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Frontiers in Cardiovascular Medicine 2022Diabetic cardiomyopathy (DCM), characterized by cardiomyopathy with the absence of coronary artery disease, hypertension, and valvular heart disease in patients with...
OBJECTIVE
Diabetic cardiomyopathy (DCM), characterized by cardiomyopathy with the absence of coronary artery disease, hypertension, and valvular heart disease in patients with diabetes, significantly increases the risk of heart failure. Galectin-3 (Gal-3) has been shown to regulate cardiac inflammation and fibrosis, but its role in DCM remains unclear. This study aimed to determine whether Gal-3 inhibition attenuates DCM and NF-κB p65 activation.
METHODS
Diabetic cardiomyopathy (DCM) was established by intraperitoneal (IP) injection of streptozotocin for 5 consecutive days in mice. Myocardial injury markers, such as creatine kinase isoenzyme (CK-BM) and lactate dehydrogenase, were detected using ELISA. We used non-invasive transthoracic echocardiography to examine cardiac structure and function. Histological staining was used to explore myocardial morphology and fibrosis. Profibrotic markers and inflammatory cytokines were detected by ELISA and real-time PCR . The terminal deoxyribonucleotide transferasemediated dUTP nick end-labeling (TUNEL) and immunofluorescence assays were conducted to examine myocardial apoptosis and oxidative stress. Inflammatory cytokines induced by high glucose (HG) were also found in RAW264.7 macrophages. The underlying molecular mechanisms were determined using immunofluorescence and Western blotting analyses.
RESULTS
The Gal-3 knockdown was observed to ameliorate myocardial apoptosis, oxidative stress, inflammatory cytokines release, macrophage infiltration, and fibrosis, thus, decreasing cardiac dysfunction in DCM mice. In addition, the silence of Gal-3 could suppress macrophage infiltration and inflammatory cytokine release induced by HG. Finally, a Gal-3/NF-κB p65 regulatory network was clarified in the pathogenesis of DCM.
CONCLUSION
The Gal-3 may promote myocardial apoptosis, oxidative stress, inflammation, and fibrosis and by the mechanism of reduction of NF-κB p65 activation.
PubMed: 35557520
DOI: 10.3389/fcvm.2022.868372 -
Cell Reports Jul 2023The protein kinase ATR is essential for replication stress responses in all eukaryotes. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotide...
The protein kinase ATR is essential for replication stress responses in all eukaryotes. Ribonucleotide reductase (RNR) catalyzes the formation of deoxyribonucleotide (dNTP), the universal building block for DNA replication and repair. However, the relationship between ATR and RNR is not well understood. Here, we show that ATR promotes the protein stability of RNR in Arabidopsis. Through an activation tagging-based genetic screen, we found that overexpression of TSO2, a small subunit of RNR, partially suppresses the hypersensitivity of the atr mutant to replication stress. Biochemically, TSO2 interacts with PRL1, a central subunit of the Cullin4-based E3 ubiquitin ligase CRL4, which polyubiquitinates TSO2 and promotes its degradation. ATR inhibits CRL4 to attenuate TSO2 degradation. Our work provides an important insight into the replication stress responses and a post-translational regulatory mechanism for RNR. Given the evolutionary conservation of the proteins involved, the ATR-PRL1-RNR module may act across eukaryotes.
Topics: Arabidopsis; Arabidopsis Proteins; Ataxia Telangiectasia Mutated Proteins; DNA Damage; DNA Replication; Ribonucleotide Reductases; Ubiquitin-Protein Ligases
PubMed: 37354461
DOI: 10.1016/j.celrep.2023.112685 -
Yi Chuan = Hereditas Feb 2022As an important precursor for DNA synthesis, the four deoxyribonucleoside triphosphates (dATP, dTTP, dGTP, and dCTP) are necessary raw materials for DNA replication,...
As an important precursor for DNA synthesis, the four deoxyribonucleoside triphosphates (dATP, dTTP, dGTP, and dCTP) are necessary raw materials for DNA replication, recombination, and repair in cells. The correct synthesis and integrity of DNA are important manifestations of the genome stability, so the stability of the dNTP library state is essential to maintain the stability of the genome and the cell. In terms of the quality of the dNTP library, the incorporation of some heterogeneous dNTPs, such as oxidized dNTPs, into DNA can easily cause base substitutions and even DNA breaks and rearrangements, which will greatly damage the stability of the genome. At the same time, the cell has also evolved the corresponding NTP pyrophosphatase to remove it, and to correct the damaged DNA and repair the DNA gap by forming a DNA damage repair network. In terms of the number of dNTP libraries, the imbalance of the dNTP concentration and ratio will also cause base and frameshift mutations, which will also cause genome instability. As a result, cells have evolved a huge enzyme-controlled network to carry them out under precise control. This article mainly reviews the potential harm of damage to dNTP library components in cells, the clearance of damaged dNTPs, the regulation on the balance between dNTP library components, and finally discusses clinical diseases related to dNTP library homeostasis. It provides insights on the research of the correlation between the stability of the cellular dNTP library and the genome, and finally provides some theoretical basis for the treatment of related diseases.
Topics: DNA Replication; Deoxyribonucleotides; Genome; Genomic Instability; Homeostasis; Humans
PubMed: 35210212
DOI: 10.16288/j.yczz.21-211 -
Pharmacology Research & Perspectives Dec 2020SARS-CoV-2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and...
SARS-CoV-2, a member of the coronavirus family, has caused a global public health emergency. Based on our analysis of hepatitis C virus and coronavirus replication, and the molecular structures and activities of viral inhibitors, we previously reasoned that the FDA-approved hepatitis C drug EPCLUSA (Sofosbuvir/Velpatasvir) should inhibit coronaviruses, including SARS-CoV-2. Here, using model polymerase extension experiments, we demonstrate that the active triphosphate form of Sofosbuvir is incorporated by low-fidelity polymerases and SARS-CoV RNA-dependent RNA polymerase (RdRp), and blocks further incorporation by these polymerases; the active triphosphate form of Sofosbuvir is not incorporated by a host-like high-fidelity DNA polymerase. Using the same molecular insight, we selected 3'-fluoro-3'-deoxythymidine triphosphate and 3'-azido-3'-deoxythymidine triphosphate, which are the active forms of two other anti-viral agents, Alovudine and AZT (an FDA-approved HIV/AIDS drug) for evaluation as inhibitors of SARS-CoV RdRp. We demonstrate the ability of two of these HIV reverse transcriptase inhibitors to be incorporated by SARS-CoV RdRp where they also terminate further polymerase extension. Given the 98% amino acid similarity of the SARS-CoV and SARS-CoV-2 RdRps, we expect these nucleotide analogues would also inhibit the SARS-CoV-2 polymerase. These results offer guidance to further modify these nucleotide analogues to generate more potent broad-spectrum anti-coronavirus agents.
Topics: Antiviral Agents; Betacoronavirus; COVID-19; Carbamates; Coronavirus Infections; Dideoxynucleotides; Drug Combinations; Heterocyclic Compounds, 4 or More Rings; Humans; Pandemics; Pneumonia, Viral; RNA-Dependent RNA Polymerase; SARS-CoV-2; Sofosbuvir; Thymine Nucleotides; Zidovudine
PubMed: 33124786
DOI: 10.1002/prp2.674 -
Database : the Journal of Biological... Nov 2021Protein domains are functional and structural units of proteins. They are responsible for a particular function that contributes to protein's overall role. Because of...
Protein domains are functional and structural units of proteins. They are responsible for a particular function that contributes to protein's overall role. Because of this essential role, the majority of the genetic variants occur in the domains. In this study, the somatic mutations across 21 cancer types were mapped to the individual protein domains. To map the mutations to the domains, we employed the whole human proteome to predict the domains in each protein sequence and recognized about 149 668 domains. A novel Perl-API program was developed to convert the protein domain positions into genomic positions, and users can freely access them through GitHub. We determined the distribution of protein domains across 23 chromosomes with the help of these genomic positions. Interestingly, chromosome 19 has more number of protein domains in comparison with other chromosomes. Then, we mapped the cancer mutations to all the protein domains. Around 46-65% of mutations were mapped to their corresponding protein domains, and significantly mutated domains for all the cancer types were determined using the local false discovery ratio (locfdr). The chromosome positions for all the protein domains can be verified using the cross-reference ensemble database. Database URL: https://dcmp.vit.ac.in/.
Topics: Deoxycytidine Monophosphate; Humans; Mutant Proteins; Neoplasms; Protein Domains; Proteome
PubMed: 34791106
DOI: 10.1093/database/baab066 -
Cancer Research Communications Aug 2023Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the synthesis of deoxyribonucleosides and is required for DNA replication. Multiple types of cancer,...
UNLABELLED
Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the synthesis of deoxyribonucleosides and is required for DNA replication. Multiple types of cancer, including Ewing sarcoma tumors, are sensitive to RNR inhibitors or a reduction in the levels of either the RRM1 or RRM2 subunits of RNR. However, the polypharmacology and off-target effects of RNR inhibitors have complicated the identification of the mechanisms that regulate sensitivity and resistance to this class of drugs. Consequently, we used a conditional knockout (CRISPR/Cas9) and rescue approach to target RRM1 in Ewing sarcoma cells and identified that loss of the RRM1 protein results in the upregulation of the expression of multiple members of the activator protein-1 (AP-1) transcription factor complex, including c-Jun and c-Fos, and downregulation of c-Myc. Notably, overexpression of c-Jun and c-Fos in Ewing sarcoma cells is sufficient to inhibit cell growth and downregulate the expression of the c-Myc oncogene. We also identified that the upregulation of AP-1 is mediated, in part, by SLFN11, which is a replication stress response protein that is expressed at high levels in Ewing sarcoma. In addition, small-molecule inhibitors of RNR, including gemcitabine, and histone deacetylase inhibitors, which reduce the level of the RRM1 protein, also activate AP-1 signaling and downregulate the level of c-Myc in Ewing sarcoma. Overall, these results provide novel insight into the critical pathways activated by loss of RNR activity and the mechanisms of action of inhibitors of RNR.
SIGNIFICANCE
RNR is the rate-limiting enzyme in the synthesis of deoxyribonucleotides. Although RNR is the target of multiple chemotherapy drugs, polypharmacology and off-target effects have complicated the identification of the precise mechanism of action of these drugs. In this work, using a knockout-rescue approach, we identified that inhibition of RNR upregulates AP-1 signaling and downregulates the level of c-Myc in Ewing sarcoma tumors.
Topics: Humans; Sarcoma, Ewing; Transcription Factor AP-1; Neuroectodermal Tumors, Primitive, Peripheral; Signal Transduction; Proto-Oncogene Proteins c-fos; Ribonucleotide Reductases; Craniocerebral Trauma; DNA Replication; Nuclear Proteins
PubMed: 37599787
DOI: 10.1158/2767-9764.CRC-23-0268 -
The ISME Journal Jul 2023Phages are prevalent in diverse environments and play major ecological roles attributed to their tremendous diversity and abundance. Among these viruses, transposable...
Phages are prevalent in diverse environments and play major ecological roles attributed to their tremendous diversity and abundance. Among these viruses, transposable phages (TBPs) are exceptional in terms of their unique lifestyle, especially their replicative transposition. Although several TBPs have been isolated and the life cycle of the representative phage Mu has been extensively studied, the diversity distribution and ecological functions of TBPs on the global scale remain unknown. Here, by mining TBPs from enormous microbial genomes and viromes, we established a TBP genome dataset (TBPGD), that expands the number of accessible TBP genomes 384-fold. TBPs are prevalent in diverse biomes and show great genetic diversity. Based on taxonomic evaluations, we propose the categorization of TBPs into four viral groups, including 11 candidate subfamilies. TBPs infect multiple bacterial phyla, and seem to infect a wider range of hosts than non-TBPs. Diverse auxiliary metabolic genes (AMGs) are identified in the TBP genomes, and genes related to glycoside hydrolases and pyrimidine deoxyribonucleotide biosynthesis are highly enriched. Finally, the influences of TBPs on their hosts are experimentally examined by using the marine bacterium Shewanella psychrophila WP2 and its infecting transposable phage SP2. Collectively, our findings greatly expand the genetic diversity of TBPs, and comprehensively reveal their potential influences in various ecosystems.
Topics: Bacteriophages; Ecosystem; Genome, Viral; Viruses; Bacteria
PubMed: 37069234
DOI: 10.1038/s41396-023-01414-z -
Sub-cellular Biochemistry 2022Herein we present a multidisciplinary discussion of ribonucleotide reductase (RNR), the essential enzyme uniquely responsible for conversion of ribonucleotides to... (Review)
Review
Herein we present a multidisciplinary discussion of ribonucleotide reductase (RNR), the essential enzyme uniquely responsible for conversion of ribonucleotides to deoxyribonucleotides. This chapter primarily presents an overview of this multifaceted and complex enzyme, covering RNR's role in enzymology, biochemistry, medicinal chemistry, and cell biology. It further focuses on RNR from mammals, whose interesting and often conflicting roles in health and disease are coming more into focus. We present pitfalls that we think have not always been dealt with by researchers in each area and further seek to unite some of the field-specific observations surrounding this enzyme. Our work is thus not intended to cover any one topic in extreme detail, but rather give what we consider to be the necessary broad grounding to understand this critical enzyme holistically. Although this is an approach we have advocated in many different areas of scientific research, there is arguably no other single enzyme that embodies the need for such broad study than RNR. Thus, we submit that RNR itself is a paradigm of interdisciplinary research that is of interest from the perspective of the generalist and the specialist alike. We hope that the discussions herein will thus be helpful to not only those wanting to tackle RNR-specific problems, but also those working on similar interdisciplinary projects centering around other enzymes.
Topics: Animals; Deoxyribonucleotides; Mammals; Oxidoreductases; Ribonucleotide Reductases; Ribonucleotides
PubMed: 36151376
DOI: 10.1007/978-3-031-00793-4_5 -
Chembiochem : a European Journal of... Jun 2020Synthetic mRNAs are promising candidates for a new class of transformative drugs that provide genetic information for patients' cells to develop their own cure. One key...
Synthetic mRNAs are promising candidates for a new class of transformative drugs that provide genetic information for patients' cells to develop their own cure. One key advancement to develop so-called druggable mRNAs was the preparation of chemically modified mRNAs, by replacing standard bases with modified bases, such as uridine with pseudouridine, which can ameliorate the immunogenic profile and translation efficiency of the mRNA. Thus the introduction of modified nucleobases was the foundation for the clinical use of such mRNAs. Herein we describe modular and simple methods to chemoenzymatically modify mRNA. Alkyne- and/or azide-modified nucleotides are enzymatically incorporated into mRNA and subsequently conjugated to fluorescent dyes using click chemistry. This allows visualization of the labeled mRNA inside cells. mRNA coding for the enhanced green fluorescent protein (eGFP) was chosen as a model system and the successful expression of eGFP demonstrated that our modified mRNA is accepted by the translation machinery.
Topics: Azides; Cell-Free System; Click Chemistry; DNA; DNA-Directed RNA Polymerases; Deoxyuracil Nucleotides; Deoxyuridine; Dideoxyadenosine; Green Fluorescent Proteins; Humans; Protein Biosynthesis; Pseudouridine; RNA, Messenger; Transcription, Genetic; Uridine; Viral Proteins
PubMed: 31943671
DOI: 10.1002/cbic.201900718 -
ELife Feb 2022Antibiotic-resistant ) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active...
Antibiotic-resistant ) are an emerging public health threat due to increasing numbers of multidrug resistant (MDR) organisms. We identified two novel orally active inhibitors, PTC-847 and PTC-672, that exhibit a narrow spectrum of activity against including MDR isolates. By selecting organisms resistant to the novel inhibitors and sequencing their genomes, we identified a new therapeutic target, the class Ia ribonucleotide reductase (RNR). Resistance mutations in map to the N-terminal cone domain of the α subunit, which we show here is involved in forming an inhibited αβ state in the presence of the β subunit and allosteric effector dATP. Enzyme assays confirm that PTC-847 and PTC-672 inhibit RNR and reveal that allosteric effector dATP potentiates the inhibitory effect. Oral administration of PTC-672 reduces infection in a mouse model and may have therapeutic potential for treatment of that is resistant to current drugs.
Topics: Allosteric Regulation; Animals; Anti-Bacterial Agents; Deoxyadenine Nucleotides; Disease Models, Animal; Drug Resistance, Bacterial; Escherichia coli; Female; Gonorrhea; Humans; Mice; Mice, Inbred BALB C; Microbial Sensitivity Tests; Neisseria gonorrhoeae; Pyridines; Ribonucleotide Reductases
PubMed: 35137690
DOI: 10.7554/eLife.67447