Search Results (961)

The transcription factor homeodomain-only protein X (HOPX) is the smallest member of the homeodomain protein family. Lacking a DNA-binding domain, it acts as a co-effector, interacting with other transcription factors such as serum response factor (SRF) and GATA-binding factor 6 (GATA6) to regulate the differentiation and development of the heart and lung. HOPX exerts a tumor-suppressive function in various types of epithelial-derived carcinoma, while it promotes oncogenic effects in mesenchymal-derived sarcoma, indicating a distinct role of HOPX in the two major types of the malignancy. In addition, accumulating evidence shows that HOPX is expressed in the immune system and involved in the differentiation of immune cells. Recently, the emerging role of HOPX in metabolism has gained attention. This review describes the identification of HOPX in various tissues and discusses its role in carcinogenesis, as well as its functions in tissue differentiation, lipid metabolism, immunity, and the tumor microenvironment. The participation of HOPX in carcinogenesis and immunity implies that it may serve as a potential enhancer in tumor immunotherapy. Full article

Background: Point mutations in mitochondrial DNA (mtDNA) cause a range of neurometabolic disorders that currently have no curative treatments. The m.8993T>G mutation in the Homo sapiens MT-ATP6 gene leads to neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) when heteroplasmy exceeds approximately 70%. Methods: We engineered a split DddA-derived cytosine base editor (DdCBE), each half fused to programmable TALE DNA-binding domains and a mitochondrial targeting sequence, to correct the m.8993T>G mutation in patient-derived induced pluripotent stem cells (iPSCs). Seven days after plasmid delivery, deep amplicon sequencing showed 35 ± 3% on-target C•G→T•A conversion at position 8993, reducing mutant heteroplasmy from 80 ± 2% to 45 ± 3% with less than 0.5% editing at ten predicted off-target loci. Results: Edited cells exhibited a 25% increase in basal oxygen consumption rate, a 50% improvement in ATP-linked respiration, and a 2.3-fold restoration of ATP synthase activity. Directed neural differentiation yielded 85 ± 2% Nestin-positive progenitors compared to 60 ± 2% in unedited controls. Conclusions: Edits remained stable over 30 days in culture. These results establish mitochondrial base editing as a precise and durable strategy to ameliorate biochemical and cellular defects in NARP patient cells. Full article

Mycobacteria possess carbon monoxide dehydrogenase (CO-DH) to utilize CO as an energy source and to resist host defense mechanisms. The expression of the CO-DH gene is regulated by CutR, a LysR-type transcriptional regulator (LTTR) that exhibits unique characteristics, suggesting that it functions as a dimer rather than the typical tetramer. Size-exclusion chromatography revealed that CutR forms a stable dimer. Electrophoretic mobility shift assays demonstrated that dimeric CutR specifically binds to an inverted repeat sequence (IR1) containing T-n12-A motifs located upstream of the cutB gene, which encodes the medium subunit of CO-DH. Crystal structure determination at 1.8 Å resolution revealed that CutR consists of an N-terminal DNA-binding domain with a winged helix-turn-helix motif and a C-terminal ligand-binding domain comprising two regulatory subdomains (RD1 and RD2), forming a unique two-fold symmetrical homodimer. This dimer is stabilized through four interfaces, including an extensive 12-stranded antiparallel β-sheet formed between RD1 subdomains via intertwining C-terminal β11 strands. This represents the first symmetric dimeric LTTR structure with tightly associated ligand-binding domains. The recognition helices are spaced closer together than they are in typical DNA-bound LTTRs, despite binding longer T-n12-A sequences, suggesting that a conformational change is required to enhance DNA-binding affinity. A putative ligand-binding site was identified between the RD1 and RD2 subdomains, where glycerol binding induced local conformational changes. Comparative genomic analysis revealed conservation of CutR and the IR1 sequence across Mycobacterium species, supporting the dimeric regulatory mechanism and providing new insights into LTTR diversity. Full article

Currently, a major challenge exists in CRISPR-mediated genome editing research in sheep: the low efficiency of exogenous large DNA fragment targeted integration without drug selection or fluorescence enrichment. This restriction significantly impedes the use of precise genome editing in sheep for agricultural, biological, and biomedical purposes. In this study, we employed the strategy of increasing the local concentration of the homologous repair template at the site of the DNA double-strand break (DSB). We achieved this by fusing the DNA binding domain (BD) of the Gal4 protein (Gal4-BD) to the N-terminal end of the SpCas9 protein using a 32-amino acid (aa) flexible linker. Additionally, we incorporated a 17 bp UAS at the 3′ end of the donor template, which can be specifically recognized and bound by Gal-BD. As a result, we observed a significant improvement in the knock-in efficiency of the exogenous large DNA fragment (2997 bp) in sheep fetal fibroblasts (SFFs), increasing it from 5.30% (8/151) to 16.67% (32/192), providing a comparatively efficient and user-friendly method to promote CRISPR-mediated gene knock-in in sheep. Full article

Background: Breast and ovarian cancers (BC and OC) are prevalent malignancies in women globally, with germline variants in the BRCA2 gene significantly increasing the risk of developing these cancers. Despite extensive studies, the frequency and impact of BRCA2 variants in women from Jalisco, Mexico, remain underexplored. Objective: The aim of this study was to identify and characterize BRCA2 gene variants in Mexican women diagnosed with BC and OC and to assess their functional and structural consequences using computational analyses. Methodology: Genomic DNA from 140 Mexican women with BC and/or OC, selected based on clinical criteria suggestive of BRCA2 variants, was sequenced using NGS targeting BRCA2 coding regions. Functional effects were predicted with Ensembl VEP, SIFT, and PolyPhen-2. Structural impacts of missense variants were assessed using HOPE and AlphaFold models. Results: BRCA2 variants were identified in 12.86% of patients, with higher frequency in OC (21.05%) than BC (12%). Several mapped to key functional domains, including BRC repeats and the DNA-binding domain. Many were predicted as deleterious or probably damaging, though clinical classifications were often conflicting. Structural analysis indicated potential disruptions in protein stability or interactions for most missense variants. Clinically, BRCA2-positive BC patients were younger at diagnosis and showed a trend toward lower complete response. Conclusion: BRCA2 variants were found in 12.86% of patients, including six VUSs not reported in other populations. Several affected key functional domains with predicted deleterious effects. Findings support the need for genetic panels tailored to the Mexican population. Full article

Background/Objectives: Mutations in NACHT, LRR and PYD domain-containing protein 2 (NLRP2) and NLRP7 genes, members of the NOD-like receptor (NLR) family of innate immune sensors, result in recurrent miscarriages and reproductive wastage in women. These genes have been identified to be maternal effect genes in humans and mice regulating early embryo development. Previous research in vitro suggests that NLRP2 and NLRP7 regulate DNA methylation and/or immune signaling through inflammasome formation. However, the exact mechanisms underlying NLRP2 and NLRP7 function are not well defined. Methods: To determine the interacting proteins required for NLRP2/NLRP7-mediated regulation of DNA methylation, yeast 2-hybrid screens, coimmunoprecipitation, and FRET studies were performed and verified the ability of novel protein interactions to affect global DNA methylation by 5-methylcytosine-specific ELISA. Results: Various methodologies employed in this research demonstrate a novel protein interaction between human ErbB3-binding protein 1 (EBP1, also known as proliferation-associated protein 2G4 (PA2G4) and NLRP2 or NLRP7. In addition, NLRP2 and NLRP7 regulate EBP1 gene expression. Functionally, global DNA methylation levels appeared to decrease further when NLRP2 and NLRP7 were co-expressed with EBP1, although additional studies may need to confirm the significance of this effect. Conclusions: Since EBP1 is implicated in apoptosis, cell proliferation, DNA methylation, and differentiation, our discovery significantly advances our understanding of how mutations in NLRP2 or NLRP7 may contribute to reproductive wastage in women through EBP1. Full article

Argonaute (Ago) proteins are ubiquitous across all domains of life. Some prokaryotic Agos (pAgos) function as endonucleases that utilize short nucleic acid guides to recognize and cleave complementary targets. Yet, considerable diversity within pAgos leaves many of their biochemical and functional features insufficiently understood. This study characterizes CalAgo, an pAgo from the mesophilic bacterium Cohnella algarum, which demonstrates DNA-guided DNA endonuclease and RNA endonuclease activities at physiological temperatures. CalAgo’s cleavage activity depends on Mn²⁺ and Mg²⁺ ions and remains effective across a wide range of temperatures and pH levels. CalAgo utilizes only short guides ranging from 15 to 21 nucleotides (nt) in length, in contrast to other reported pAgos that target both DNA and RNA, which often exhibit broad guide selectivity. CalAgo preferentially loads 5′-phosphorylated guides and shows no significant preference among guides with different 5′-end nucleotides. CalAgo is sensitive to guide–target mismatches, and introducing a single mismatch at positions 12 or 15 of the guide strand abolished detectable activity. Structural modeling suggests that this unique guide specificity may originate from structural features in its PAZ domain involved in 3′-guide binding. In summary, this study deepens insight into mesophilic pAgos and supports their potential utility in nucleic acid-based applications. Full article

The genome of the herpesvirus is a linear double-stranded DNA. The viral genome replicates in the host cell to form a concatemeric DNA, which is then cleaved to produce a unit-length genome. This unit-length genome is packaged into procapsid to produce mature virus particles. The terminase large subunit, pUL15, mediates the cleavage and packaging of viral concatemeric genomes. Duck plague virus (DPV) is a member of the α herpesvirus subfamily. Previous studies have demonstrated that the C-terminal region of DPV pUL15 exhibits non-sequence-specific DNA cleavage activity in vitro, but the characteristics of DPV full-length pUL15 remain unclear. In this study, it was determined that the full-length pUL15 exerted non-sequence-specific nuclease activity. Additionally, full-length pUL15 was capable of binding to DNA and hydrolyzing ATP. To analyze the functional domain of DPV pUL15, pUL15 mutants were constructed, expressed, and purified. The results revealed that DNA-binding and ATPase functions of pUL15 were primarily mediated by its N-terminal region, and the nuclease activity was conducted by its C-terminus. The loss of the nuclease activity did not effect on the DNA-binding and ATPase activity. Taken together, this study’s findings demonstrated that DPV pUL15 is a multifunctional enzyme with ATPase, nuclease, and DNA-binding activities. These results will provide important clues for subsequent studies on the function of terminase and the process of viral genome packaging, and provide a foundational basis for the development of broad-spectrum anti-herpesviral drugs targeting the conserved terminase complex, with direct relevance to veterinary medicine. Full article

Colorectal carcinoma (CRC) is characterized by mutations in p53 and the Wnt signaling pathway, and immunotherapy has shown limited efficacy in microsatellite-stable CRC. Here, CEABP1, a binding protein for the CRC biomarker carcinoembryonic antigen (CEA), was designed de novo through the AI-based computational generation methods RFDiffusion/ProteinMPNN and stringent in silico selection, for targeted delivery of purified p53 protein and transcription factor T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) transcription factor decoy (TFD) DNA into CRC cells. The cell-penetrating peptide (CPP) p28 was employed to deliver the p28-p53-CEABP1 protein, which significantly enhanced p53’s inhibition of CRC cell proliferation and xenograft tumor growth. Codelivery of the p14ARF protein together with p53 prolonged the effective antitumor duration of p53. In addition, the DNA binding domain of Max was fused with CPP and CEABP1 to deliver TCF/LEF TFD DNA, comprising concatenated consensus binding motifs for TCF/LEF and Max, into CRC cells to inhibit Wnt target gene transcription, leading to marked suppression of CRC cell proliferation and xenograft tumor growth. These findings paved the way for the development of precision anticancer therapeutics using designed binding proteins of tumor biomarkers for targeted delivery of tumor suppressor proteins and TFD DNA. Full article

Bacteria employ transcriptional regulators, such as those belonging to the Xenobiotic Response Element (XRE) family, to regulate metabolic processes. These regulators often exhibit autoregulatory properties and function as dimers to recognize palindromic DNA motifs. However, the binding motifs of the XRE family transcriptional regulators in bacteria have not yet been well characterized on a large scale. To identify potential XRE transcriptional regulator recognition motifs efficiently, we developed a computational approach combining structural alignment, sequence scanning, and motif clustering. We first identified the potential motifs of XRE regulators using computational methods. Using the helix–turn–helix (HTH) domain of XRE family regulators as a template, we collected 27,732 proteins containing the domain from bacterial databases. By extracting upstream sequences of these proteins and employing bioinformatics tools like MEME and motifStack to search potential motifs, 5622 motifs were identified and subsequently clustered into 223 clusters. These clusters can be classified into 7 main types based on the base conservation patterns observed in motifs. Interaction models between representative proteins and their corresponding motifs were predicted using AlphaFold. Subsequently, we conducted experimental validation via electrophoretic mobility shift assays (EMSAs) and confirmed the feasibility of our approach, as nine out of ten tested interactions showed clear protein–DNA binding. However, due to limitations in experimental conditions, the remaining predicted motifs have not yet undergone experimental validation. Since conserved sequences and well-predicted structures cannot replace real-world scenarios, there are limitations to relying solely on computational predictions, and experimental validation remains necessary. In summary, our study establishes a reliable framework for identifying XRE family transcriptional regulator recognition motifs and provides valuable insights into bacterial regulation. Full article

Bacillus subtilis DinG/XPD-like paralogues, DinG and YpvA, have been implicated in overcoming replication stress. DinG possesses a DEDD exonuclease and DNA helicase domains, whereas YpvA lacks the DEDD exonuclease domain. We report that DinG·Mg²⁺ (hereafter referred to as DinG) degrades linear single-stranded (lss) DNA with 3′→5′ polarity and binds lssDNA with higher affinity than its exonuclease-deficient mutant DinG D10A E12A. DinG’s ssDNA-dependent ATPase activity neither stimulates nor inhibits DNA degradation. When bound to the 3′-end of forked DNA, DinG destabilises and degrades the substrate; however, in the presence of ATP, DinG dissociates before reaching the duplex junction. DinG degrades the RNA strand within RNA–DNA hybrids but does not cleave lssRNA unless complexed with Mn²⁺. DinG removes genomic R-loops, as RnhC and PcrA do. DinG physically interacts with RecA and PolA and functions in the same pathway as translesion synthesis (TLS) DNA polymerases (DNAPs) to respond to both spontaneous and methyl methanesulphonate (MMS)-induced mutagenesis. DinG-mGold forms spontaneous foci at or near replication forks, which become enriched following MMS or rifampicin treatment. We propose that DinG contributes to mitigating replication stress by degrading R-loop barriers and facilitating TLS, potentially via RecA-linked mechanisms. Full article

Pediatric tumors such as neuroblastoma are characterized by a genome-wide ‘transcriptional burden’, surmising the involvement of multiple alterations of gene expression. Search for master regulators of transcription whose inactivation is lethal for tumor cells identified the non-POU domain-containing octamer-binding protein (NONO), a member of the Drosophila Behavior/Human Splicing family known for the ability to form complexes with macromolecules. NONO emerges as an essential mechanism in normal neurogenesis as well as in tumor biology. In particular, NONO interactions with RNAs, largely with long non-coding MYCN transcripts, have been attributed to the aggressiveness of neuroblastoma. Broadening its significance beyond MYCN regulation, NONO guards a subset of transcription factors that comprise a core regulatory circuit, a self-sustained loop that maintains transcription. As a component of protein–protein complexes, NONO has been implicated in the control of cell cycle progression, double-strand DNA repair, and, generally, in cell survival. Altogether, the pro-oncogenic roles of NONO justify the need for its inactivation as a therapeutic strategy. However, considering NONO as a therapeutic target, its druggability is a challenge. Recent advances in the inactivation of NONO and downstream signaling with small molecular weight compounds make promising the development of pharmacological antagonists of NONO pathway(s) for neuroblastoma treatment. Full article

BACH1 (BTB And CNC Homology 1) and NRF2 (Nuclear Factor Erythroid 2-related Factor 2) are transcription factors that regulate antioxidant and iron metabolism genes by competing for binding to cis-regulatory Maf-binding elements (CsMBEs) as heterodimers with small Maf proteins (sMafs). To dissect the mechanisms underlying this competition, we developed a chimeric tethering system where the DNA-binding domains of BACH1 or NRF2 were covalently linked to sMafG via a flexible, cleavable linker. This design enables efficient heterodimer formation on DNA and circumvents kinetic barriers to partner exchange in the solution. The site-specific fluorescent labelling of proteins allowed for the tracking of complex compositions by electrophoretic mobility shift assays. Both BACH1/sMafG and NRF2/sMafG heterodimers bind CsMBEs with similar affinities. Notably, DNA binding by BACH1 was impaired in a C574-dependent, redox-sensitive manner and promoted the exchange of heterodimer partners. Competition assays demonstrated that BACH1 and NRF2 can displace each other from preformed DNA-bound complexes, with greater efficiency when presented as preassembled heterodimers with sMafG. These findings reveal a redox-sensitive mechanism for regulating transcriptional switches at CsMBEs and highlight how preformed heterodimers facilitate the rapid displacement at target promoters. Full article

Background/Objectives: Orofacial clefts (OFCs) are among the most common birth defects globally, sometimes exacerbated by adverse drug reactions (ADRs) from corticosteroids and antiepileptics. Comprehending the pharmacogenomic and pharmacogenetic elements that lead to ADRs is essential for enhancing precision medicine and clinical outcomes. This study examines rare genetic variants in drug-metabolizing and drug-transporting genes among Ghanaian and Nigerian families with a history of OFCs, intending to assess their pathogenicity and functional implications. Methods: We recruited 104 Ghanaian families and 26 Nigerian families, generating whole-genome sequencing (WGS) data from 390 individuals (130 case-parent trios). DNA isolated from saliva and buccal swab samples underwent WGS, and subsequent WGS data were analyzed through extensive bioinformatics analyses. Variants were called and annotated using the GATK workflow. The HOPE in silico modeling tool evaluated the structural impact of genetic variants on encoded proteins, while molecular docking using PyRx examined alterations in ligand binding affinity. Results: Our study revealed pathogenic variants in vital genes associated with drug metabolism and transport, specifically CYP1A2, CYP2C18, CYP27A1, CYP2B6, SLC6A2, and ABCC3. Structural modeling research demonstrated substantial size, charge, conformation, and hydrophobicity variations between wildtype and mutant proteins. Variants positioned near conserved regions or within functional domains were anticipated to be deleterious, potentially compromising protein function and ligand interactions. Molecular docking studies verified changes in binding affinities between wildtype and mutant proteins for common ligands. The identified variations were linked to the metabolism of frequently used pharmaceuticals in Africa, such as caffeine, ketoconazole, efavirenz, carbamazepine, and artemether. Conclusions: These findings highlight the need for pharmacogenetic screening to inform personalized medicine, diminish ADRs, and enhance the clinical care of OFCs in Sub-Saharan Africa. Full article

RNA polymerases are essential enzymes that catalyze DNA transcription into RNA, vital for protein synthesis, gene expression regulation, and cellular responses. Non-template-dependent RNA polymerases, which synthesize RNA without a template, are valuable in biological research due to their flexibility in producing RNA without predefined sequences. However, their substrate polymerization mechanisms are not well understood. This study examines Poly (A) polymerase (PAP), a nucleotide transferase superfamily member, to explore its substrate selectivity using computational methods. Previous research shows PAP’s polymerization efficiency for nucleoside triphosphates (NTPs) ranks ATP > GTP > CTP > UTP, though the reasons remain unclear. Using 500 ns Gaussian accelerated molecular dynamics simulations, stability analysis, secondary structure analysis, MM-PBSA calculations, and Markov state modeling, we investigate PAP’s differential polymerization efficiencies. Results show that ATP binding enhances PAP’s structural flexibility and increases solvent-accessible surface area, likely strengthening protein–substrate or protein–solvent interactions and affinity. In contrast, polymerization of other NTPs leads to a more open conformation of PAP’s two domains, facilitating substrate dissociation from the active site. Additionally, ATP binding induces a conformational shift in residues 225–230 of the active site from a loop to an α-helix, enhancing regional rigidity and protein stability. Both ATP and GTP form additional π–π stacking interactions with PAP, further stabilizing the protein structure. This theoretical study of PAP polymerase’s substrate selectivity mechanisms aims to clarify the molecular basis of substrate recognition and selectivity in its catalytic reactions. These findings offer valuable insights for the targeted engineering and optimization of polymerases and provide robust theoretical support for developing novel polymerases for applications in drug discovery and related fields. Full article