DNA Binding Research Articles

Structure characterization, DNA binding, molecular docking and antitumor activity of manganese isothiocyanate complex with neocuproine ligand

Structure characterization, DNA binding, molecular docking and antitumor activity of manganese isothiocyanate complex with neocuproine ligand

Amyotrophic lateral sclerosis caused by FUS mutations: advances with broad implications.

Autosomal dominant mutations in the gene encoding the DNA and RNA binding protein FUS are a cause of amyotrophic lateral sclerosis (ALS), and about 0·3-0·9% of patients with ALS are FUS mutation carriers. FUS-mutation-associated ALS (FUS-ALS) is characterised by early onset and rapid progression, compared with other forms of ALS. However, different pathogenic mutations in FUS can result in markedly different age at symptom onset and rate of disease progression. Most FUS mutations disrupt its nuclear localisation, leading to its cytoplasmic accumulation in the CNS. FUS also forms inclusions in around 5% of patients with the related neurodegenerative condition frontotemporal dementia. However, there are key differences between the two diseases at the genetic and neuropathological level, which suggest distinct pathogenic processes. Experimental models have uncovered potential pathogenic mechanisms in FUS-ALS and informed therapeutic strategies that are currently in development, including the silencing of FUS expression using an intrathecally administered antisense oligonucleotide.

The functions of papillomavirus E2 proteins.

All papillomaviruses encode an E2 protein and it is essential for the viral life cycle. E2 has three domains; a carboxyl-terminal DNA binding and dimerization domain, an amino-terminal protein interaction domain and a hinge region linking these two. Following homo-dimerization human papillomavirus E2 binds to four 12bp palindromic DNA sequences located in the non-coding long control region (LCR) of the viral genome. E2 has three main roles during the viral life cycle. It regulates transcription from the host, and potentially the viral, genome. It initiates viral replication via recruitment of the helicase E1 to the origin of replication. It segregates the viral genome during mitosis to ensure that viral genomes reside in daughter nuclei. This review will describe all of these functions and the mechanisms and interacting partners E2 uses to achieve them. It will also describe a potential role for E2 in mediating HPV cancer therapeutic outcomes.

Dysprosium complex with 1,10-phenanthroline-5,6-dione: Synthesis, structure, DNA binding profile, and in vitro cytotoxic evaluation against HT29 cancer cell lines

Dysprosium complex with 1,10-phenanthroline-5,6-dione: Synthesis, structure, DNA binding profile, and in vitro cytotoxic evaluation against HT29 cancer cell lines

Role of TRIM29 in disease: What is and is not known.

Tripartite motif-containing proteins (TRIMs), comprising the greatest subfamily of E3 ubiquitin ligases with approximately 80 members of this family, are widely distributed in mammalian cells. TRIMs actively participate in ubiquitination of target proteins, a type of post-translational modification associated with protein degradation and other functions. Tripartite motif-containing protein 29 (TRIM29), a member of the TRIM family, differs from other members of this family in that it lacks the RING finger structural domain containing cysteine and histidine residues that mediates DNA binding, protein-protein interactions, and ubiquitin ligase, at its N-terminus. The expression of TRIM29 was initially found to be associated with cancer and diabetic nephropathy progression, and antiviral immunity which is triggered by virus-derived nucleic acids binding to pattern recognition receptors (PRRs) on immune cells. Recently, TRIM29 has also been explored as a diagnostic biomarker and therapeutic target for some immune-related diseases. Here, we review the functions of TRIM29 in the progression of diseases and the inherent mechanisms, as well as the remaining gaps in the literature. A thorough understanding of the detailed regulatory mechanisms of TRIM29 will ultimately facilitate the development of different therapeutic strategies for various diseases.

Single-molecule microscopy reveals that importin α slides along DNA while transporting cargo molecules.

Importin α is a crucial player in the nucleocytoplasmic transport of nuclear localization signal (NLS)-containing cargo proteins and is suggested to bind to DNA directly. We hypothesized that importin α, after binding to DNA, may move along DNA via sliding or hopping. We investigated the movement dynamics of importin αs fused to AcGFP along DNA using single-molecule fluorescence microscopy and single-tethered DNA arrays. Single-molecule data demonstrated importin α diffuses along DNA in fast and slow mobility modes. The diffusion by importin α in the fast mobility mode did not depend on salt concentration, suggesting sliding motion with continuous contact with DNA. The sliding was supported by restricted diffusion of importin α in Cas9 obstacles bound to DNA. Next, we tested whether importin α can transport a cargo molecule along DNA. Two-color imaging data established that importin α co-slides along DNA with SV40 TAg-NLS as a model cargo. We found that importin β1 together with RanGTP significantly enhanced the DNA binding of importin α and the recruitment of a model cargo TRIM28 to DNA, suggesting that importin β1/RanGTP are involved in the switching of importin α/cargo from the nuclear transport pathway to DNA sliding. Single-molecule and in vivo immunofluorescence assay demonstrates importin α assists in accumulating TRIM28 within nuclear chromatin regions. Thus, we present novel findings on the sliding dynamics and the cargo transport of importin α along DNA. The relatively faster sliding by importin α allows efficient delivery of cargo proteins to their target sites, even on long genomic DNA.

LEF3 phosphorylation attenuates the replication of Bombyx mori nucleopolyhedrovirus by suppressing its interaction with alkaline nuclease.

Late expression factor 3 (LEF3), a multifunctional single-stranded DNA binding protein encoded by baculoviruses, is indispensable for viral DNA replication and plays a pivotal role in viral infection. Our previous quantitative analysis of phosphorylomics revealed that the phosphorylation levels of two serine residues (S8 and S25) located in LEF3 nuclear localization sequence were significantly up-regulated after Bombyx mori nucleopolyhedrovirus (BmNPV) infection, but the underlying mechanism remained unknown. To investigate the impact of phosphorylation on BmNPV infection, site-direct mutagenesis was performed on LEF3 to obtain phosphorylated mimic (S/D) or dephosphorylated mimic (S/A) mutants. The results demonstrated that the viral replication and proliferation were inhibited by phosphorylation of S8 or S25. Furthermore, we found that the N-terminal 125 amino acids region was responsible for interacting with virus-encoded alkaline nuclease, but this interaction could be suppressed by the phosphorylation. Our findings indicated that phosphorylation may serve as an antiviral strategy for host.

Beyond small molecules: advancing MYC-targeted cancer therapies through protein engineering.

Protein engineering has emerged as a powerful approach toward the development of novel therapeutics targeting the MYC/MAX/E-box network, an active driver of >70% of cancers. The MYC/MAX heterodimer regulates numerous genes in our cells by binding the Enhancer box (E-box) DNA site and activating the transcription of downstream genes. Traditional small molecules that inhibit MYC face significant limitations that include toxic effects, drug delivery challenges, and resistance. Recent advances in protein engineering offer promising alternatives by creating protein-based drugs that directly disrupt the MYC/MAX dimerization interface and/or MYC/MAX's binding to specific DNA targets. Designed DNA binding proteins like Omomyc, DuoMyc, ME47, MEF, and Mad inhibit MYC activity through specific dimerization, sequestration, and DNA-binding mechanisms. Compared to small molecules, these engineered proteins can offer superior specificity and efficacy and provide a potential pathway for overcoming the limitations of traditional cancer therapies. The success of these protein therapeutics highlights the importance of protein engineering in developing cancer treatments.

A Biomimetic Sweeping Microrobot for Active Therapy of Ulcerative Colitis

AbstractOverproduction of pathogenic cell‐free DNA (cfDNA) and reactive oxygen species (ROS) plays crucial roles in the onset and perpetuation of ulcerative colitis (UC). Inspired by sweeping robots, a magnesium@polylactic acid‐glycolic acid copolymer@polyethylenimine (Mg@PLGA@PEI) microswimmer capable of cleaning off deleterious disease triggers along its path of progress is designed. Mg@PLGA@PEI is successfully synthesized by adopting a core‐shell structure with a small opening which allows for Mg‐water reaction. The distinctive motility performance resulting from sustained detachment of the produced hydrogen not only contributes to strengthened hydrogen diffusion concomitant with potentiated ROS neutralization, but also facilitates the contacting probability with microenvironmental cfDNA and thus enhances the DNA binding efficiency. By integrating these merits, the developed Mg@PLGA@PEI confers desirable curative efficacy in a classical DSS‐induced acute colitis mouse model. Enema administration of Mg@PLGA@PEI microswimmers substantially alleviates the manifestations related to UC, as evidenced by the body weight recovery, colon length retention, colon tissue protection, and attenuated intestinal inflammation, which is attributed to the active scavenging of cfDNA and ROS. This work provides a paradigm for a drug‐free strategy competent in spontaneously eliminating causative triggers with minimal side effects, which presents a promising alternative for the active therapy of UC or other cfDNA‐ and ROS‐related diseases in the clinic.

Conformational dynamics in specialized C2H2 zinc finger domains enable zinc-responsive gene repression in S. pombe.

Loz1 is a zinc-responsive transcription factor in fission yeast that maintains cellular zinc homeostasis by repressing the expression of genes required for zinc uptake in high zinc conditions. Previous deletion analysis of Loz1 found a region containing two tandem C2H2 zinc-fingers and an upstream "accessory domain" rich in histidine, lysine, and arginine residues to be sufficient for zinc-dependent DNA binding and gene repression. Here we report unexpected biophysical properties of this pair of seemingly classical C2H2 zinc fingers. Isothermal titration calorimetry and NMR spectroscopy reveal two distinct zinc binding events localized to the zinc fingers. NMR spectra reveal complex dynamic behavior in this zinc-responsive region spanning time scales from fast 10-12-10-10 to slow >100 s. Slow exchange due to cis-trans isomerization of the TGERP linker results in the doubling of many signals in the protein. Conformational exchange on the 10-3 s timescale throughout the first zinc finger distinguishes it from the second and is linked to a weaker affinity for zinc. These findings reveal a mechanism of zinc sensing by Loz1 and illuminate how the protein's rough free-energy landscape enables zinc sensing, DNA binding and regulated gene expression.

Rs762855 single nucleotide polymorphism modulates the risk for diffuse-type gastric cancer in females: a genome-wide association study in the Korean population.

Intestinal-type gastric cancer (IGC) and diffuse-type gastric cancer (DGC) exhibit different prevalence rates between sexes. While environmental factors like Helicobacter pylori infection and alcohol consumption contribute to these differences, they do not fully account for them, suggesting a role for host genetic factors. We conducted a meta-analysis to explore associations between single nucleotide polymorphisms (SNPs) and the risk of IGC or DGC. The analysis included the SNUBH cohort (998 participants: 159 DGCs, 303 IGCs, 4,962,361 variants) and the GC_HC cohort (6,233 participants: 389 DGCs, 405 IGCs, 4,541,617 variants). Significant variants were validated in the SNUBH2_AA cohort (5,511 participants: 40 DGCs, 49 IGCs, 3,668,632 variants). The meta-analysis identified that rs762855 (chr4:3,074,795; hg19) is significantly associated with DGC risk in females (OR [95% CI]: 1.758 [1.438-2.150], P = 3.91 × 10-8), a finding replicated in the SNUBH2_AA datasets (OR [95% CI]: 3.356 [1.031-10.92], P = 4.43 × 10-2). Gene-set and transcriptomic analyses revealed that the Myb/SANT DNA Binding Domain Containing 1 (MSANTD1) gene is significantly linked to DGC susceptibility in females. In addition, Mendelian randomization analyses suggested that increased serum total protein and non-albumin protein (NAP) levels elevate DGC risk in females (P < 0.05), but not in males. The rs762855 SNP, MSANTD1, and serum NAP levels are associated with DGC risk in Korean females.

Inhibition of HMGA2 binding to AT-rich DNA by its negatively charged C-terminus.

The mammalian high mobility group protein AT-hook 2 (HMGA2) is a small DNA-binding protein that specifically targets AT-rich DNA sequences. Structurally, HMGA2 is an intrinsically disordered protein (IDP), comprising three positively charged 'AT-hooks' and a negatively charged C-terminus. HMGA2 can form homodimers through electrostatic interactions between its 'AT-hooks' and C-terminus. This suggests that the negatively charged C-terminus may inhibit DNA binding by interacting with the positively charged 'AT-hooks.' In this paper, we demonstrate that the C-terminus significantly influences HMGA2's DNA-binding properties. For example, the C-terminal deletion mutant HMGA2Δ95-108 binds more tightly to the AT-rich DNA oligomer FL814 than wild-type HMGA2. Additionally, a synthetic peptide derived from the C-terminus (the C-terminal motif peptide or CTMP) strongly inhibits HMGA2's binding to FL814, likely by interacting with the 'AT-hooks,' as shown by various biochemical and biophysical assays. Molecular modeling demonstrates that electrostatic interactions and hydrogen bonding are the primary forces driving CTMP's binding to the 'AT-hooks.' Intriguingly, we found that hydration does not play a role in HMGA2-DNA binding. These results suggest that the highly negatively charged C-terminus of HMGA2 plays a critical role in regulating its DNA-binding capacity through autoinhibition, likely facilitating the target search process for AT-rich DNA sequences.

An alternatively translated isoform of PPARG proposes AF-1 domain inhibition as an insulin sensitization target.

PPARγ is the pharmacological target of thiazolidinediones (TZDs), potent insulin sensitizers that prevent metabolic disease morbidity but are accompanied by side effects such as weight gain, in part due to non-physiological transcriptional agonism. Using high throughput genome engineering, we targeted nonsense mutations to every exon of PPARG, finding an ATG in Exon 2 (chr3:12381414, CCDS2609 c.A403) that functions as an alternative translational start site. This downstream translation initiation site gives rise to a PPARγ protein isoform (M135), preferentially generated from alleles containing nonsense mutations upstream of c.A403. PPARγ M135 retains the DNA and ligand binding domains of full-length PPARγ but lacks the N-terminal AF-1 domain. Despite being truncated, PPARγ M135 shows increased transactivation of target genes, but only in the presence of agonists. Accordingly, human missense mutations disrupting AF-1 domain function actually increase agonist-induced cellular PPARγ activity compared to wild-type (WT), and carriers of these AF-1 disrupting variants are protected from metabolic syndrome. Thus, we propose the existence of PPARγ M135 as a fully functional, alternatively translated isoform that may be therapeutically generated to treat insulin resistance-related disorders.

Epigenetic Threads of Neurodegeneration: TFAM's Intricate Role in Mitochondrial Transcription.

There is a myriad of activities that involve mitochondria that are crucial for maintaining cellular equilibrium and genetic stability. In the pathophysiology of neurodegenerative illnesses, mitochondrial transcription influences mitochondrial equilibrium, which in turn affects their biogenesis and integrity. Among the crucial proteins for keeping the genome in optimal repair is mitochondrial transcription factor A, more commonly termed TFAM. TFAM's non-specific DNA binding activity demonstrates its involvement in the control of mitochondrial DNA (mtDNA) transcription. The role of TFAM in controlling packing, stability, and replication when assessing the quantity of the mitochondrial genome is well recognised. Despite mounting evidence linking lower mtDNA copy numbers to various age-related diseases, the correlation between TFAM abundance and neurodegenerative disease remains insufficient. This review delves into the link between neurodegeneration and mitochondrial dysfunction caused by oxidative stress. Additionally, the article will go into detail about how TFAM controls mitochondrial transcription, which is responsible for encoding key components of the oxidative phosphorylation (OXPHOS) system.

Evolution of transcription factor-containing superfamilies in Eukaryotes.

Regulation of gene expression helps determine various phenotypes in most cellular life forms. It is orchestrated at different levels and at the point of transcription initiation by transcription factors (TFs). TFs bind to DNA through domains that are evolutionarily related, by shared membership of the same superfamilies (TF-SFs), to those found in other nucleic acid binding and protein-binding functions (nTFs for non-TFs). Here we ask how TF DNA binding sequence families in eukaryotes have evolved in relation to their nTF relatives. TF numbers scale by power law with the total number of protein-coding genes differently in different clades, with fungi usually showing sub-linear powers whereas chordates show super-linear scaling. The LECA probably encoded a complex regulatory machinery with both TFs and nTFs, but with an excess of nTFs when compared to the relative distribution of TFs and nTFs in extant organisms. Losses drive the evolution of TFs and nTFs, with the possible exception of TFs in animals for some tree topologies. TFs are highly dynamic in evolution, showing higher gain and loss rates than nTFs though both are conserved to similar extents. Gains of TFs and nTFs are driven by the appearance of a large number of new sequence clusters in a small number of nodes, which determine the presence of as many as a third of extant TFs and nTFs as well as the relative presence of TFs and nTFs. Whereas nodes showing explosion of TF numbers belong to multicellular clades, those for nTFs lie among the fungi and the protists.

Nanoparticle-assisted PCR: fundamentals, mechanisms, and forensic implications.

Polymerase Chain Reaction (PCR) has transformed forensic DNA analysis butis still limited when dealing with compromised trace or inhibitor-containing samples. Nanotechnology has been integrated into nanoPCR (nanoparticle-assisted PCR) to overcome these obstacles. Nanomaterials improve PCR sensitivity, selectivity, and efficiency. Examples of these materials are semiconductor quantum dots and metal nanoparticles. They enhance DNA binding to primers, stabilize enzymes, and function as effective heat conductors, making accurate amplification possible even with tainted samples. The developments in nanoPCR have potential uses in forensics, as they allow for the more sensitive analysis of smaller, polluted, or deteriorated samples. Nevertheless, there are methodological and ethical issues. To provide credible and legitimate forensic evidence, rigorous validation and standardization of NanoPCR techniques are vital. The article addresses the relevant ethical and methodological aspects in forensic casework while examining the integration of nanotechnology into PCR.

TDP43 augments astrocyte inflammatory activity through mtDNA-cGAS-STING axis in NMOSD

Abnormality in transactivating response region DNA binding protein 43 (TDP43) is well-recognized as the pathological hallmark of neurodegenerative diseases. However, the role of TDP43 in neuromyelitis optica spectrum disorder (NMOSD) remains unknown. Here, our observations demonstrate an upregulation of TDP43 in both in vitro and in vivo models of NMOSD, as well as in biological samples from NMOSD patients. Single-nucleus RNA sequencing revealed that NMOSD induced A1-like reactive astrocytes and astrocyte mitochondrial dysfunction in mice. We further found that NMOSD provoked the translocation of TDP43 to mitochondria and the release of mitochondrial DNA (mtDNA) into the cytoplasm. NMOSD caused activation of mtDNA/cyclic GMP-AMP synthase (cGAS) / stimulator of interferon genes (STING) pathway and A1-type inflammatory activation in astrocytes. Crucially, the knockdown of TDP43 markedly ameliorated NMOSD-induced mitochondrial dysfunction and the activation of the cGAS/STING pathway in astrocytes. Conversely, overexpression of TDP43 exacerbated these pathological changes. Specific silencing astrocytic TDP43 ameliorated NMOSD-induced injury in mice, and conversely, TDP43 overexpression intensified the injury. Meanwhile, both cGAS and STING inhibitors attenuated NMOSD-induced injury in mice. In summary, our data suggest that TDP43 exacerbates inflammatory activation of astrocytes in NMOSD through upregulating the mtDNA/cGAS/STING signaling pathway. Therefore, targeting TDP43 represents a compelling therapeutic strategy for NMOSD.

Functional validation of a novel STAT3 'variant of unknown significance' identifies a new case of STAT3 GOF Syndrome and reveals broad immune cell defects.

STAT3 orchestrates crucial immune responses through its pleiotropic functions as a transcription factor. Patients with germline monoallelic dominant negative or hypermorphic STAT3 variants, who present with immunodeficiency and/or immune dysregulation, have revealed the importance of balanced STAT3 signaling in lymphocyte differentiation and function, and immune homeostasis. Here, we report a novel missense variant of unknown significance in the DNA binding domain of STAT3 in a patient who experienced hypogammaglobulinemia, lymphadenopathy, hepatosplenomegaly, immune thrombocytopenia, eczema and enteropathy over a 35-year period. In vitro demonstration of prolonged STAT3 activation due to delayed de-phosphorylation, and enhanced transcriptional activity, confirmed this to be a novel pathogenic STAT3 gain-of-function variant. Peripheral blood lymphocytes from this patient, and patients with confirmed STAT3 Gain-of-function Syndrome, were collected to investigate mechanisms of disease pathogenesis. B cell dysregulation was evidenced by a loss of class-switched memory B cells and a significantly expanded CD19hiCD21lo B cell population, likely influenced by a skewed CXCR3+ TFH population. Interestingly, unlike STAT3 dominant negative variants, cytokine secretion by activated peripheral blood STAT3 GOF CD4+ T cells and frequencies of Treg cells were intact, suggesting CD4+ T cell dysregulation likely occurs at sites of disease rather than the periphery. This study provides an in-depth case study in confirming a STAT3 gain-of-function variant and identifies lymphocyte dysregulation in peripheral blood of patients with STAT3 Gain-of-function Syndrome. Identifying cellular biomarkers of disease provide a flow cytometric based screen to guide validation of additional novel STAT3 gain-of-function variants as well as provide insights into putative mechanisms of disease pathogenesis.

Apaf-1 is an evolutionarily conserved DNA sensor that switches the cell fate between apoptosis and inflammation

Apoptotic protease activating factor 1 (Apaf-1) was traditionally defined as a scaffold protein in mammalian cells for assembling a caspase activation platform known as the ‘apoptosome’ after its binding to cytochrome c. Although Apaf-1 structurally resembles animal NOD-like receptor (NLR) and plant resistance (R) proteins, whether it is directly involved in innate immunity is still largely unknown. Here, we found that Apaf-1-like molecules from lancelets, fruit flies, mice, and humans have conserved DNA sensing functionality. Mechanistically, mammalian Apaf-1 recruits receptor-interacting protein 2 (RIP2, also known as RIPK2) via its WD40 repeat domain and promotes RIP2 oligomerization to initiate NF-κB-driven inflammation upon cytoplasmic DNA recognition. Furthermore, DNA binding of Apaf-1 determines cell fate by switching the cellular processes between intrinsic stimuli-activated apoptosis and inflammation. These findings suggest that Apaf-1 is an evolutionarily conserved DNA sensor and may serve as a cell fate checkpoint, which determines whether cells initiate inflammation or undergo apoptosis by distinct ligand binding.

Genetic susceptibility and potential therapeutic targets of unruptured intracranial aneurysms: A genome-wide study based on Mendelian randomization.

At present, although some studies have offered certain insights into the genetic factors related to unruptured intracranial aneurysms (uIAs), the potential genetic targets associated with uIAs remain largely unknown. Thus, this research adopted Mendelian randomization (MR) analysis to study two genome-wide association studies on uIAs, aiming to determine the reliable genetic susceptibility and potential therapeutic targets for uIAs. This study summarizes the data of expression quantitative trait loci (eQTL) as exposure data. The outcome data of uIAs were derived from the study by Bakker et al. and the FinnGen Biobank (version R10). The reliable genetic susceptibility and potential therapeutic targets of uIAs were identified by means of Mendelian randomization (MR) methods, with the inverse variance weighting (IVW) method as the primary analytical approach. Simultaneously, sensitivity and pleiotropy analyses were carried out, and the results were visualized. Subsequently, drug predictions and molecular docking were conducted for the potential gene targets to verify their reliability. The MR analysis of the training cohort identified 100 targets related to uIAs. Then, these 100 gene targets and eQTL data were verified by MR Analysis again with the testing cohort. Finally, 7 gene targets were selected, namely MTMR3, SERINC1, CITED2, NKX3-1, ATOX1, MYADM and SLC20A1-DT.GO/KEGG enrichment analysis confirmed that the 7 gene targets mainly participate in the process Biological functions and pathways such as art development, cellular response to hypoxia, male Gonad development, RNA polymerase II specific DNA binding transcription factor binding, DNA binding transcription factor binding, Mineral absorption, Inositol phase metabolism, Photoshatidylinositol signaling system, etc.The protein-protein interaction（PPI） network describes the interactions between seven gene targets and related proteins.The molecular docking diagram shows good binding between candidate drugs and proteins related to gene targets. The study identified 7 reliable gene susceptibility and potential therapeutic targets associated with uIAs, offering new insights for clinical diagnosis and treatment of uIAs, and suggesting novel research directions for understanding the etiology and molecular mechanisms of uIAs.