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MSystems Apr 2023Ribonucleotide reductases (RNRs) are key enzymes which catalyze the synthesis of deoxyribonucleotides, the monomers needed for DNA replication and repair. RNRs are...
Ribonucleotide reductases (RNRs) are key enzymes which catalyze the synthesis of deoxyribonucleotides, the monomers needed for DNA replication and repair. RNRs are classified into three classes (I, II, and III) depending on their overall structure and metal cofactors. Pseudomonas aeruginosa is an opportunistic pathogen which harbors all three RNR classes, increasing its metabolic versatility. During an infection, P. aeruginosa can form a biofilm to be protected from host immune defenses, such as the production of reactive oxygen species by macrophages. One of the essential transcription factors needed to regulate biofilm growth and other important metabolic pathways is AlgR. AlgR is part of a two-component system with FimS, a kinase that catalyzes its phosphorylation in response to external signals. Additionally, AlgR is part of the regulatory network of cell RNR regulation. In this study, we investigated the regulation of RNRs through AlgR under oxidative stress conditions. We determined that the nonphosphorylated form of AlgR is responsible for class I and II RNR induction after an HO addition in planktonic culture and during flow biofilm growth. We observed similar RNR induction patterns upon comparing the P. aeruginosa laboratory strain PAO1 with different P. aeruginosa clinical isolates. Finally, we showed that during Galleria mellonella infection, when oxidative stress is high, AlgR is crucial for transcriptional induction of a class II RNR gene (). Therefore, we show that the nonphosphorylated form of AlgR, in addition to being crucial for infection chronicity, regulates the RNR network in response to oxidative stress during infection and biofilm formation. The emergence of multidrug-resistant bacteria is a serious problem worldwide. Pseudomonas aeruginosa is a pathogen that causes severe infections because it can form a biofilm that protects it from immune system mechanisms such as the production of oxidative stress. Ribonucleotide reductases are essential enzymes which synthesize deoxyribonucleotides used in the replication of DNA. RNRs are classified into three classes (I, II, and III), and P. aeruginosa harbors all three of these classes, increasing its metabolic versatility. Transcription factors, such as AlgR, regulate the expression of RNRs. AlgR is involved in the RNR regulation network and regulates biofilm growth and other metabolic pathways. We determined that AlgR induces class I and II RNRs after an HO addition in planktonic culture and biofilm growth. Additionally, we showed that a class II RNR is essential during Galleria mellonella infection and that AlgR regulates its induction. Class II RNRs could be considered excellent antibacterial targets to be explored to combat P. aeruginosa infections.
Topics: Pseudomonas aeruginosa; Hydrogen Peroxide; Oxidative Stress; Reactive Oxygen Species; Deoxyribonucleotides
PubMed: 36794960
DOI: 10.1128/msystems.01005-22 -
Journal of the American Chemical Society Jul 2019Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base...
Previously, we reported the creation of a semi-synthetic organism (SSO) that stores and retrieves increased information by virtue of stably maintaining an unnatural base pair (UBP) in its DNA, transcribing the corresponding unnatural nucleotides into the codons and anticodons of mRNAs and tRNAs, and then using them to produce proteins containing noncanonical amino acids (ncAAs). Here we report a systematic extension of the effort to optimize the SSO by exploring a variety of deoxy- and ribonucleotide analogues. Importantly, this includes the first in vivo structure-activity relationship (SAR) analysis of unnatural ribonucleoside triphosphates. Similarities and differences between how DNA and RNA polymerases recognize the unnatural nucleotides were observed, and remarkably, we found that a wide variety of unnatural ribonucleotides can be efficiently transcribed into RNA and then productively and selectively paired at the ribosome to mediate the synthesis of proteins with ncAAs. The results extend previous studies, demonstrating that nucleotides bearing no significant structural or functional homology to the natural nucleotides can be efficiently and selectively paired during replication, to include each step of the entire process of information storage and retrieval. From a practical perspective, the results identify the most optimal UBP for replication and transcription, as well as the most optimal unnatural ribonucleoside triphosphates for transcription and translation. The optimized SSO is now, for the first time, able to efficiently produce proteins containing multiple, proximal ncAAs.
Topics: Base Pairing; Deoxyribonucleotides; Genetic Code; Nucleotides; Protein Biosynthesis; Synthetic Biology; Transcription, Genetic
PubMed: 31241334
DOI: 10.1021/jacs.9b02075 -
Anti-cancer Drugs Aug 2022Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is an important mitochondrial protein, while its function in endometrial cancer remains unknown. This study...
Leucine zipper/EF hand-containing transmembrane-1 (LETM1) is an important mitochondrial protein, while its function in endometrial cancer remains unknown. This study aimed to explore the function of LETM1 in endometrial cancer and reveal the underlying mechanisms involving carboxy-terminal modulator protein (CTMP). Immunohistochemistry was performed to detect the expression of LETM1 and CTMP in normal, atypical hyperplastic and endometrial cancer endometrial tissues. LETM1 and CTMP were silenced in two endometrial cancer cell lines (ISK and KLE), which were verified by western blot. Cell viability, colony number, migration and invasion were detected by cell counting kit-8, colony formation, wound healing and trans-well assays, respectively. A xenograft mouse model was established to determine the antitumor potential of LETM1/CTMP silencing in vivo . In addition, CTMP was overexpressed to evaluate its regulatory relationship with LETM1 in endometrial cancer cells. The expression of LETM1 and CTMP proteins were higher in endometrial cancer tissues than atypical hyperplastic tissues and were higher in atypical hyperplastic tissues than normal tissues. LETM1 and CTMP were also upregulated in ISK and KLE cells. Silencing of LETM1 or CTMP could decrease the viability, colony number, migration and invasion of endometrial cancer cells and the weight and volume of tumor xenografts. In addition, CTMP was downregulated by LETM1 silencing in KLE cells, and its overexpression enhanced the malignant characteristics of si-LETM1-transfected KLE cells. Silencing of LETM1 inhibits the malignant progression of endometrial cancer through downregulating CTMP.
Topics: Animals; Calcium-Binding Proteins; Carrier Proteins; Cell Line, Tumor; Endometrial Neoplasms; Female; Humans; Membrane Proteins; Mice; Mitochondrial Proteins; Nucleotides, Cyclic; Palmitoyl-CoA Hydrolase; Thymidine Monophosphate
PubMed: 35324530
DOI: 10.1097/CAD.0000000000001301 -
Cells May 2022Diseases that affect the mitochondrial electron transport chain (ETC) often manifest as threshold effect disorders, meaning patients only become symptomatic once a...
Diseases that affect the mitochondrial electron transport chain (ETC) often manifest as threshold effect disorders, meaning patients only become symptomatic once a certain level of ETC dysfunction is reached. Cells can invoke mechanisms to circumvent reaching their critical ETC threshold, but it is an ongoing challenge to identify such processes. In the nematode , severe reduction of mitochondrial ETC activity shortens life, but mild reduction actually extends it, providing an opportunity to identify threshold circumvention mechanisms. Here, we show that removal of ATL-1, but not ATM-1, worm orthologs of ATR and ATM, respectively, key nuclear DNA damage checkpoint proteins in human cells, unexpectedly lessens the severity of ETC dysfunction. Multiple genetic and biochemical tests show no evidence for increased mutation or DNA breakage in animals exposed to ETC disruption. Reduced ETC function instead alters nucleotide ratios within both the ribo- and deoxyribo-nucleotide pools, and causes stalling of RNA polymerase, which is also known to activate ATR. Unexpectedly, mutants confronted with mitochondrial ETC disruption maintain normal levels of oxygen consumption, and have an increased abundance of translating ribosomes. This suggests checkpoint signaling by ATL-1 normally dampens cytoplasmic translation. Taken together, our data suggest a model whereby ETC insufficiency in results in nucleotide imbalances leading to the stalling of RNA polymerase, activation of ATL-1, dampening of global translation, and magnification of ETC dysfunction. The loss of ATL-1 effectively reverses the severity of ETC disruption so that animals become phenotypically closer to wild type.
Topics: Animals; Ataxia Telangiectasia Mutated Proteins; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Cell Respiration; Mitochondria; Nuclear Proteins; Nucleotides
PubMed: 35681427
DOI: 10.3390/cells11111731 -
Biochemistry Aug 2023Ras proteins in the mitogen-activated protein kinase (MAPK) signaling pathway represent one of the most frequently mutated oncogenes in cancer. Ras binds guanosine...
Ras proteins in the mitogen-activated protein kinase (MAPK) signaling pathway represent one of the most frequently mutated oncogenes in cancer. Ras binds guanosine nucleotides and cycles between active (GTP) and inactive (GDP) conformations to regulate the MAPK signaling pathway. Guanosine and other nucleotides exist in cells as either 2'-hydroxy or 2'-deoxy forms, and imbalances in the deoxyribonucleotide triphosphate pool have been associated with different diseases, such as diabetes, obesity, and cancer. However, the biochemical properties of Ras bound to dGNP are not well understood. Herein, we use native mass spectrometry to monitor the intrinsic GTPase activity of H-Ras and N-Ras oncogenic mutants, revealing that the rate of 2'-deoxy guanosine triphosphate (dGTP) hydrolysis differs compared to the hydroxylated form, in some cases by seven-fold. Moreover, K-Ras expressed from HEK293 cells exhibited a higher than anticipated abundance of dGNP, despite the low abundance of dGNP in cells. Additionally, the GTPase and dGTPase activity of K-Ras was found to be accelerated by 10.2- and 3.8-fold in the presence of small molecule covalent inhibitors, which may open opportunities for the development of Pan-Ras inhibitors. The molecular assemblies formed between H-Ras and N-Ras, including mutant forms, with the catalytic domain of SOS (SOS) were also investigated. The results show that the different mutants of H-Ras and N-Ras not only engage SOS differently, but these assemblies are also dependent on the form of guanosine triphosphate bound to Ras. These findings bring to the forefront a new perspective on the nucleotide-dependent biochemical properties of Ras that may have implications for the activation of the MAPK signaling pathway and Ras-driven cancers.
Topics: Humans; HEK293 Cells; ras Proteins; Guanosine Triphosphate; Hydrolysis; Proto-Oncogene Proteins p21(ras); GTP Phosphohydrolases; Deoxyguanine Nucleotides
PubMed: 37487239
DOI: 10.1021/acs.biochem.3c00258 -
Journal of Bacteriology Oct 2023Ribonucleotides frequently contaminate DNA and, if not removed, cause genomic instability. Consequently, all organisms are equipped with RNase H enzymes to remove...
Ribonucleotides frequently contaminate DNA and, if not removed, cause genomic instability. Consequently, all organisms are equipped with RNase H enzymes to remove RNA-DNA hybrids (RDHs). lacking RNase HI () and RNase HII () enzymes, the ∆ ∆ double mutant, accumulates RDHs in its DNA. These RDHs can convert into RNA-containing DNA lesions (R-lesions) of unclear nature that compromise genomic stability. The ∆ double mutant has severe phenotypes, like growth inhibition, replication stress, sensitivity to ultraviolet radiation, SOS induction, increased chromosomal fragmentation, and defects in nucleoid organization. In this study, we found that RNase HI deficiency also alters wild-type levels of DNA supercoiling. Despite these severe chromosomal complications, ∆ double mutant survives, suggesting that dedicated pathways operate to avoid or repair R-lesions. To identify these pathways, we systematically searched for mutants synthetic lethal (colethal) with the defect using an unbiased color screen and a candidate gene approach. We identified both novel and previously reported -colethal and -coinhibited mutants, characterized them, and sorted them into avoidance or repair pathways. These mutants operate in various parts of nucleic acid metabolism, including replication fork progression, R-loop prevention and removal, nucleoid organization, tRNA modification, recombinational repair, and chromosome-dimer resolution, demonstrating the pleiotropic nature of RNase H deficiency. IMPORTANCE Ribonucleotides (rNs) are structurally very similar to deoxyribonucleotides. Consequently, rN contamination of DNA is common and pervasive across all domains of life. Failure to remove rNs from DNA has severe consequences, and all organisms are equipped with RNase H enzymes to remove RNA-DNA hybrids. RNase H deficiency leads to complications in bacteria, yeast, and mouse, and diseases like progressive external ophthalmoplegia (mitochondrial defects in RNASEH1) and Aicardi-Goutières syndrome (defects in RNASEH2) in humans. mutant, deficient in RNases H, has severe chromosomal complications. Despite substantial problems, nearly half of the mutant population survives. We have identified novel and previously confirmed pathways in various parts of nucleic acid metabolism that ensure survival with RNase H deficiency.
Topics: Humans; Animals; Mice; Escherichia coli; Ultraviolet Rays; DNA; Genomic Instability; Ribonuclease H; RNA; Ribonucleotides
PubMed: 37819120
DOI: 10.1128/jb.00280-23 -
Redox Biology Jun 2021Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of...
Oxidatively generated damage to DNA has been implicated in the pathogenesis of a wide variety of diseases. Increasingly, interest is also focusing upon the effects of damage to the other nucleic acids, RNA and the (2'-deoxy-)ribonucleotide pools, and evidence is growing that these too may have an important role in disease. LC-MS/MS has the ability to provide absolute quantification of specific biomarkers, such as 8-oxo-7,8-dihydro-2'-deoxyGuo (8-oxodG), in both nuclear and mitochondrial DNA, and 8-oxoGuo in RNA. However, significant quantities of tissue are needed, limiting its use in human biomonitoring studies. In contrast, the comet assay requires much less material, and as little as 5 μL of blood may be used, offering a minimally invasive means of assessing oxidative stress in vivo, but this is restricted to nuclear DNA damage only. Urine is an ideal matrix in which to non-invasively study nucleic acid-derived biomarkers of oxidative stress, and considerable progress has been made towards robustly validating these measurements, not least through the efforts of the European Standards Committee on Urinary (DNA) Lesion Analysis. For urine, LC-MS/MS is considered the gold standard approach, and although there have been improvements to the ELISA methodology, this is largely limited to 8-oxodG. Emerging DNA adductomics approaches, which either comprehensively assess the totality of adducts in DNA, or map DNA damage across the nuclear and mitochondrial genomes, offer the potential to considerably advance our understanding of the mechanistic role of oxidatively damaged nucleic acids in disease.
Topics: 8-Hydroxy-2'-Deoxyguanosine; Biomarkers; Chromatography, Liquid; DNA Damage; Deoxyguanosine; Humans; Nucleic Acids; Oxidative Stress; Tandem Mass Spectrometry
PubMed: 33579665
DOI: 10.1016/j.redox.2021.101872 -
Biomedicine & Pharmacotherapy =... Apr 2023Elevated myocardial intracellular sodium ([Na]) was shown to decrease mitochondrial calcium ([Ca]) via mitochondrial sodium/calcium exchanger (NCX), resulting in...
BACKGROUND
Elevated myocardial intracellular sodium ([Na]) was shown to decrease mitochondrial calcium ([Ca]) via mitochondrial sodium/calcium exchanger (NCX), resulting in decreased mitochondrial ATP synthesis. The sodium-glucose co-transporter 2 inhibitor (SGLT2i) ertugliflozin (ERTU) improved energetic deficit and contractile dysfunction in a mouse model of high fat, high sucrose (HFHS) diet-induced diabetic cardiomyopathy (DCMP). As SGLT2is were shown to lower [Na] in isolated cardiomyocytes, we hypothesized that energetic improvement in DCMP is at least partially mediated by a decrease in abnormally elevated myocardial [Na].
METHODS
Forty-two eight-week-old male C57BL/6J mice were fed a control or HFHS diet for six months. In the last month, a subgroup of HFHS-fed mice was treated with ERTU. At the end of the study, left ventricular contractile function and energetics were measured simultaneously in isolated beating hearts by P NMR (Nuclear Magnetic Resonance) spectroscopy. A subset of untreated HFHS hearts was perfused with vehicle vs. CGP 37157, an NCX inhibitor. Myocardial [Na] was measured by Na NMR spectroscopy.
RESULTS
HFHS hearts showed diastolic dysfunction, decreased contractile reserve, and impaired energetics as reflected by decreased phosphocreatine (PCr) and PCr/ATP ratio. Myocardial [Na] was elevated > 2-fold in HFHS (vs. control diet). ERTU reversed the impairments in HFHS hearts to levels similar to or better than control diet and decreased myocardial [Na] to control levels. CGP 37157 normalized the PCr/ATP ratio in HFHS hearts.
CONCLUSIONS
Elevated myocardial [Na] contributes to mitochondrial and contractile dysfunction in DCMP. Targeting myocardial [Na] and/or NCX may be an effective strategy in DCMP and other forms of heart disease associated with elevated myocardial [Na].
Topics: Mice; Male; Animals; Diabetic Cardiomyopathies; Sodium-Glucose Transporter 2 Inhibitors; Sodium; Calcium; Deoxycytidine Monophosphate; Myocardial Contraction; Mice, Inbred C57BL; Myocardium; Adenosine Triphosphate; Diabetes Mellitus
PubMed: 36731341
DOI: 10.1016/j.biopha.2023.114310 -
Journal of Pharmaceutical Analysis Feb 2022Endogenous ribonucleotides (RNs) and deoxyribonucleotides (dRNs) are important metabolites related to the pathogenesis of many diseases. In light of their physiological...
Endogenous ribonucleotides (RNs) and deoxyribonucleotides (dRNs) are important metabolites related to the pathogenesis of many diseases. In light of their physiological and pathological significances, a novel and sensitive pre-column derivatization method with N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was developed to determine RNs and dRNs in human cells using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). A one-step extraction of cells with 85% methanol followed by a simple derivatization reaction within 5 min at room temperature contributed to shortened analysis time. The derivatives of 22 nucleoside mono-, di- and triphosphates were retained on the typical C column and eluted by ammonium acetate and acetonitrile in 9 min. Under these optimal conditions, good linearity was achieved in the tested calibration ranges. The lower limit of quantitation (LLOQ) was determined to be 0.1-0.4 μM for the tested RNs and 0.001-0.1 μM for dRNs. In addition, the precision (CV) was <15% and the RSD of stability was lower than 10.4%. Furthermore, this method was applied to quantify the endogenous nucleotides in human colorectal carcinoma cell lines HCT 116 exposed to 10-hydroxycamptothecin. In conclusion, our method has proven to be simple, rapid, sensitive, and reliable. It may be used for specific expanded studies on intracellular pharmacology in vitro.
PubMed: 35573880
DOI: 10.1016/j.jpha.2021.01.001 -
ACS Chemical Biology Sep 2021While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers,...
While alarmone nucleotides guanosine-3',5'-bisdiphosphate (ppGpp) and guanosine-5'-triphosphate-3'-diphosphate (pppGpp) are archetypical bacterial second messengers, their adenosine analogues ppApp (adenosine-3',5'-bisdiphosphate) and pppApp (adenosine-5'-triphosphate-3'-diphosphate) are toxic effectors that abrogate bacterial growth. The alarmones are both synthesized and degraded by the members of the RelA-SpoT Homologue (RSH) enzyme family. Because of the chemical and enzymatic liability of (p)ppGpp and (p)ppApp, these alarmones are prone to degradation during structural biology experiments. To overcome this limitation, we have established an efficient and straightforward procedure for synthesizing nonhydrolysable (p)ppNupp analogues starting from 3'-azido-3'-deoxyribonucleotides as key intermediates. To demonstrate the utility of (p)ppGpp as a molecular tool, we show that (i) as an HD substrate mimic, ppGpp competes with ppGpp to inhibit the enzymatic activity of human MESH1 Small Alarmone Hyrolase, SAH; and (ii) mimicking the allosteric effects of (p)ppGpp, (p)ppGpp acts as a positive regulator of the synthetase activity of long ribosome-associated RSHs Rel and RelA. Finally, by solving the structure of the N-terminal domain region (NTD) of Rel complexed with pppGpp, we show that as an HD substrate mimic, the analogue serves as a orthosteric regulator that promotes the same intra-NTD structural rearrangements as the native substrate.
Topics: Adenine Nucleotides; Allosteric Site; Bacillus subtilis; Bacterial Proteins; Deoxyribonucleotides; Escherichia coli; Gene Expression Regulation, Bacterial; Ligases; Protein Binding; Protein Conformation; Pyrophosphatases
PubMed: 34477366
DOI: 10.1021/acschembio.1c00398