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Biosensors Oct 2021The level of pyrophosphatase (PPase) expression has been suggested as a potential biomarker of various cancers, and its prognostic value has been evaluated in patients...
The level of pyrophosphatase (PPase) expression has been suggested as a potential biomarker of various cancers, and its prognostic value has been evaluated in patients suffering from lung cancer, colorectal cancer, and hyperthyroidism. However, the detection of PPase usually needs specific materials that require complicated, time-consuming reactions with restricted linear range and sensitivity, limiting their application in early clinical diagnosis. Herein, we developed a DNAzyme-based biosensor for the detection of PPase. In the presence of PPase, pyrophosphate (PPi) and Cu ions released from the PPi-Cu-PPi complex induce the cleavage of the DNAzyme and the corresponding substrate. An apurinic/apyrimidinic (AP) site was elaborately designed within substrates that could encase the fluorophore 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND). The fluorescence of ATMND was initially quenched but restored when the DNAzyme/substrate complex was hydrolyzed with the release of ATMND. In this way, the PPase activity can be estimated by detecting the increased fluorescence of the released ATMND. Under optimized conditions, the activity of PPase could be analyzed at concentrations from 0.5 to 1000 mU, with the lowest detectable concentration being 0.5 mU. This work lays a foundation for developing a DNAzyme-amplified fluorescent biosensor with a high sensitivity, a wide linear range, and single-step operation for use as an easy diagnostic for PPase analysis.
Topics: Biosensing Techniques; DNA, Catalytic; Fluorescent Dyes; Humans; Pyrophosphatases
PubMed: 34821638
DOI: 10.3390/bios11110422 -
ACS Omega Jun 2022Terpenoids form a large pool of highly diverse organic compounds possessing several economically important properties, including nutritional, aromatic, and...
Terpenoids form a large pool of highly diverse organic compounds possessing several economically important properties, including nutritional, aromatic, and pharmacological properties. The 1-deoxy-d-xylulose 5-phosphate (DXP) pathway's end enzyme, nuclear distribution protein (NudF), interacting with isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), is critical for the synthesis of isoprenol/prenol/downstream compounds. The enzyme is yet to be thoroughly investigated to increase the overall yield of terpenoids in the , which is widely used in industry and is generally regarded as a safe (GRAS) bacterium. The study aims to analyze the evolutionary conservation across the active site for mapping the key residues for mutagenesis studies. The 37-sequence data set, extracted from 103 entries, shows a high phylogenetic divergence, and only six one-motif sequences ASB92783.1, ASB69297.1, ASB56714.1, AOR97677.1, AOL97023.1, and OAZ71765.1 show a monophyly relationship, unlike a complete polyphyly relationship between the other 31 three-motif sequences. Furthermore, only 47 of 179 residues of the representative sequence CUB50584.1 are observed to be significantly conserved. Docking analysis suggests a preferential bias of adenosine diphosphate (ADP)-ribose pyrophosphatase toward IPP, and a nearly threefold energetic difference is observed between IPP and DMAPP. The loops are hereby shown to play a regulatory role in guiding the promiscuity of NudF toward a specific ligand. Computational saturation mutagenesis of the seven hotspot residues identifies two key positions LYS78 and PHE116, orderly encoded within loop1 and loop7, majorly interacting with the ligands DMAPP and IPP, and their mutants K78I/K78L and PHE116D/PHE116E are found to stabilize the overall conformation. Molecular dynamics analysis shows that the IPP complex is significantly more stable than the DMAPP complex, and the NudF structure is very unstable. Besides showing a promiscuous binding of NudF with ligands, the analysis suggests its rate-limiting nature. The study would allow us to customize the metabolic load toward the synthesis of any of the downstream molecules. The findings would pave the way for the development of catalytically improved NudF mutants for the large-scale production of specific terpenoids with significant nutraceutical or commercial value.
PubMed: 35721994
DOI: 10.1021/acsomega.2c01677 -
Journal of Fungi (Basel, Switzerland) Sep 2022Inositol pyrophosphates (IPPs) comprise a specific class of signaling molecules that regulate central biological processes in eukaryotes. The conserved Vip1/PPIP5K...
Inositol pyrophosphates (IPPs) comprise a specific class of signaling molecules that regulate central biological processes in eukaryotes. The conserved Vip1/PPIP5K family controls intracellular IP levels, the highest phosphorylated form of IPPs present in yeasts, as it has both inositol kinase and pyrophosphatase activities. Previous studies have shown that the fission yeast Vip1/PPIP5K family member Asp1 impacts chromosome transmission fidelity via the modulation of spindle function. We now demonstrate that an IP analogue is targeted by endogenous Asp1 and that cellular IP is subject to cell cycle control. Mitotic entry requires Asp1 kinase function and IP levels are increased at the G2/M transition. In addition, the kinetochore, the conductor of chromosome segregation that is assembled on chromosomes is modulated by IP. Members of the yeast CCAN kinetochore-subcomplex such as Mal2/CENP-O localize to the kinetochore depending on the intracellular IP-level: higher than wild-type IP levels reduce Mal2 kinetochore targeting, while a reduction in IP has the opposite effect. As our perturbations of the inositol polyphosphate and IPP pathways demonstrate that kinetochore architecture depends solely on IP and not on other IPPs, we conclude that chromosome transmission fidelity is controlled by IP via an interplay between entry into mitosis, kinetochore architecture, and spindle dynamics.
PubMed: 36135658
DOI: 10.3390/jof8090933 -
Cells Oct 2019Many facets of ribosome biogenesis and function, including ribosomal RNA (rRNA) transcription, 70S assembly and protein translation, are negatively impacted upon... (Review)
Review
Many facets of ribosome biogenesis and function, including ribosomal RNA (rRNA) transcription, 70S assembly and protein translation, are negatively impacted upon induction of a nutrient stress-sensing signalling pathway termed the stringent response. This stress response is mediated by the alarmones guanosine tetra- and penta-phosphate ((p)ppGpp), the accumulation of which leads to a massive cellular response that slows growth and aids survival. The 70S bacterial ribosome is an intricate structure, with assembly both complex and highly modular. Presiding over the assembly process is a group of P-loop GTPases within the TRAFAC (Translation Factor Association) superclass that are crucial for correct positioning of both early and late stage ribosomal proteins (r-proteins) onto the rRNA. Often described as 'molecular switches', members of this GTPase superfamily readily bind and hydrolyse GTP to GDP in a cyclic manner that alters the propensity of the GTPase to carry out a function. TRAFAC GTPases are considered to act as checkpoints to ribosome assembly, involved in binding to immature sections in the GTP-bound state, preventing further r-protein association until maturation is complete. Here we review our current understanding of the impact of the stringent response and (p)ppGpp production on ribosome maturation in prokaryotic cells, focusing on the inhibition of (p)ppGpp on GTPase-mediated subunit assembly, but also touching upon the inhibition of rRNA transcription and protein translation.
Topics: Amino Acid Sequence; Bacterial Proteins; GTP Phosphohydrolases; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Guanosine Triphosphate; Models, Molecular; Prokaryotic Cells; Protein Binding; Protein Biosynthesis; Pyrophosphatases; Ribosomal Proteins; Ribosomes
PubMed: 31653044
DOI: 10.3390/cells8111313 -
Mediators of Inflammation 2023Ulcerative colitis (UC) is an inflammatory bowel disease of unknown cause that typically affects the colon and rectum. Innate intestinal immunity, including macrophages,...
Ulcerative colitis (UC) is an inflammatory bowel disease of unknown cause that typically affects the colon and rectum. Innate intestinal immunity, including macrophages, plays a significant role in the pathological development of UC. Using the CIBERSORT algorithm, we observed elevated levels of 22 types of immune cell infiltrates, as well as increased M1 and decreased M2 macrophages in UC compared to normal colonic mucosa. Weighted gene coexpression network analysis (WGCNA) was used to identify modules associated with macrophages and UC, resulting in the identification of 52 macrophage-related genes (MRGs) that were enriched in macrophages at single-cell resolution. Consensus clustering based on these 52 MRGs divided the integrated UC cohorts into three subtypes. Machine learning algorithms were used to identify ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), sodium- and chloride-dependent neutral and basic amino acid transporter B(0+) (SLC6A14), and 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) in the training set, and their diagnostic value was validated in independent validation sets. Gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA) revealed the main biological effects, and that interleukin-17 was one of several signaling pathways enriched by the three genes. We also constructed a competitive endogenous RNA (CeRNA) network reflecting a potential posttranscriptional regulatory mechanism. Expression of diagnostic markers was validated in vivo and in biospecimens, and our immunohistochemistry (IHC) results confirmed that HMGCS2 gradually decreased during the transformation of UC to colorectal cancer. In conclusion, ENPP1, SLC6A14, and HMGCS2 are associated with macrophages and the progression of UC pathogenesis and have good diagnostic value for patients with UC.
Topics: Humans; Colitis, Ulcerative; Rectum; Macrophages; Intestinal Mucosa
PubMed: 38633005
DOI: 10.1155/2023/4373840 -
Scientific Reports Apr 2021Proton-translocating inorganic pyrophosphatases (H-PPases) are an ancient family of membrane bound enzymes that couple pyrophosphate (PPi) hydrolysis to H translocation...
Proton-translocating inorganic pyrophosphatases (H-PPases) are an ancient family of membrane bound enzymes that couple pyrophosphate (PPi) hydrolysis to H translocation across membranes. In this study, we conducted a molecular characterization of two isoenzymes (PdVP1 and PdVP2) located in respectively the alveolar sacs and in the membranes of the intracellular vacuoles of a scuticociliate parasite (Philasterides dicentrarchi) of farmed turbot. We analyzed the genetic expression of the isoenzymes after administration of antiparasitic drugs and after infection in the host. PdVP1 and PdVP2 are encoded by two genes of 2485 and 3069 bp, which respectively contain 3 and 11 exons and express proteins of 746 and 810 aa of molecular mass 78.9 and 87.6 kDa. Topological predictions from isoenzyme sequences indicate the formation of thirteen transmembrane regions (TMRs) for PdVP1 and seventeen TMRs for PdVP2. Protein structure modelling indicated that both isoenzymes are homodimeric, with three Mg binding sites and an additional K binding site in PdVP2. The levels of identity and similarity between the isoenzyme sequences are respectively 33.5 and 51.2%. The molecular weights of the native proteins are 158 kDa (PdVP1) and 178 kDa (PdVP2). The isoenzyme sequences are derived from paralogous genes that form a monophyletic grouping with other ciliate species. Genetic expression of the isoenzymes is closely related to the acidification of alveolar sacs (PdVP1) and intracellular vacuoles (PdVP2): antiparasitic drugs inhibit transcription, while infection increases transcription of both isoenzymes. The study findings show that P. dicentrarchi possesses two isoenzymes with H-PPase activity which are located in acidophilic cell compartment membranes and which are activated during infection in the host and are sensitive to antiparasitic drugs. The findings open the way to using molecular modelling to design drugs for the treatment of scuticociliatosis.
Topics: Amino Acid Sequence; Animals; Exons; Fish Diseases; Flatfishes; Gene Expression Regulation; Inorganic Pyrophosphatase; Isoenzymes; Parasites; Transcription, Genetic; Vacuoles
PubMed: 33875762
DOI: 10.1038/s41598-021-88102-0 -
Journal of Bone and Mineral Research :... Sep 2022Biallelic ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency induces vascular/soft tissue calcifications in generalized arterial calcification of...
Biallelic ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) deficiency induces vascular/soft tissue calcifications in generalized arterial calcification of infancy (GACI), and low bone mass with phosphate-wasting rickets in GACI survivors (autosomal hypophosphatemic rickets type-2). ENPP1 haploinsufficiency induces early-onset osteoporosis and mild phosphate wasting in adults. Both conditions demonstrate the unusual combination of reduced accrual of skeletal mineral, yet excess and progressive heterotopic mineralization. ENPP1 is the only enzyme that generates extracellular pyrophosphate (PPi), a potent inhibitor of both bone and heterotopic mineralization. Life-threatening vascular calcification in ENPP1 deficiency is due to decreased plasma PPi; however, the mechanism by which osteopenia results is not apparent from an understanding of the enzyme's catalytic activity. To probe for catalysis-independent ENPP1 pathways regulating bone, we developed a murine model uncoupling ENPP1 protein signaling from ENPP1 catalysis, Enpp1 mice. In contrast to Enpp1 mice, which lack ENPP1, Enpp1 mice have normal trabecular bone microarchitecture and favorable biomechanical properties. However, both models demonstrate low plasma Pi and PPi, increased fibroblast growth factor 23 (FGF23), and by 23 weeks, osteomalacia demonstrating equivalent phosphate wasting in both models. Reflecting findings in whole bone, calvarial cell cultures from Enpp1 mice demonstrated markedly decreased calcification, elevated transcription of Sfrp1, and decreased nuclear β-catenin signaling compared to wild-type (WT) and Enpp1 cultures. Finally, the decreased calcification and nuclear β-catenin signaling observed in Enpp1 cultures was restored to WT levels by knockout of Sfrp1. Collectively, our findings demonstrate that catalysis-independent ENPP1 signaling pathways regulate bone mass via the expression of soluble Wnt inhibitors such as secreted frizzled-related protein 1 (SFRP1), whereas catalysis dependent pathways regulate phosphate homeostasis through the regulation of plasma FGF23. © 2022 American Society for Bone and Mineral Research (ASBMR).
Topics: Animals; Bone and Bones; Catalysis; Familial Hypophosphatemic Rickets; Fibroblast Growth Factors; Mammals; Mice; Phosphates; Phosphoric Diester Hydrolases; Pyrophosphatases; Vascular Calcification; beta Catenin
PubMed: 35773783
DOI: 10.1002/jbmr.4640 -
Journal of Medicinal Food Sep 2020Higher bone mineral density (BMD) is often associated with greater consumption of black tea (BT). However, the dose-response of BT on mineralization in an osteoblast...
Higher bone mineral density (BMD) is often associated with greater consumption of black tea (BT). However, the dose-response of BT on mineralization in an osteoblast cell model has not yet been studied. The study objective was to determine the dose-dependent response of BT in Saos-2 cells and investigate changes to several proteins involved in the mineralization process. Mineralization was induced in the presence of BT at concentrations that represent levels likely achieved through daily consumption (0.1, 0.5, 0.75, 1 g gallic acid equivalents [GAE]/mL) or through supplementation (2, 5, or 10 g GAE/mL). BT exerted a positive dose-response on bone mineralization, peaking at 1 g GAE/mL of BT ( < .05). Cellular activity was significantly greater than control with exposure to 2-10 g GAE/mL of BT (at 24 h) ( < .05) and 1-10 g GAE/mL (at 48 h) ( < .05), with a peak at 5 g GAE/mL at 24 and 48 h ( < .05). Protein expression of alkaline phosphatase and ectonucleotide pyrophosphatase/phosphodiesterase-1 were unchanged, whereas a moderate dose of BT (0.75 g GAE/mL) resulted in greater expression of osteopontin compared with the highest dose (10 g GAE/mL) ( < .05). Doses of BT from 0.5 to 10 g GAE/mL resulted in higher antioxidant capacity compared with control ( < .05). In summary, the higher antioxidant capacity, enhanced cell viability, and upregulated mineralization suggest that consumption of BT may have a positive effect on BMD at levels obtained through consumption of tea.
Topics: Antioxidants; Calcification, Physiologic; Cell Line, Tumor; Dose-Response Relationship, Drug; Humans; Osteoblasts; Osteopontin; Tea
PubMed: 32208048
DOI: 10.1089/jmf.2020.0010 -
The New Phytologist Feb 2020Two types of tonoplast proton pumps, H -pyrophosphatase (V-PPase) and the H -ATPase (V-ATPase), establish the proton gradient that powers molecular traffic across the...
Two types of tonoplast proton pumps, H -pyrophosphatase (V-PPase) and the H -ATPase (V-ATPase), establish the proton gradient that powers molecular traffic across the tonoplast thereby facilitating turgor regulation and nutrient homeostasis. However, how proton pumps regulate development remains unclear. In this study, we investigated the function of two types of proton pumps in Arabidopsis embryo development and pattern formation. While disruption of either V-PPase or V-ATPase had no obvious effect on plant embryo development, knocking out both resulted in severe defects in embryo pattern formation from the early stage. While the first division in wild-type zygote was asymmetrical, a nearly symmetrical division occurred in the mutant, followed by abnormal pattern formation at all stages of embryo development. The embryonic defects were accompanied by dramatic differences in vacuole morphology and distribution, as well as disturbed localisation of PIN1. The development of mutant cotyledons and root, and the auxin response of mutant seedlings supported the hypothesis that mutants lacking tonoplast proton pumps were defective in auxin transport and distribution. Taking together, we proposed that two tonoplast proton pumps are required for vacuole morphology and PIN1 localisation, thereby controlling vacuole and auxin-related developmental processes in Arabidopsis embryos and seedlings.
Topics: Arabidopsis; Arabidopsis Proteins; Embryonic Development; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Gravitropism; Inorganic Pyrophosphatase; Membrane Transport Proteins; Mutation; Naphthols; Phthalimides; Plant Roots; Protein Transport; Proton-Translocating ATPases
PubMed: 31569267
DOI: 10.1111/nph.16231 -
MBio Feb 2022Inositol pyrophosphate (IPP) dynamics govern expression of the fission yeast phosphate homeostasis regulon via their effects on lncRNA-mediated transcription...
Inositol pyrophosphate (IPP) dynamics govern expression of the fission yeast phosphate homeostasis regulon via their effects on lncRNA-mediated transcription interference. The growth defects (ranging from sickness to lethality) elicited by fission yeast mutations that inactivate IPP pyrophosphatase enzymes are exerted via the agonistic effects of too much 1,5-IP8 on RNA 3'-processing and transcription termination. To illuminate determinants of IPP toxicosis, we conducted a genetic screen for spontaneous mutations that suppressed the sickness of Asp1 pyrophosphatase mutants. We identified a missense mutation, C823R, in the essential Cft1 subunit of the cleavage and polyadenylation factor complex that suppresses even lethal Asp1 IPP pyrophosphatase mutations, thereby fortifying the case for 3'-processing/termination as the target of IPP toxicity. The suppressor screen also identified Gde1 and Spx1 (SPAC6B12.07c), both of which have an IPP-binding SPX domain and both of which are required for lethality elicited by Asp1 mutations. A survey of other SPX proteins in the proteome identified the Vtc4 and Vtc2 subunits of the vacuolar polyphosphate polymerase as additional agents of IPP toxicosis. Gde1, Spx1, and Vtc4 contain enzymatic modules (glycerophosphodiesterase, RING finger ubiquitin ligase, and polyphosphate polymerase, respectively) fused to their IPP-sensing SPX domains. Structure-guided mutagenesis of the IPP-binding sites and the catalytic domains of Gde1 and Spx1 indicated that both modules are necessary to elicit IPP toxicity. Whereas Vtc4 polymerase catalytic activity is required for IPP toxicity, its IPP-binding site is not. Epistasis analysis, transcriptome profiling, and assays of Pho1 expression implicate Spx1 as a transducer of IP8 signaling to the 3'-processing/transcription termination machinery. Impeding the catabolism of the inositol pyrophosphate (IPP) signaling molecule IP8 is cytotoxic to fission yeast. Here, by performing a genetic suppressor screen, we identified several cellular proteins required for IPP toxicosis. Alleviation of IPP lethality by a missense mutation in the essential Cft1 subunit of the cleavage and polyadenylation factor consolidates previous evidence that toxicity results from IP8 action as an agonist of RNA 3'-processing and transcription termination. Novel findings are that IP8 toxicity depends on IPP-sensing SPX domain proteins with associated enzymatic functions: Gde1 (glycerophosphodiesterase), Spx1 (ubiquitin ligase), and Vtc2/4 (polyphosphate polymerase). The effects of Spx1 deletion on phosphate homeostasis imply a role for Spx1 in communicating an IP8-driven signal to the transcription and RNA processing apparatus.
Topics: Diphosphates; Fungal Proteins; Inositol Phosphates; Ligases; mRNA Cleavage and Polyadenylation Factors; Polyphosphates; Pyrophosphatases; RNA; Schizosaccharomyces; Ubiquitins
PubMed: 35012333
DOI: 10.1128/mbio.03476-21