expression patterns in different potato tissues. In addition, the expression of three StNPR1 genes was significantly downregulated after being infected by Ralstonia solanacearum (RS), while the difference in the expression of StNPR2/3 was insignificant. We also established potato StNPR1 overexpression lines that showed a significantly increased resistance to R. solanacearum and elevated activities of chitinase, β-1,3-glucanase, and phenylalanine deaminase. Increased peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT) activities, as well as decreased hydrogen peroxide, regulated the dynamic balance of reactive oxygen species (ROS) in the StNPR1 overexpression lines. The transgenic plants activated the expression of the genes associated with the Salicylic acid (SA) defense response but suppressed the expression of the genes associated with Jasmonic acid (JA) signaling. This resulted in resistance to Ralstonia solanacearum. Full article

Epidemiological evidence supports an association between cow’s milk consumption and the risk of diffuse large B-cell lymphoma (DLBCL), the most common non-Hodgkin lymphoma worldwide. This narrative review intends to elucidate the potential impact of milk-related agents, predominantly milk-derived exosomes (MDEs) and their microRNAs (miRs) in lymphomagenesis. Upregulation of PI3K-AKT-mTORC1 signaling is a common feature of DLBCL. Increased expression of B cell lymphoma 6 (BCL6) and suppression of B lymphocyte-induced maturation protein 1 (BLIMP1)/PR domain-containing protein 1 (PRDM1) are crucial pathological deviations in DLBCL. Translational evidence indicates that during the breastfeeding period, human MDE miRs support B cell proliferation via epigenetic upregulation of BCL6 (via miR-148a-3p-mediated suppression of DNA methyltransferase 1 (DNMT1) and miR-155-5p/miR-29b-5p-mediated suppression of activation-induced cytidine deaminase (AICDA) and suppression of BLIMP1 (via MDE let-7-5p/miR-125b-5p-targeting of PRDM1). After weaning with the physiological termination of MDE miR signaling, the infant’s BCL6 expression and B cell proliferation declines, whereas BLIMP1-mediated B cell maturation for adequate own antibody production rises. Because human and bovine MDE miRs share identical nucleotide sequences, the consumption of pasteurized cow’s milk in adults with the continued transfer of bioactive bovine MDE miRs may de-differentiate B cells back to the neonatal “proliferation-dominated” B cell phenotype maintaining an increased BLC6/BLIMP1 ratio. Persistent milk-induced epigenetic dysregulation of BCL6 and BLIMP1 expression may thus represent a novel driving mechanism in B cell lymphomagenesis. Bovine MDEs and their miR cargo have to be considered potential pathogens that should be removed from the human food chain. Full article

The existence of the thiouridine synthetase, methyltransferase and pseudouridine synthase (THUMP) domain was originally predicted by a bioinformatic study. Since the prediction of the THUMP domain more than two decades ago, many tRNA modification enzymes containing the THUMP domain have been identified. According to their enzymatic activity, THUMP-related tRNA modification enzymes can be classified into five types, namely 4-thiouridine synthetase, deaminase, methyltransferase, a partner protein of acetyltransferase and pseudouridine synthase. In this review, I focus on the functions and structures of these tRNA modification enzymes and the modified nucleosides they produce. Biochemical, biophysical and structural studies of tRNA 4-thiouridine synthetase, tRNA methyltransferases and tRNA deaminase have established the concept that the THUMP domain captures the 3′-end of RNA (in the case of tRNA, the CCA-terminus). However, in some cases, this concept is not simply applicable given the modification patterns observed in tRNA. Furthermore, THUMP-related proteins are involved in the maturation of other RNAs as well as tRNA. Moreover, the modified nucleosides, which are produced by the THUMP-related tRNA modification enzymes, are involved in numerous biological phenomena, and the defects of genes for human THUMP-related proteins are implicated in genetic diseases. In this review, these biological phenomena are also introduced. Full article

Booroola fecundity (FecB) gene, a mutant of bone morphogenetic protein 1B (BMPR-1B) that was discovered in Booroola Merino, was the first prolificacy gene identified in sheep related to increased ovulation rate and litter size. The mechanism of FecB impact on reproduction is unclear. Methods: In this study, adult Han ewes with homozygous FecB(B)/FecB(B) mutations (Han BB group) and ewes with FecB(+)/FecB(+) wildtype (Han ++ group) were selected. Methylated DNA immunoprecipitation and high-throughput sequencing (MeDIP-seq) was used to identify differences in methylated genes in ovary tissue. Results: We examined differences in DNA methylation patterns between HanBB and Han ++ sheep. In both sheep, methylated reads were mainly distributed at the gene body regions, CpG islands and introns. The differentially methylated genes were enriched in neurotrophy in signaling pathway, Gonadotropin Releasing Hormone (GnRH) signaling pathway, Wnt signaling pathway, oocyte meiosis, vascular endothelial growth factor (VEGF) signaling pathway, etc. Differentially-methylated genes were co-analyzed with differentially-expressed mRNAs. Several genes which could be associated with female reproduction were identified, such as FOXP3 (forkhead box P3), TMEFF2 (Transmembrane Protein with EGF Like and Two Follistatin Like Domains 2) and ADAT2 (Adenosine Deaminase TRNA Specific 2). Conclusions: We constructed a MeDIP-seq based methylomic study to investigate the ovarian DNA methylation differences between Small-Tail Han sheep with homozygous FecB mutant and wildtype, and successfully identified FecB gene-associated differentially-methylated genes. This study has provided information with which to understand the mechanisms of FecB gene-induced hyperprolificacy in sheep. Full article

Growth factors are mostly secreted proteins that play key roles in an organism’s biophysical processes through binding to specific receptors on the cell surface. The mollusk-like growth factor (MLGF) is a novel cell signaling protein in the adenosine deaminase-related growth factor (ADGF) subfamily. In this study, the MLGF gene was cloned and characterized from the digestive gland tissue of Pacific abalone and designated as Hdh-MLGF. The transcribed full-length sequence of Hdh-MLGF was 1829 bp long with a 1566 bp open reading frame (ORF) encoding 521 amino acids. The deduced amino acid sequence contained a putative signal peptide and two conserved adenosine deaminase domains responsible for regulating molecular function. Fluorescence in situ hybridization localized Hdh-MLGF in the submucosa layer of digestive tubules in the digestive gland. The mRNA expression analysis indicated that Hdh-MLGF expression was restricted to the digestive gland in the adult Pacific abalone. However, Hdh-MLGF mRNA expressions were observed in all stages of embryonic and larval development, suggesting Hdh-MLGF might be involved in the Pacific abalone embryonic and larval development. This is the first study describing Hdh-MLGF and its involvement in the Pacific abalone embryonic and larval development. Full article

APOBEC3 enzymes are polynucleotide deaminases, converting cytosine to uracil on single-stranded DNA (ssDNA) and RNA as part of the innate immune response against viruses and retrotransposons. APOBEC3G is a two-domain protein that restricts HIV. Although X-ray single-crystal structures of individual catalytic domains of APOBEC3G with ssDNA as well as full-length APOBEC3G have been solved recently, there is little structural information available about ssDNA interaction with the full-length APOBEC3G or any other two-domain APOBEC3. Here, we investigated the solution-state structures of full-length APOBEC3G with and without a 40-mer modified ssDNA by small-angle X-ray scattering (SAXS), using size-exclusion chromatography (SEC) immediately prior to irradiation to effect partial separation of multi-component mixtures. To prevent cytosine deamination, the target 2′-deoxycytidine embedded in 40-mer ssDNA was replaced by 2′-deoxyzebularine, which is known to inhibit APOBEC3A, APOBEC3B and APOBEC3G when incorporated into short ssDNA oligomers. Full-length APOBEC3G without ssDNA comprised multiple multimeric species, of which tetramer was the most scattering species. The structure of the tetramer was elucidated. Dimeric interfaces significantly occlude the DNA-binding interface, whereas the tetrameric interface does not. This explains why dimers completely disappeared, and monomeric protein species became dominant, when ssDNA was added. Data analysis of the monomeric species revealed a full-length APOBEC3G–ssDNA complex that gives insight into the observed “jumping” behavior revealed in studies of enzyme processivity. This solution-state SAXS study provides the first structural model of ssDNA binding both domains of APOBEC3G and provides data to guide further structural and enzymatic work on APOBEC3–ssDNA complexes. Full article

Adenosine deaminase acting on RNA 1 (ADAR1) is an enzyme responsible for double-stranded RNA (dsRNA)-specific adenosine-to-inosine RNA editing, which is estimated to occur at over 100 million sites in humans. ADAR1 is composed of two isoforms transcribed from different promoters: p150 and N-terminal truncated p110. Deletion of ADAR1 p150 in mice activates melanoma differentiation-associated protein 5 (MDA5)-sensing pathway, which recognizes endogenous unedited RNA as non-self. In contrast, we have recently demonstrated that ADAR1 p110-mediated RNA editing does not contribute to this function, implying that a unique Z-DNA/RNA-binding domain α (Zα) in the N terminus of ADAR1 p150 provides specific RNA editing, which is critical for preventing MDA5 activation. In addition, a mutation in the Zα domain is identified in patients with Aicardi–Goutières syndrome (AGS), an inherited encephalopathy characterized by overproduction of type I interferon. Accordingly, we and other groups have recently demonstrated that Adar1 Zα-mutated mice show MDA5-dependent type I interferon responses. Furthermore, one such mutant mouse carrying a W197A point mutation in the Zα domain, which inhibits Z-RNA binding, manifests AGS-like encephalopathy. These findings collectively suggest that Z-RNA binding by ADAR1 p150 is essential for proper RNA editing at certain sites, preventing aberrant MDA5 activation. Full article