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Mutation Research 2023Barth syndrome is a rare disease affecting mitochondria structure and function in males. In our previous study, we have shown a new mutation (c.83T>A, p.Val28Glu) in the...
Analysis of tafazzin and deoxyribonuclease 1 like 1 transcripts and X chromosome sequencing in the evaluation of the effect of mosaicism in the TAZ gene on phenotypes in a family affected by Barth syndrome.
Barth syndrome is a rare disease affecting mitochondria structure and function in males. In our previous study, we have shown a new mutation (c.83T>A, p.Val28Glu) in the TAZ gene in two affected patients with congenital cardiomyopathy. Furthermore, women in this family had no mutations in their blood cells, whereas they only had mutations in the oral epithelial cells. The objective of the project was to evaluate the effect of intertissue mosaicisms on the Barth syndrome phenotypes, searching for another disease-related loci on chromosome X and finally to assess the consequences of the mutation. We conducted the advanced genetic study including cytogenetic research (constitutional karyotyping in blood and fibroblasts), NGS sequencing (with custom chromosome X sequencing together with the evaluation of loss of heterozygosity (LOH) and aberrations (CNV) in the whole genome) in four different tissues and sequencing of tafazzin and deoxyribonuclease 1 like 1 transcripts. The presence of deletions within the 5'untranslated region of the TAZ gene and/or the noncoding regions of the DNASE1L1 gene were detected in several tissues. Whereas, there is no intertissue mosaicism regarding point mutation in TAZ gene in all investigated tissues in female carriers. Only the male patient presented biochemical markers and neurological symptoms of Barth syndrome. All the female carriers are healthy and have normal tafazzin and deoxyribonuclease 1 like 1 transcripts in 2 analyzed tissues. The conclusion of this study is that we cannot rule out or confirm mosaicism in the noncoding regions of TAZ or DNASE1L1 genes, but this is not clinically relevant in female carriers because they are healthy. Finally, it has been proven that mutation (c.83T>A, p.Val28Glu) is responsible for disease in males in this family.
Topics: Female; Humans; Male; Acyltransferases; Barth Syndrome; Deoxyribonucleases; Mosaicism; Phenotype; Transcription Factors; X Chromosome
PubMed: 36628843
DOI: 10.1016/j.mrfmmm.2022.111812 -
EMBO Molecular Medicine Jan 2024The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of...
The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients.
Topics: Humans; Animals; Mice; CRISPR-Cas Systems; Deoxyribonuclease I; Gene Editing; Hyperoxaluria, Primary
PubMed: 38182795
DOI: 10.1038/s44321-023-00008-8 -
Biomolecules Sep 2015Deamination of adenine occurs in DNA, RNA, and their precursors via a hydrolytic reaction and a nitrosative reaction. The generated deaminated products are potentially... (Review)
Review
Deamination of adenine occurs in DNA, RNA, and their precursors via a hydrolytic reaction and a nitrosative reaction. The generated deaminated products are potentially mutagenic because of their structural similarity to natural bases, which in turn leads to erroneous nucleotide pairing and subsequent disruption of cellular metabolism. Incorporation of deaminated precursors into the nucleic acid strand occurs during nucleotide synthesis by DNA and RNA polymerases or base modification by DNA- and/or RNA-editing enzymes during cellular functions. In such cases, removal of deaminated products from DNA and RNA by a nuclease might be required depending on the cellular function. One such enzyme, endonuclease V, recognizes deoxyinosine and cleaves 3' end of the damaged base in double-stranded DNA through an alternative excision repair mechanism in Escherichia coli, whereas in Homo sapiens, it recognizes and cleaves inosine in single-stranded RNA. However, to explore the role of endonuclease V in vivo, a detailed analysis of cell biology is required. Based on recent reports and developments on endonuclease V, we discuss the potential functions of endonuclease V in DNA repair and RNA metabolism.
Topics: Animals; DNA Repair; Deamination; Deoxyribonuclease (Pyrimidine Dimer); Humans; RNA Editing
PubMed: 26404388
DOI: 10.3390/biom5042194 -
Bioorganic & Medicinal Chemistry Letters May 2021The use of DNA-encoded libraries (DELs) has increased greatly over the last decade, and today a majority of pharmaceutical companies employ the technology. The... (Review)
Review
The use of DNA-encoded libraries (DELs) has increased greatly over the last decade, and today a majority of pharmaceutical companies employ the technology. The technology may be applied to most soluble and purified targets. However, standard DEL technology has limitations; some targets are challenging to purify, and it is not possible to directly screen for cellular or biochemical activity. Numerous creative methods have been reported to overcome these limitations and expand DEL target scope. Reported proof-of-concept experiments include DEL selections of cell surfaces, and inside of living cells. Additional alternatives include the construction and biochemical screening of one-bead-one-compound (OBOC) DELs using picoliter aqueous droplets or microfabricated wells as containers. In these cases, the small-molecule moiety of the library member is liberated from its DNA barcode, and able to interact freely with the desired target. Lastly, patent literature suggests the ability to conduct cellular functional screens using OBOC DELs.
Topics: Animals; Cell Line; DNA; Deoxyribonucleases; Drug Evaluation, Preclinical; Humans; Molecular Structure; Small Molecule Libraries
PubMed: 33631371
DOI: 10.1016/j.bmcl.2021.127851 -
MBio Feb 2023Many Gram-negative bacteria deploy a type VI secretion system (T6SS) to inject toxins into target cells to promote their survival and replication in complex...
Many Gram-negative bacteria deploy a type VI secretion system (T6SS) to inject toxins into target cells to promote their survival and replication in complex environments. Here, we report that Acinetobacter baumannii uses its T6SS to kill fungi and that the effector TafE () is responsible for such killing. Although ectopically expressed TafE is toxic to both Escherichia coli and Saccharomyces cerevisiae, deletion of only affects the antifungal activity of A. baumannii. We demonstrate that TafE is a DNase capable of targeting the nuclei of yeast cells and that an Ntox15 domain is essential for its ability to degrade DNA. Furthermore, our findings show that A. baumannii is protected from the toxicity of TafE by elaborating the immunity protein TaeI (), which antagonizes the activity of the effector by direct binding. The discovery of A. baumannii T6SS effectors capable of killing multiple taxonomically distinct microbes has shed light on a mechanism of the high-level fitness of this pathogen in environments characterized by scarce nutrients and the potential presence of diverse microorganisms. Acinetobacter baumannii is an increasing important nosocomial pathogen that is difficult to combat due to its ability to survive in harsh environments and the emergence of isolates that are resistant to multiple antibiotics. A better understanding of the mechanism underlying the toughness of A. baumannii may identify its Achilles' heel, which will facilitate the development of novel preventive and treatment measures. In this study, our findings show that A. baumannii kills fungi with the DNase effector TafE injected into competitor cells by its type VI secretion system. A. baumannii is protected from the activity of TafE by the immunity protein TaeI, which inactivates the effector by direct binding. Our results suggest that inactivation of its T6SS or effectors may reduce the fitness of A. baumannii and increase the effectiveness of treatment by means such as antibiotics. Furthermore, our finding suggests that targeted degradation of TaeI may be an effective strategy to kill A. baumannii.
Topics: Acinetobacter baumannii; Type VI Secretion Systems; Deoxyribonucleases; Anti-Bacterial Agents; Fungi; Bacterial Proteins
PubMed: 36625573
DOI: 10.1128/mbio.03420-22 -
Cell Reports Jul 2023Pioneer transcription factors interact with nucleosomes to scan silent, compact chromatin, enabling cooperative events that modulate gene activity. While at a subset of...
Pioneer transcription factors interact with nucleosomes to scan silent, compact chromatin, enabling cooperative events that modulate gene activity. While at a subset of sites pioneer factors access chromatin by assisted loading with other transcription factors, the nucleosome-binding properties of pioneer factors enable them to initiate zygotic genome activation, embryonic development, and cellular reprogramming. To better understand nucleosome targeting in vivo, we assess whether pioneer factors FoxA1 and Sox2 target stable or unstable nucleosomes and find that they target DNase-resistant, stable nucleosomes, whereas HNF4A, a non-nucleosome binding factor, targets open, DNase-sensitive chromatin. Despite FOXA1 and SOX2 targeting similar proportions of DNase-resistant chromatin, using single-molecule tracking, we find that FOXA1 uses lower nucleoplasmic diffusion and longer residence times while SOX2 uses higher nucleoplasmic diffusion and shorter residence times to scan compact chromatin, while HNF4 scans compact chromatin much less efficiently. Thus, pioneer factors target compact chromatin through distinct processes.
Topics: Chromatin; Deoxyribonucleases; Nucleosomes; Protein Binding; Transcription Factors; Hepatocyte Nuclear Factor 3-alpha; SOXB1 Transcription Factors
PubMed: 37405916
DOI: 10.1016/j.celrep.2023.112748 -
The Journal of Investigative Dermatology Jan 2023Hidradenitis suppurativa (HS) is a debilitating inflammatory skin disorder characterized by abscess-like nodules and boils resulting in fistulas and tissue scarring. We...
Hidradenitis suppurativa (HS) is a debilitating inflammatory skin disorder characterized by abscess-like nodules and boils resulting in fistulas and tissue scarring. We previously reported evidence of an autoimmune signature in HS, characterized by enhanced neutrophil extracellular trap (NET) infiltration in HS skin lesions and dysregulation of the adaptive immune system characterized by the presence of autoantibodies. Timely removal of NETs is critical for tissue homeostasis to prevent a dysregulated generation of modified autoantigens and tissue damage. DNases 1 and 1L3 play important roles in proper NET removal. We tested the hypothesis that NETs in patients with HS are not effectively cleared owing to the presence of antibodies against DNase 1 and DNase 1L3. We report that HS serum poorly degraded NETs. Addition of exogenous DNase 1 restored NET degradation capabilities in a subset of HS samples. DNase 1 activity was significantly decreased in HS sera. Anti‒DNase 1 and ‒DNase 1L3 antibodies were detected in serum samples and skin lesions from patients with HS. Purified IgGs from HS decreased DNase 1 activity and NET degradation. Taken together, this identification of neutralizing antibodies against nucleases in HS expands the understanding of the pathogenesis of this disease to support an autoimmune mechanism in its underlying pathogenesis.
Topics: Humans; Extracellular Traps; Hidradenitis Suppurativa; Deoxyribonucleases; Deoxyribonuclease I; Autoantibodies
PubMed: 35934056
DOI: 10.1016/j.jid.2022.06.024 -
Journal of Investigative Surgery : the... Dec 2023Hepatic ischemia-reperfusion injury (IRI) is a common innate immune-mediated sterile inflammatory response in liver transplantation and liver tumor resection. Neutrophil...
BACKGROUND
Hepatic ischemia-reperfusion injury (IRI) is a common innate immune-mediated sterile inflammatory response in liver transplantation and liver tumor resection. Neutrophil extracellular traps (NETs) can aggravate liver injury and activates innate immune response in the process of liver IRI. However, Curcumin (Cur) can reverse this damage and reduce NETs formation. Nevertheless, the specific regulatory mechanism is still unclear in liver IRI. This study aimed to explore the potential mechanisms that how does Cur alleviate hepatic IRI by inhibits NETs production and develop novel treatment regimens.
METHODS
We established a hepatic IRI model by subjecting C57BL/6J mice to 60 min of ischemia, followed by reperfusion for 2 h, 6 h, 12 h, and 24 h respectively. Subsequently, we were separated into 5 groups, namely the I/R group, Cur group, DNase-1 group, Cur + DNase1 group and sham operation group. Serum alanine aminotransferase (ALT) and aspartate transaminase (AST), Hematoxylin-eosin staining, immunofluorescence, and TUNEL analysis were applied to assess liver injury degree and NETs levels. Western blot assay was used to detect the protein levels of apoptosis-related proteins and MEK pathway proteins.
RESULTS
Cur could alleviate hepatic IRI by inhibiting the generation of NETs via suppressing the MEK/ERK pathway. In addition, this study also revealed that DNase-1 is vital for alleviating hepatic IRI by reducing the generation of NETs.
CONCLUSIONS
Cur combined with DNase-1 was more effective than the two drugs administered alone in alleviating hepatic IRI by inhibiting the generation of NETs. These results also suggested that curcumin combined with DNase-1 was a potential therapeutic strategy to mitigate hepatic IRI.
Topics: Mice; Animals; Curcumin; Extracellular Traps; Mice, Inbred C57BL; Liver; Reperfusion Injury; Inflammation; Deoxyribonucleases; Mitogen-Activated Protein Kinase Kinases
PubMed: 36603844
DOI: 10.1080/08941939.2022.2164813 -
Scientific Reports Jun 2022The human genome contains four DNase1 and two DNase2 genes. The origin and functional specialization of this repertoire are not fully understood. Here we use genomics...
The human genome contains four DNase1 and two DNase2 genes. The origin and functional specialization of this repertoire are not fully understood. Here we use genomics and transcriptomics data to infer the evolutionary history of DNases and investigate their biological significance. Both DNase1 and DNase2 families have expanded in vertebrates since ~ 650 million years ago before the divergence of jawless and jawed vertebrates. DNase1, DNase1L1, and DNase1L3 co-existed in jawless fish, whereas DNase1L2 originated in amniotes by tandem duplication of DNase1. Among the non-human DNases, DNase1L4 and newly identified DNase1L5 derived from early duplications that were lost in terrestrial vertebrates. The ancestral gene of the DNase2 family, DNase2b, has been conserved in synteny with the Uox gene across 700 million years of animal evolution,while DNase2 originated in jawless fish. DNase1L1 acquired a GPI-anchor for plasma membrane attachment in bony fishes, and DNase1L3 acquired a C-terminal basic peptide for the degradation of microparticle DNA in jawed vertebrates. The appearance of DNase1L2, with a distinct low pH optimum and skin localization, is among the amniote adaptations to life on land. The expansion of the DNase repertoire in vertebrates meets the diversified demand for DNA debris removal in complex multicellular organisms.
Topics: Animals; DNA; Deoxyribonuclease I; Deoxyribonucleases; Evolution, Molecular; Fishes; Gene Duplication; Humans; Phylogeny; Synteny; Vertebrates
PubMed: 35725583
DOI: 10.1038/s41598-022-14133-w -
Nucleic Acids Research Jul 2014This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss 'Type II'... (Review)
Review
This article continues the series of Surveys and Summaries on restriction endonucleases (REases) begun this year in Nucleic Acids Research. Here we discuss 'Type II' REases, the kind used for DNA analysis and cloning. We focus on their biochemistry: what they are, what they do, and how they do it. Type II REases are produced by prokaryotes to combat bacteriophages. With extreme accuracy, each recognizes a particular sequence in double-stranded DNA and cleaves at a fixed position within or nearby. The discoveries of these enzymes in the 1970s, and of the uses to which they could be put, have since impacted every corner of the life sciences. They became the enabling tools of molecular biology, genetics and biotechnology, and made analysis at the most fundamental levels routine. Hundreds of different REases have been discovered and are available commercially. Their genes have been cloned, sequenced and overexpressed. Most have been characterized to some extent, but few have been studied in depth. Here, we describe the original discoveries in this field, and the properties of the first Type II REases investigated. We discuss the mechanisms of sequence recognition and catalysis, and the varied oligomeric modes in which Type II REases act. We describe the surprising heterogeneity revealed by comparisons of their sequences and structures.
Topics: DNA; Deoxyribonucleases, Type II Site-Specific; Evolution, Molecular; History, 20th Century; History, 21st Century; Protein Engineering; Restriction Mapping
PubMed: 24878924
DOI: 10.1093/nar/gku447