Impact of ITPA gene polymorphism for predicting anemia and treatment outcome in HCV infected patients taking Sofosbuvir Ribavirin therapy.

BACKGROUND: Globally, 80 million people are suffering from chronic Hepatitis C virus (HCV) infection. Sofosbuvir ribavirin-based anti-HCV therapy is associated with anemia and other adverse effects. Polymorphisms of Inosine triphosphatase (ITPA) gene may cause functional impairment in the Inosine triphosphate pyrophosphatase enzyme, resulting in enhanced sustained viral response (SVR) and protection from ribavirin-associated anemia in patients on therapy. The study objective was to investigate the effect of Inosine triphosphatase gene polymorphism on SVR achievement, hemoglobin decline and ribavirin dose reduction in patients on therapy. METHODS: This prospective cohort study was of 170 hepatitis C infected patients received 6-month sofosbuvir ribavirin therapy. Patient viral load, reduction in ribavirin amount, liver function test, and complete blood count were noted monthly. Inosine triphosphatase variants rs1127354 and rs7270101 were assessed through the restriction fragment length polymorphism and confirmed using Sanger sequencing. The impact of polymorphism on cumulative reduction of ribavirin, and anti-HCV therapy outcome were studied. RESULTS: A total of 74.3% of patients had ITPA rs1127354 CC genotype, 25.7% were CA and AA 0%. The frequency of ITPA genotype rs7270101-AA was 95%, AC 5%, and CC was 0%. ITPA rs1127354-CA had a notably positive impact on SVR achievement with a zero-relapse rate. ITPA rs1127354-CA genotype was significantly (P ˂0.05) protective against ≥ 2 g/dl Hb reduction from baseline to 1st, 2nd and 6th months of therapy. During treatment, Hb reduction ≥ 10 g/dl was frequently observed in rs1127354-CC genotype and rs7270101-AA genotype patients. Ribavirin dose reduction was significantly (P ˂0.05) high in rs1127354-CC genotype as compared to genotype CA whereas no significant difference was observed in ribavirin dose reduction in rs7270101 AA and non-AA genotype. Patient baseline characteristics such as age, body mass index, rs1127354-CC genotype, and baseline Hb were significantly associated with significant Hb reduction. CONCLUSION: Pretreatment evaluation of ITPA polymorphism can be a diagnostic tool to find out patients at risk of anemia and improve treatment adherence. ITPA genotype rs1127354-CA contributes to improved compliance with ribavirin dose and protects against hemoglobin decline in HCV patients while taking ribavirin-based therapy. However, ITPA rs1127354, rs7270101 polymorphism have no significant impact on SVR achievement.

Structural and biochemical characterization of active sites mutant in human inorganic pyrophosphatase.

Inorganic pyrophosphatases (PPases) are enzymes that catalyze the conversion of inorganic pyrophosphate (PPi) into phosphate (Pi). Human inorganic pyrophosphatase 1 (Hu-PPase) exhibits high expression levels in a variety of tumors and plays roles in cell proliferation, apoptosis, invasion and metastasis, making it a promising prognostic biomarker and a target for cancer therapy. Despite its widespread presence, the catalytic mechanism of Hu-PPase in humans remains inadequately understood. The signature motif amino acid sequence (DXDPXD) within the active sites of PPases is preserved across different species. In this research, an enzymatic activity assay revealed that mutations led to a notable reduction in enzymatic function, although the impact of the four amino acids on the activity of the pocket varied. To investigate the influence of these residues on the substrate binding and enzymatic function of PPase, the crystal structure of the Hu-PPase-ED quadruple mutant (D116A/D118A/P119A/D121A) was determined at 1.69 Å resolution. The resulting structure maintained a barrel-like shape similar to that of the wild-type, albeit lacking Mg2+ ions. Molecular docking analysis demonstrated a decreased ability of Hu-PPase-ED to bind to PPi. Further, molecular dynamics simulation analysis indicated that the mutation rendered the loop of Mg2+ ion-binding residues less stable. Therefore, the effect on enzyme activity did not result from a change in the gross protein structure but rather from a mutation that abolished the Mg2+-coordinating groups, thereby eliminating Mg2+ binding and leading to the loss of enzyme activity.

Insight into small-molecule inhibitors targeting extracellular nucleotide pyrophosphatase/phosphodiesterase1 for potential multiple human diseases.

Extracellular nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as a type II transmembrane glycoprotein. It plays a crucial role in various biological processes, such as bone mineralization, cancer cell proliferation, and immune regulation. Consequently, ENPP1 has garnered attention as a promising target for pharmacological interventions. Despite its potential, the development of clinical-stage ENPP1 inhibitors for solid tumors, diabetes, and silent rickets remains limited. However, there are encouraging findings from preclinical trials involving small molecules exhibiting favorable therapeutic effects and safety profiles. This perspective aims to shed light on the structural properties, biological functions and the relationship between ENPP1 and diseases. Additionally, it focuses on the structure-activity relationship of ENPP1 inhibitors, with the intention of guiding the future development of new and effective ENPP1 inhibitors.

Emergence of a multilocus mutator genotype in mutator Escherichia coli experimental populations under repeated lethal selection.

Mutator alleles, which confer increased mutation rates, are known to spontaneously emerge and "hitchhike" to fixation in evolving asexual populations. Theory predicts that in an evolving asexual mutator population, a second mutator allele may spontaneously arise and hitchhike to fixation. Here, we describe an empirical test of the hypothesis of repeated hitchhiking. The starting population was a clonal strain of mutL-Escherichia coli whose mutation rate was 100-fold higher than wild type. We exposed the mutL- strain to a series of three antibiotics in increasing order of selective strength: fosfomycin, rifampicin, and streptomycin. Two independent replicates of the experiment were performed. As predicted, elevated mutation rates and enrichment for multilocus mutators (which bear more than one mutator allele) were observed in the end point populations of both experiments. DNA sequencing revealed an identical spontaneous 1-bp insertion in the mutator gene mutT in both end point populations. In the multilocus mutators, the causal relationship between the mutT- mutations and the increase in mutation rate was supported with mutT+ plasmid complementation tests. Surprisingly, when the experiment was repeated with the antibiotics deployed in decreasing order of selective strength, enrichment for multilocus mutators was not observed. Our data support the likelihood that the mutT- mutations rose to fixation in both populations, consistent with the hypothesis of repeated mutator hitchhiking. The escalation of mutation rates in asexual populations is relevant to multiple biological scenarios, including antibiotic resistance, host-pathogen interactions, and carcinogenesis.

Discovery of Pyrido[2,3-d]pyrimidin-7-one Derivatives as Highly Potent and Efficacious Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) Inhibitors for Cancer Treatment.

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is an extracellular enzyme responsible for hydrolyzing cyclic guanosine monophosphate-adenosine monophosphate (cGAMP), the endogenous agonist for the stimulator of interferon genes (STING) pathway. Inhibition of ENPP1 can trigger STING and promote antitumor immunity, offering an attractive therapeutic target for cancer immunotherapy. Despite progress in the discovery of ENPP1 inhibitors, the diversity in chemical structures and the efficacy of the agents are far from desirable, emphasizing the demand for novel inhibitors. Herein, we describe the design, synthesis, and biological evaluation of a series of ENPP1 inhibitors based on the pyrido[2,3-d]pyrimidin-7-one scaffold. Optimization efforts led to compound 31 with significant potency in both ENPP1 inhibition and STING pathway stimulation in vitro. Notably, 31 demonstrated in vivo efficacy in a syngeneic 4T1 mouse triple negative breast cancer model. These findings provide a promising lead compound with a novel scaffold for further drug development in cancer immunotherapy.

NUDT16 regulates CtIP PARylation to dictate homologous recombination repair.

CtIP initiates DNA end resection and mediates homologous recombination (HR) repair. However, the underlying mechanisms of CtIP regulation and how the control of its regulation affects DNA repair remain incompletely characterized. In this study, NUDT16 loss decreases CtIP protein levels and impairs CtIP recruitment to double-strand breaks (DSBs). Furthermore, overexpression of a catalytically inactive NUDT16 mutant is unable to rescue decreased CtIP protein and impaired CtIP recruitment to DSBs. In addition, we identified a novel posttranslational modification of CtIP by ADP-ribosylation that is targeted by a PAR-binding E3 ubiquitin ligase, RNF146, leading to CtIP ubiquitination and degradation. These data suggest that the hydrolase activity of NUDT16 plays a major role in controlling CtIP protein levels. Notably, ADP-ribosylation of CtIP is required for its interaction with NUDT16, its localization at DSBs, and for HR repair. Interestingly, NUDT16 can also be ADP-ribosylated. The ADP-ribosylated NUDT16 is critical for CtIP protein stability, CtIP recruitment to DSBs, and HR repair in response to DNA damage. In summary, we demonstrate that NUDT16 and its PARylation regulate CtIP stability and CtIP recruitment to DSBs, providing new insights into our understanding of the regulation of CtIP-mediated DNA end resection in the HR repair pathway.

Differential effects of the lipidic and ionic microenvironment on NPP1's phosphohydrolase and phosphodiesterase activities.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is an enzyme present in matrix vesicles (MV). NPP1 participates on the regulation of bone formation by producing pyrophosphate (PPi) from adenosine triphosphate (ATP). Here, we have used liposomes bearing dipalmitoylphosphatidylcholine (DPPC), sphingomyelin (SM), and cholesterol (Chol) harboring NPP1 to mimic the composition of MV lipid rafts to investigate ionic and lipidic influence on NPP1 activity and mineral propagation. Atomic force microscopy (AFM) revealed that DPPC-liposomes had spherical and smooth surface. The presence of SM and Chol elicited rough and smooth surface, respectively. NPP1 insertion produced protrusions in all the liposome surface. Maximum phosphodiesterase activity emerged at 0.082 M ionic strength, whereas maximum phosphomonohydrolase activity arose at low ionic strength. Phosphoserine-Calcium Phosphate Complex (PS-CPLX) and amorphous calcium-phosphate (ACP) induced mineral propagation in DPPC- and DPPC:SM-liposomes and in DPPC:Chol-liposomes, respectively. Mineral characterization revealed the presence of bands assigned to HAp in the mineral propagated by NPP1 harbored in DPPC-liposomes without nucleators or in DPPC:Chol-liposomes with ACP nucleators. These data show that studying how the ionic and lipidic environment affects NPP1 properties is important, especially for HAp obtained under controlled conditions in vitro.

The novel phosphatase NUDT5 is a critical regulator of triple-negative breast cancer growth.

BACKGROUND: The most aggressive form of breast cancer is triple-negative breast cancer (TNBC), which lacks expression of the estrogen receptor (ER) and progesterone receptor (PR), and does not have overexpression of the human epidermal growth factor receptor 2 (HER2). Treatment options for women with TNBC tumors are limited, unlike those with ER-positive tumors that can be treated with hormone therapy, or those with HER2-positive tumors that can be treated with anti-HER2 therapy. Therefore, we have sought to identify novel targeted therapies for TNBC. In this study, we investigated the potential of a novel phosphatase, NUDT5, as a potential therapeutic target for TNBC. METHODS: The mRNA expression levels of NUDT5 in breast cancers were investigated using TCGA and METABRIC (Curtis) datasets. NUDT5 ablation was achieved through siRNA targeting and NUDT5 inhibition with the small molecule inhibitor TH5427. Xenograft TNBC animal models were employed to assess the effect of NUDT5 inhibition on in vivo tumor growth. Proliferation, death, and DNA replication assays were conducted to investigate the cellular biological effects of NUDT5 loss or inhibition. The accumulation of 8-oxo-guanine (8-oxoG) and the induction of γH2AX after NUDT5 loss was determined by immunofluorescence staining. The impact of NUDT5 loss on replication fork was assessed by measuring DNA fiber length. RESULTS: In this study, we demonstrated the significant role of an overexpressed phosphatase, NUDT5, in regulating oxidative DNA damage in TNBCs. Our findings indicate that loss of NUDT5 results in suppressed growth of TNBC both in vitro and in vivo. This growth inhibition is not attributed to cell death, but rather to the suppression of proliferation. The loss or inhibition of NUDT5 led to an increase in the oxidative DNA lesion 8-oxoG, and triggered the DNA damage response in the nucleus. The interference with DNA replication ultimately inhibited proliferation. CONCLUSIONS: NUDT5 plays a crucial role in preventing oxidative DNA damage in TNBC cells. The loss or inhibition of NUDT5 significantly suppresses the growth of TNBCs. These biological and mechanistic studies provide the groundwork for future research and the potential development of NUDT5 inhibitors as a promising therapeutic approach for TNBC patients.

Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 inhibitors: Research progress and prospects.

The cancer immunotherapies involved in cGAS-STING pathway have been made great progress in recent years. STING agonists exhibit broad-spectrum anti-tumor effects with strong immune response. As a negative regulator of the cGAS-STING pathway, ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) can hydrolyze extracellular 2', 3'-cGAMP and reduce extracellular 2', 3'-cGAMP concentration. ENPP1 has been validated to play important roles in diabetes, cancers, and cardiovascular disease and now become a promising target for tumor immunotherapy. Several ENPP1 inhibitors under development have shown good anti-tumor effects alone or in combination with other agents in clinical and preclinical researches. In this review, the biological profiles of ENPP1 were described, and the structures and the structure-activity relationships (SAR) of the known ENPP1 inhibitors were summarized. This review also provided the prospects and challenges in the development of ENPP1 inhibitors.

The ecto-nucleotide pyrophosphatase/phosphodiesterase 2 promotes early osteoblast differentiation and mineralization in stromal stem cells.

The phosphodiesterase enzymes mediate calcium-phosphate deposition in various tissues, although which enzymes are active in bone mineralization is unclear. Using gene array analysis, we found that a member of ecto-nucleotide pyrophosphatase/phosphodiesterase family, ENPP2, was strongly down-regulated with age in stromal stem cells that produce osteoblasts and make bone. This is in keeping with reduced bone formation in older animals. Thus, we hypothesized that ENPP2 is, at least in part, an early mediator of bone formation and thus may reflect reduced bone formation with age. Since ENPP2 has not previously been shown to have a role in osteoblast differentiation, we studied its effect on bone differentiation from stromal stem cells, verified by flow cytometry for stem cell antigens. In these remarkably uniform osteoblast precursors, we did transfection with ENPP2 DsiRNA, scrambled DsiRNA, or no transfection to make cells with normal or greatly reduced ENPP2 and analyzed osteoblast differentiation and mineralization. Osteoblast differentiation down-regulation was shown by alizarin red binding, silver staining, and alkaline phosphatase activity. Differences were confirmed by real-time PCR for alkaline phosphatase (ALPL), osteocalcin (BGLAP), and ENPP2 and by Western Blot for Enpp2. These were decreased, ∼50%, in osteoblasts transfected with ENPP2 DsiRNA compared with cells transfected with a scrambled DsiRNA or not transfected (control) cells. This finding is the first evidence for the role of ENPP2 in osteoblast differentiation and mineralization.NEW & NOTEWORTHY We report the discovery that the ecto-nucleotide pyrophosphatase/phosphodiesterase, ENPP2, is an important regulator of early differentiation of bone-forming osteoblasts.

The dePARylase NUDT16 promotes radiation resistance of cancer cells by blocking SETD3 for degradation via reversing its ADP-ribosylation.

Poly(ADP-ribosyl)ation (PARylation) is a critical posttranslational modification that plays a vital role in maintaining genomic stability via a variety of molecular mechanisms, including activation of replication stress and the DNA damage response. The nudix hydrolase NUDT16 was recently identified as a phosphodiesterase that is responsible for removing ADP-ribose units and that plays an important role in DNA repair. However, the roles of NUDT16 in coordinating replication stress and cell cycle progression remain elusive. Here, we report that SETD3, which is a member of the SET-domain containing protein (SETD) family, is a novel substrate for NUDT16, that its protein levels fluctuate during cell cycle progression, and that its stability is strictly regulated by NUDT16-mediated dePARylation. Moreover, our data indicated that the E3 ligase CHFR is responsible for the recognition and degradation of endogenous SETD3 in a PARP1-mediated PARylation-dependent manner. Mechanistically, we revealed that SETD3 associates with BRCA2 and promotes its recruitment to stalled replication fork and DNA damage sites upon replication stress or DNA double-strand breaks, respectively. Importantly, depletion of SETD3 in NUDT16-deficient cells did not further exacerbate DNA breaks or enhance the sensitivity of cancer cells to IR exposure, suggesting that the NUDT16-SETD3 pathway may play critical roles in the induction of tolerance to radiotherapy. Collectively, these data showed that NUDT16 functions as a key upstream regulator of SETD3 protein stability by reversing the ADP-ribosylation of SETD3, and NUDT16 participates in the resolution of replication stress and facilitates HR repair.

Measurement of the intracellular active metabolites of thiopurine drugs to evaluate the enzymatic activity of nudix hydrolase 15 in human blood samples.

Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 µM, 1 µM, and 2 µM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 µM) as a substrate (18.1, 14.9, and 6.43 µM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage.

Functional and structural asymmetry suggest a unifying principle for catalysis in membrane-bound pyrophosphatases.

Membrane-bound pyrophosphatases (M-PPases) are homodimeric primary ion pumps that couple the transport of Na+- and/or H+ across membranes to the hydrolysis of pyrophosphate. Their role in the virulence of protist pathogens like Plasmodium falciparum makes them an intriguing target for structural and functional studies. Here, we show the first structure of a K+-independent M-PPase, asymmetric and time-dependent substrate binding in time-resolved structures of a K+-dependent M-PPase and demonstrate pumping-before-hydrolysis by electrometric studies. We suggest how key residues in helix 12, 13, and the exit channel loops affect ion selectivity and K+-activation due to a complex interplay of residues that are involved in subunit-subunit communication. Our findings not only explain ion selectivity in M-PPases but also why they display half-of-the-sites reactivity. Based on this, we propose, for the first time, a unified model for ion-pumping, hydrolysis, and energy coupling in all M-PPases, including those that pump both Na+ and H+.

Bacteriophage T5 dUTPase: Combination of Common Enzymatic and Novel Functions.

The main function of dUTPases is to regulate the cellular levels of dUTP and dTTP, thereby playing a crucial role in DNA repair mechanisms. Despite the fact that mutant organisms with obliterated dUTPase enzymatic activity remain viable, it is not possible to completely knock out the dut gene due to the lethal consequences of such a mutation for the organism. As a result, it is considered that this class of enzymes performs an additional function that is essential for the organism's survival. In this study, we provide evidence that the dUTPase of bacteriophage T5 fulfills a supplemental function, in addition to its canonical role. We determined the crystal structure of bacteriophage T5 dUTPase with a resolution of 2.0 Å, and we discovered a distinct short loop consisting of six amino acid residues, representing a unique structural feature specific to the T5-like phages dUTPases. The removal of this element did not affect the overall structure of the homotrimer, but it had significant effects on the development of the phage. Furthermore, it was shown that the enzymatic function and the novel function of the bacteriophage T5 dUTPase are unrelated and independent from each other.

Naturally occurring quercetin and myricetin as potent inhibitors for human ectonucleotide pyrophosphatase/phosphodiesterase 1.

Ecto-nucleotide pyrophosphatases/phosphodiesterases 1 (ENPP1) is a key enzyme in purinergic signaling pathways responsible for cell-to-cell communications and regulation of several fundamental pathophysiological processes. In this study, Kyoto Green, a rapid chemical sensor of pyrophosphate, was employed to screen for effective ENPP1 inhibitors among five representative flavonoids (quercetin, myricetin, morin, kaempferol, and quercetin-3-glucoside), five nucleosides (adenosine, guanosine, inosine, uridine, and cytidine), and five deoxynucleosides (2'- and 3'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxyinosine, and 2'-deoxyuridine). Conventional colorimetric, fluorescence, and bioluminescence assays revealed that ENPP1 was effectively inhibited by quercetin (Ki ~ 4 nM) and myricetin (Ki ~ 32 nM) when ATP was used as a substrate at pH 7.4. In silico analysis indicated that the presence of a chromone scaffold, particularly one containing a hydroxyl group at the 3' position on the B ring, may promote binding to the active site pocket of ENPP1 and enhance inhibition. This study demonstrated that the naturally derived quercetin and myricetin could effectively inhibit ENPP1 enzymatic activity and may offer health benefits in arthritis management.

Antirepressor specificity is shaped by highly efficient dimerization of the staphylococcal pathogenicity island regulating repressors: Stl repressor dimerization perturbed by dUTPases.

The excision and replication, thus the life cycle of pathogenicity islands in staphylococci are regulated by Stl master repressors that form strong dimers. It has been recently shown that SaPIbov1-Stl dimers are separated during the activation of the Staphylococcus aureus pathogenicity island (SaPI) transcription via helper phage proteins. To understand the mechanism of this regulation, a quantitative analysis of the dimerization characteristics is required. Due to the highly efficient dimerization process, such an analysis has to involve specific solutions that permit relevant experiments to be performed. In the present work, we focused on two staphylococcal Stls associated with high biomedical interest, namely Stl proteins of Staphylococcus aureus bov1 and Staphylococcus hominis ShoCI794_SEPI pathogenicity islands. Exploiting the interactions of these two Stl proteins with their antirepressor-mimicking interaction partners allowed precise determination of the Stl dimerization constant in the subnanomolar range.

ENPP1 inhibits the transcription activity of the hepatitis B virus pregenomic promoter by upregulating the acetylation of LMNB1.

Current therapies for hepatitis B virus (HBV) infection can slow disease progression but cannot cure the infection, as it is difficult to eliminate or permanently silence HBV covalently closed circular DNA (cccDNA). The interaction between host factors and cccDNA is essential for their formation, stability, and transcriptional activity. Here, we focused on the regulatory role of the host factor ENPP1 and its interacting transcription factor LMNB1 in HBV replication and transcription to better understand the network of host factors that regulate HBV, which may facilitate the development of new antiviral drugs. Overexpression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in Huh7 cells decreased HBV pregenomic RNA (pgRNA) and hepatitis B core antigen (HBcAg) expression levels, whereas knockdown of ENPP1 increased them. A series of HBV promoter and mutant plasmids were constructed, and a luciferase reporter assay showed that overexpression of ENPP1 caused inhibition of the HBV promoter and its mutants. A DNA pull-down assay showed that lamin B1 (LMNB1), but not ENPP1, interacts directly with the HBV enhancer II/ basic core promoter (EnhII/BCP). ZDOCK and PyMOL software were used to predict the interaction of ENPP1 with LMNB1. Overexpression of LMNB1 inhibited the activity of the HBV promoter and its mutant. The acetylation levels at the amino acids 111K, 261K, and 483K of LMNB1 were reduced compared to the control, and an LMNB1 acetylation mutant containing 111R, 261Q, 261R, 483Q, and 483R showed increased promoter activity. In summary, ENPP1 together with LMNB1 increased the acetylation level at 111K and 261K, and LMNB1 inhibited the activity of HBV promoter and downregulated the expression of pregenomic RNA and HBcAg. Our follow-up studies will investigate the expression, clinical significance, and relevance of ENPP1 and LMNB1 in HBV patient tissues, explore the effect of LMNB1 on post-transcriptional progression, and examine whether ENPP1 can reduce cccDNA levels in the nucleus.

Cartilage tissues regulate systemic aging via ectonucleotide pyrophosphatase/phosphodiesterase 1 in mice.

Aging presents fundamental health concerns worldwide; however, mechanisms underlying how aging is regulated are not fully understood. Here, we show that cartilage regulates aging by controlling phosphate metabolism via ectonucleotide pyrophosphatase/phosphodiesterase 1 (Enpp1). We newly established an Enpp1 reporter mouse, in which an EGFP-luciferase sequence was knocked-in at the Enpp1 gene start codon (Enpp1/EGFP-luciferase), enabling detection of Enpp1 expression in cartilage tissues of resultant mice. We then established a cartilage-specific Enpp1 conditional knockout mouse (Enpp1 cKO) by generating Enpp1 flox mice and crossing them with cartilage-specific type 2 collagen Cre mice. Relative to WT controls, Enpp1 cKO mice exhibited phenotypes resembling human aging, such as short life span, ectopic calcifications, and osteoporosis, as well as significantly lower serum pyrophosphate levels. We also observed significant weight loss and worsening of osteoporosis in Enpp1 cKO mice under phosphate overload conditions, similar to global Enpp1-deficient mice. Aging phenotypes seen in Enpp1 cKO mice under phosphate overload conditions were rescued by a low vitamin D diet, even under high phosphate conditions. These findings suggest overall that cartilage tissue plays an important role in regulating systemic aging via Enpp1.

ENPP1 in Blood and Bone: Skeletal and Soft Tissue Diseases Induced by ENPP1 Deficiency.

The enzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) codes for a type 2 transmembrane glycoprotein that hydrolyzes extracellular ATP to generate pyrophosphate (PPi) and adenosine monophosphate, thereby contributing to downstream purinergic signaling pathways. The clinical phenotypes induced by ENPP1 deficiency are seemingly contradictory and include early-onset osteoporosis in middle-aged adults and life-threatening vascular calcifications in the large arteries of infants with generalized arterial calcification of infancy. The progressive overmineralization of soft tissue and concurrent undermineralization of skeleton also occur in the general medical population, where it is referred to as paradoxical mineralization to highlight the confusing pathophysiology. This review summarizes the clinical presentation and pathophysiology of paradoxical mineralization unveiled by ENPP1 deficiency and the bench-to-bedside development of a novel ENPP1 biologics designed to treat mineralization disorders in the rare disease and general medical population.

ENPP1 downregulation and FGF23 upregulation in growth-related calcification of the tibial tuberosity in rats.

During tibial tuberosity growth, superficial and deep portions can be observed; however, the deep portion is not observed after the growth period, as it develops into bone tissues. Calcification in vivo is known to be constitutively suppressed by ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) but promoted by tissue-nonspecific alkaline phosphatase (TNAP). FGF23 promotes calcification of enthesis. Gene expression of FGF23 increased rapidly at 13W in this study. Therefore, the tibial tuberosity is speculated to develop via Enpp1 downregulation and Tnap upregulation; however, the understanding of these processes remains unclear. Hence, in the present study, we aimed to explore the age-related structural changes and underlying gene expression changes in the tibial tuberosity of rats. Male Wistar rats were divided into three groups (3-, 7-, and 13-week-old; eight each). The tibial tuberosity superficial and deep portions were clearly observed in 3- and 7-week-old rats, but the presence of the deep portion was not confirmed in 13-week-old rats. The extracellular matrix of hypertrophic chondrocytes was calcified. Furthermore, the Enpp1 expression was the highest in 3-week-old rats and decreased with growth. The TNAP expression did not differ significantly among the groups. The deep portion area was significantly lower in 3-week-old rats than in 7-week-old rats. Generally, the extracellular matrix of the immature chondrocytes is not calcified. Therefore, we speculated that the cartilaginous tibial tuberosity calcifies and ossifies with growth. The Enpp1 expression decreased with growth, whereas the Tnap expression remained unchanged. Thus, we surmise that the tibial tuberosity calcifies with growth and that this process involves Enpp1 downregulation and FGF23 upregulation. As Osgood-Schlatter disease is closely related to the calcification of the tibial tuberosity, these findings may help clarify the pathogenesis of this disease.