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MBio Jun 2024Inositol pyrophosphate 1,5-IP regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes , , and , via its action as...
UNLABELLED
Inositol pyrophosphate 1,5-IP regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes , , and , via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress mRNA synthesis. 1,5-IP levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP to 1,5-IP and three inositol pyrophosphatases-the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1's substrate repertoire embraces inorganic polyphosphates, 5-IP, 1-IP, and 1,5-IP. Aps1 displays a ~twofold preference for hydrolysis of 1-IP versus 5-IP and ∆ cells have twofold higher levels of 1-IP vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an ∆ ∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP toxicosis, whereby excessive 1,5-IP drives derepression of leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an ∆ ∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of ∆ ∆ on YES by deleting . However, the severe growth defect of an ∆ strain could not be alleviated by deleting , suggesting that 1,5-IP levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity.
IMPORTANCE
Repression of the fission yeast genes , , and by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP, a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP is formed by phosphorylation of 5-IP and catabolized by inositol pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools . Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP, 1-IP, and 1,5-IP , with a ~twofold preference for 1-IP over 5-IP. ∆ cells have twofold higher levels of 1-IP than wild-type cells. An ∆ ∆ double mutation is lethal because excessive 1,5-IP triggers derepression of , leading to toxic uptake of glycerophosphocholine.
PubMed: 38940614
DOI: 10.1128/mbio.01084-24 -
PeerJ 2024Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The... (Review)
Review
Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The accumulation of PPi in the cell can result in cell death. Although the substrate is the same, there are variations in the catalysis and features of these enzymes. Two enzyme forms have been identified in bacteria: cytoplasmic or soluble pyrophosphatases and membrane-bound pyrophosphatases, which play major roles in cell bioenergetics. In eukaryotic cells, cytoplasmic enzymes are the predominant form of PPases (c-PPases), while membrane enzymes (m-PPases) are found only in protists and plants. The study of bacterial cytoplasmic and membrane-bound pyrophosphatases has slowed in recent years. These enzymes are central to cell metabolism and physiology since phospholipid and nucleic acid synthesis release important amounts of PPi that must be removed to allow biosynthesis to continue. In this review, two aims were pursued: first, to provide insight into the structural features of PPases known to date and that are well characterized, and to provide examples of enzymes with novel features. Second, the scientific community should continue studying these enzymes because they have many biotechnological applications. Additionally, in this review, we provide evidence that there are m-PPases present in fungi; to date, no examples have been characterized. Therefore, the diversity of PPase enzymes is still a fruitful field of research. Additionally, we focused on the roles of H/Na pumps and m-PPases in cell bioenergetics. Finally, we provide some examples of the applications of these enzymes in molecular biology and biotechnology, especially in plants. This review is valuable for professionals in the biochemistry field of protein structure-function relationships and experts in other fields, such as chemistry, nanotechnology, and plant sciences.
Topics: Inorganic Pyrophosphatase; Bacteria; Fungi; Diphosphates
PubMed: 38938619
DOI: 10.7717/peerj.17496 -
Pharmaceuticals (Basel, Switzerland) Jun 2024Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In...
Calcium pyrophosphate dehydrate (CPPD) crystals are found in the synovial fluid of patients with articular chondrocalcinosis or sometimes with osteoarthritis. In inflammatory conditions, the synovial membrane (SM) is subjected to transient hypoxia, especially during movement. CPPD formation is supported by an increase in extracellular inorganic pyrophosphate (ePPi) levels, which are mainly controlled by the transporter Ank and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We demonstrated previously that transforming growth factor (TGF)-β1 increased ePPi production by inducing Ank and Enpp1 expression in chondrocytes. As the TGF-β1 level raises in synovial fluid under hypoxic conditions, we investigated whether hypoxia may transform SM as a major source of ePPi production. Synovial fibroblasts and SM explants were exposed to 10 ng/mL of TGF-β1 in normoxic or hypoxic (5% O) culture conditions. Ank and Enpp1 expression were assessed by quantitative PCR, Western blot and immunohistochemistry. ePPi was quantified in culture supernatants. RNA silencing was used to define the respective roles of and in TGF-β1-induced ePPi generation. The molecular mechanisms involved in hypoxia were investigated using an promoter reporter plasmid for transactivation studies, as well as gene overexpression and RNA silencing, the respective role of hypoxia-induced factor (HIF)-1 and HIF-2. Our results showed that TGF-β1 increased Ank, Enpp1, and therefore ePPi production in synovial fibroblasts and SM explants. Ank was the major contributor in ePPi production compared to ENPP1. Hypoxia increased ePPi levels on its own and enhanced the stimulating effect of TGF-β1. Hypoxic conditions enhanced promoter transactivation in an HIF-1-dependent/HIF-2-independent fashion. We demonstrated that under hypoxia, SM is an important contributor to ePPi production in the joint through the induction of and . These findings are of interest as a rationale for the beneficial effect of anti-inflammatory drugs on SM in crystal depositions.
PubMed: 38931405
DOI: 10.3390/ph17060738 -
BMC Infectious Diseases Jun 2024Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from...
BACKGROUND
Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes.
METHODS
Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm).
RESULTS
We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M.S. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and HO. Since schistosomes are damaged by HO, we determined if SmNPP5 could impede HO production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M.S.
CONCLUSIONS
The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes.
Topics: Riboflavin; Flavin Mononucleotide; Animals; Flavin-Adenine Dinucleotide; Schistosoma mansoni; Mice; Humans; Schistosomiasis mansoni
PubMed: 38918706
DOI: 10.1186/s12879-024-09538-z -
Journal of Cancer 2024DNA damage-inducible transcript 3 (DDIT3) is a transcription factor central to apoptosis, differentiation, and stress response. DDIT3 has been extensively studied in...
DNA damage-inducible transcript 3 (DDIT3) is a transcription factor central to apoptosis, differentiation, and stress response. DDIT3 has been extensively studied in cancer biology. However, its precise implications in breast cancer progression and its interaction with the immune microenvironment are unclear. In this study, we utilized a novel multi-omics integration strategy, combining bulk RNA sequencing, single-cell sequencing, spatial transcriptomics and immunohistochemistry, to explore the role of DDIT3 in breast cancer and establish the correlation between DDIT3 and poor prognosis in breast cancer patients. We identified a robust prognostic signature, including six genes (unc-93 homolog B1, TLR signaling regulator, anti-Mullerian hormone, DCTP pyrophosphatase 1, mitochondrial ribosomal protein L36, nuclear factor erythroid 2, and Rho GTPase activating protein 39), associated with DDIT3. This signature stratified the high-risk patient groups, characterized by increased infiltration of the regulatory T cells and M2-like macrophages and fibroblast growth factor (FGF)/FGF receptor signaling activation. Notably, the high-risk patient group demonstrated enhanced sensitivity to immunotherapy, presenting novel therapeutic opportunities. Integrating multi-omics data helped determine the spatial expression pattern of DDIT3 in the tumor microenvironment and its correlation with immune cell infiltration. This multi-dimensional analysis provided a comprehensive understanding of the intricate interplay between DDIT3 and the immune microenvironment in breast cancer. Overall, our study not only facilitates understanding the role of DDIT3 in breast cancer but also offers innovative insights for developing prognostic models and therapeutic strategies. Identifying the DDIT3-related prognostic signature and its association with the immune microenvironment provided a promising avenue for personalized breast cancer treatment.
PubMed: 38911383
DOI: 10.7150/jca.96491 -
The Pharmacogenomics Journal Jun 2024Thiopurines, an effective therapy for Crohn's disease (CD), often lead to adverse events (AEs). Gene polymorphisms affecting thiopurine metabolism may predict AEs. This...
Thiopurines, an effective therapy for Crohn's disease (CD), often lead to adverse events (AEs). Gene polymorphisms affecting thiopurine metabolism may predict AEs. This retrospective study in CD patients (n = 114) with TPMT activity > 5 Units/Red Blood Cells analyzed TPMT (c.238 G > C, c.460 G > A, c.719 A > G), ITPA (c.94 C > A, IVS2 + 21 A > C), and NUDT15 (c.415 C > T) polymorphisms. All patients received azathioprine (median dose 2.2 mg/kg) with 41.2% experiencing AEs, mainly myelotoxicity (28.1%). No NUDT15 polymorphisms were found, 7% had TPMT, and 31.6% had ITPA polymorphisms. AEs led to therapy modifications in 41.2% of patients. Multivariate analysis identified advanced age (OR 1.046, p = 0.007) and ITPA IVS2 + 21 A > C (OR 3.622, p = 0.015) as independent predictors of AEs. IVS2 + 21 A > C was also associated with myelotoxicity (OR 2.863, p = 0.021). These findings suggest that ITPA IVS2 + 21 A > C polymorphism and advanced age predict AEs during thiopurine therapy for CD with intermediate-normal TPMT activity.
Topics: Humans; Crohn Disease; Pyrophosphatases; Female; Male; Adult; Retrospective Studies; Azathioprine; Methyltransferases; Middle Aged; Young Adult; Immunosuppressive Agents; Adolescent; Pharmacogenomic Variants; Polymorphism, Single Nucleotide; Polymorphism, Genetic; Mercaptopurine; Multivariate Analysis; Aged; Risk Factors; Nudix Hydrolases; Inosine Triphosphatase
PubMed: 38906864
DOI: 10.1038/s41397-024-00341-2 -
MBio Jun 2024Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising...
Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes , , and ) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP levels derepress the regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP toxicity is alleviated by deletion of , the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating () allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of hyper-repressed expression in phosphate-replete cells; suppressed the derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious lncRNA termination); and delayed induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the and promoters, but not for the promoters that drive the synthesis of the -repressive lncRNAs. Transcription profiling of ∆ and () cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast genes , , and by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic alleles derepress the genes via the action of IP as an agonist of precocious lncRNA 3'-processing/termination. IP toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP levels on termination. In this study, a forward genetic screen revealed that IP toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of -activating transcription factor Pho7 to its binding sites in the mRNA promoters.
PubMed: 38899862
DOI: 10.1128/mbio.01252-24 -
International Journal of Molecular... May 2024Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of...
Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation.
Topics: Cystathionine beta-Synthase; Mutation; Protein Binding; Mutagenesis, Site-Directed; Adenine Nucleotides; Protein Domains; Pyrophosphatases; Adenosine Diphosphate; Adenosine Triphosphate; Bacterial Proteins; Inorganic Pyrophosphatase; Models, Molecular; Binding Sites
PubMed: 38891956
DOI: 10.3390/ijms25115768 -
Advanced Science (Weinheim,... Jun 2024Radiation enteritis is the most common complication of pelvic radiotherapy, but there is no effective prevention or treatment drug. Apoptotic T cells and their products...
Radiation enteritis is the most common complication of pelvic radiotherapy, but there is no effective prevention or treatment drug. Apoptotic T cells and their products play an important role in regulating inflammation and maintaining physiological immune homeostasis. Here it is shown that systemically infused T cell-derived apoptotic extracellular vesicles (ApoEVs) can target mice irradiated intestines and alleviate radiation enteritis. Mechanistically, radiation elevates the synthesis of intestinal 2'3' cyclic GMP-AMP (cGAMP) and activates cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) proinflammatory pathway. After systemic infusion of ApoEVs, the ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) enriches on the surface of ApoEVs hydrolyze extracellular cGAMP, resulting in inhibition of the cGAS-STING pathway activated by irradiation. Furthermore, after ApoEVs are phagocytosed by phagocytes, ENPP1 on ApoEVs hydrolyzed intracellular cGAMP, which serves as an intracellular cGAMP hydrolyzation mode, thereby alleviating radiation enteritis. The findings shed light on the intracellular and extracellular hydrolysis capacity of ApoEVs and their role in inflammation regulation.
PubMed: 38888507
DOI: 10.1002/advs.202401634 -
Bioorganic & Medicinal Chemistry Letters Jun 2024The STING (stimulator of interferon genes) pathway is one of the pathways that regulate innate immunity, and the extracellular hydrolytic enzyme ecto-nucleotide...
The STING (stimulator of interferon genes) pathway is one of the pathways that regulate innate immunity, and the extracellular hydrolytic enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as its dominant negative regulator. Since activation of the innate immune system is a promising strategy for the treatment of various infectious diseases and cancers, ENPP1 inhibitors have attracted great attention as candidate drugs. We have previously identified small-molecule ENPP1 inhibitors having a [1,2,4]triazolo[1,5-a]pyrimidine scaffold by means of chemical screening using a fluorescence probe, TG-mAMP. In this study, we evaluated the structure-activity relationships of the hit and lead compounds in detail, and succeeded in developing compounds that strongly and selectively inhibit ENPP1 not only in vitro, but also in cellular systems.
PubMed: 38851358
DOI: 10.1016/j.bmcl.2024.129820