(p)ppGpp controls stringent factors by exploiting antagonistic allosteric coupling between catalytic domains.

Amino acid starvation is sensed by Escherichia coli RelA and Bacillus subtilis Rel through monitoring the aminoacylation status of ribosomal A-site tRNA. These enzymes are positively regulated by their product-the alarmone nucleotide (p)ppGpp-through an unknown mechanism. The (p)ppGpp-synthetic activity of Rel/RelA is controlled via auto-inhibition by the hydrolase/pseudo-hydrolase (HD/pseudo-HD) domain within the enzymatic N-terminal domain region (NTD). We localize the allosteric pppGpp site to the interface between the SYNTH and pseudo-HD/HD domains, with the alarmone stimulating Rel/RelA by exploiting intra-NTD autoinhibition dynamics. We show that without stimulation by pppGpp, starved ribosomes cannot efficiently activate Rel/RelA. Compromised activation by pppGpp ablates Rel/RelA function in vivo, suggesting that regulation by the second messenger (p)ppGpp is necessary for mounting an acute starvation response via coordinated enzymatic activity of individual Rel/RelA molecules. Control by (p)ppGpp is lacking in the E. coli (p)ppGpp synthetase SpoT, thus explaining its weak synthetase activity.

Nudix hydrolase 23 post-translationally regulates carotenoid biosynthesis in plants.

Carotenoids are essential for photosynthesis and photoprotection. Plants must evolve multifaceted regulatory mechanisms to control carotenoid biosynthesis. However, the regulatory mechanisms and the regulators conserved among plant species remain elusive. Phytoene synthase (PSY) catalyzes the highly regulated step of carotenogenesis and geranylgeranyl diphosphate synthase (GGPPS) acts as a hub to interact with GGPP-utilizing enzymes for the synthesis of specific downstream isoprenoids. Here, we report a function of Nudix hydrolase 23 (NUDX23), a Nudix domain-containing protein, in post-translational regulation of PSY and GGPPS for carotenoid biosynthesis. NUDX23 expresses highly in Arabidopsis (Arabidopsis thaliana) leaves. Overexpression of NUDX23 significantly increases PSY and GGPPS protein levels and carotenoid production, whereas knockout of NUDX23 dramatically reduces their abundances and carotenoid accumulation in Arabidopsis. NUDX23 regulates carotenoid biosynthesis via direct interactions with PSY and GGPPS in chloroplasts, which enhances PSY and GGPPS protein stability in a large PSY-GGPPS enzyme complex. NUDX23 was found to co-migrate with PSY and GGPPS proteins and to be required for the enzyme complex assembly. Our findings uncover a regulatory mechanism underlying carotenoid biosynthesis in plants and offer promising genetic tools for developing carotenoid-enriched food crops.

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.

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.

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.

Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

Pthlha and mechanical force control early patterning of growth zones in the zebrafish craniofacial skeleton.

Secreted signals in patterning systems often induce repressive signals that shape their distributions in space and time. In developing growth plates (GPs) of endochondral long bones, Parathyroid hormone-like hormone (Pthlh) inhibits Indian hedgehog (Ihh) to form a negative-feedback loop that controls GP progression and bone size. Whether similar systems operate in other bones and how they arise during embryogenesis remain unclear. We show that Pthlha expression in the zebrafish craniofacial skeleton precedes chondrocyte differentiation and restricts where cells undergo hypertrophy, thereby initiating a future GP. Loss of Pthlha leads to an expansion of cells expressing a novel early marker of the hypertrophic zone (HZ), entpd5a, and later HZ markers, such as ihha, whereas local Pthlha misexpression induces ectopic entpd5a expression. Formation of this early pre-HZ correlates with onset of muscle contraction and requires mechanical force; paralysis leads to loss of entpd5a and ihha expression in the pre-HZ, mislocalized pthlha expression and no subsequent ossification. These results suggest that local Pthlh sources combined with force determine HZ locations, establishing the negative-feedback loop that later maintains GPs.

ENPP1 variants in patients with GACI and PXE expand the clinical and genetic heterogeneity of heritable disorders of ectopic calcification.

Pseudoxanthoma elasticum (PXE) and generalized arterial calcification of infancy (GACI) are clinically distinct genetic entities of ectopic calcification associated with differentially reduced circulating levels of inorganic pyrophosphate (PPi), a potent endogenous inhibitor of calcification. Variants in ENPP1, the gene mutated in GACI, have not been associated with classic PXE. Here we report the clinical, laboratory, and molecular evaluations of ten GACI and two PXE patients from five and two unrelated families registered in GACI Global and PXE International databases, respectively. All patients were found to carry biallelic variants in ENPP1. Among ten ENPP1 variants, one homozygous variant demonstrated uniparental disomy inheritance. Functional assessment of five previously unreported ENPP1 variants suggested pathogenicity. The two PXE patients, currently 57 and 27 years of age, had diagnostic features of PXE and had not manifested the GACI phenotype. The similarly reduced PPi plasma concentrations in the PXE and GACI patients in our study correlate poorly with their disease severity. This study demonstrates that in addition to GACI, ENPP1 variants can cause classic PXE, expanding the clinical and genetic heterogeneity of heritable ectopic calcification disorders. Furthermore, the results challenge the current prevailing concept that plasma PPi is the only factor governing the severity of ectopic calcification.

ENPP1's regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating STING signaling.

The metazoan innate immune second messenger 2'3'-cGAMP is present both inside and outside cells. However, only extracellular cGAMP can be negatively regulated by the extracellular hydrolase ENPP1. Here, we determine whether ENPP1's regulation of extracellular cGAMP is a ubiquitous mechanism of attenuating stimulator of interferon genes (STING) signaling. We identified ENPP1H362A, a point mutation that cannot degrade the 2'-5' linkage in cGAMP while maintaining otherwise normal function. The selectivity of this histidine is conserved down to bacterial nucleotide pyrophosphatase/phosphodiesterase (NPP), allowing structural analysis and suggesting an unexplored ancient history of 2'-5' cyclic dinucleotides. Enpp1H362A mice demonstrated that extracellular cGAMP is not responsible for the devastating phenotype in ENPP1-null humans and mice but is responsible for antiviral immunity and systemic inflammation. Our data define extracellular cGAMP as a pivotal STING activator, identify an evolutionarily critical role for ENPP1 in regulating inflammation, and suggest a therapeutic strategy for viral and inflammatory conditions by manipulating ENPP1 activity.

Expression of human dCTP pyrophosphatase 1 (DCTPP1) and its association with cisplatin resistance characteristics in ovarian cancer.

Cisplatin (DDP) resistance is a major challenge in treating ovarian cancer patients. A recently discovered enzyme called dCTP pyrophosphatase 1 (DCTPP1) has been implicated in regulating cancer characteristics, including drug responses. In this study, we aimed to understand the role of DCTPP1 in cancer progression and cisplatin response. Using publicly available databases, we analysed the expression and clinical significance of DCTPP1 in ovarian cancer. Our bioinformatics analysis confirmed that DCTPP1 is significantly overexpressed in ovarian cancer and is closely associated with tumour progression and poor prognosis after cisplatin treatment. We also found that DCTPP1 located in oxidoreductase complex and may be involved in various biological processes related to cisplatin resistance, including pyrimidine nucleotide metabolism, the P53 signalling pathway and cell cycle signalling pathways. We observed higher expression of DCTPP1 in cisplatin-resistant cells (SKOV3/DDP) and samples compared to their sensitive counterparts. Additionally, we found that DCTPP1 expression was only enhanced in SKOV3/S cells when treated with cisplatin, indicating different expression patterns of DCTPP1 in cisplatin-sensitive and cisplatin-resistant cancer cells. Our study further supports the notion that cisplatin induces intracellular reactive oxygen species (ROS) and triggers cancer cell death through excessive oxidative stress. Knocking out DCTPP1 reversed the drug resistance of ovarian cancer cells by enhancing the intracellular antioxidant stress response and accumulating ROS. Based on our research findings, we conclude that DCTPP1 has prognostic value for ovarian cancer patients, and targeting DCTPP1 may be clinically significant in overcoming cisplatin resistance in ovarian cancer.

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.

Novel variant in Nudix hydrolase 15 gene influences 6-mercaptopurine toxicity in childhood acute lymphoblastic leukemia patients.

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. 6-Mercaptopurine (6-MP) is a key component of ALL treatment. Its use, however, is also associated with adverse drug reactions, particularly myelosuppression. Thiopurine S-methyltransferase (TPMT) and, more recently, Nudix hydrolase 15 (NUDT15) deficiency, due to no-function variants in their respective genes, are well known for their role in the development of this toxicity. Two novel genetic variants, rs12199316 in TPMT and rs73189762 in the NUDT15 gene, were recently identified by targeted sequencing. The latter is particularly interesting because of its potential association with 6-MP intolerance. Here, we assessed the relationship of this variant with the risk of myelosuppression and 6-MP dose intensity in 275 patients treated with Dana Farber Cancer Institute ALL protocols at the Sainte Justine University Health Center. Carriers of the NUDT15 rs73189762 variant allele were at a higher risk of myelosuppression, as shown by absolute phagocyte count reduction during consolidation II and maintenance phases of therapy. Reduction in 6-MP dose intensity was observed in patients with both rs73189762 and known no-function variants in the NUDT15 and TPMT genes. This finding supports the initial observation and suggests that 6-MP dose reduction might be beneficial for individuals with this genotype combination.

Predictive role of ITPA genetic variants in thiopurine-related myelotoxicity in Crohn's disease patients.

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.

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.

Association of NUDT15 gene polymorphism with adverse reaction, treatment efficacy, and dose of 6-mercaptopurine in patients with acute lymphoblastic leukemia: a systematic review and meta-analysis.

6-mercaptopurine (6-MP) serves as the backbone in the maintenance regimens of acute lymphoblastic leukemia (ALL). We aimed to evaluate the influence of NUDT15 gene polymorphism on the risk of myelosupression, hepatotoxicity and interruption of 6-MP, as well as treatment efficacy and dose of 6-MP in ALL patients. A total of 24 studies with 3,374 patients were included in this meta-analysis. We found 9-fold higher risk of 6-MP induced leukopenia (odds ratio [OR] =9.00, 95% confidence interval [CI]: 3.73-21.74) and 2.5-fold higher risk of 6-MP-induced neutropenia (OR=2.52, 95% CI: 1.72-3.69) for NUDT15 c.415C>T variant carriers in the dominant model. Moreover, we found that the dose intensity of 6-MP in ALL patients with one NUDT15 c.415C>T variant alleles (CT) was 19% less than that in wild-type patients (CC) (mean differences: 19.43%, 95% CI: -25.36 to -13.51). The tolerable dose intensity of 6-MP in NUDT15 c.415C>T homozygote variant (TT) and heterozygote variant (CT) carriers was 49% and 15% less than that in wild-type patients, respectively. The NUDT15 c.415C>T variant group (CT+TT) had seven times (OR=6.98, 95% CI: 2.83-17.22) higher risk of developing 6-MP intolerance than the CC group. However, NUDT15 c.415C>T polymorphism did not appear significantly associated with hepatotoxicity, treatment interruption or relapse incidence. We concluded that NUDT15 c.415C>T was a good predictor for 6-MP-induced myelosuppression in ALL patients. The dose intensity of 6-MP in ALL patients with NUDT15 c.415C>T variants was significantly lower than that in wild-type patients. This research provided a basis for further investigation into relations between NUDT15 gene and adverse reaction, treatment efficacy and dose intensity of 6-MP.

The ectoenzyme E-NPP3 negatively regulates ATP-dependent chronic allergic responses by basophils and mast cells.

Crosslinking of the immunoglobulin receptor FcεRI activates basophils and mast cells to induce immediate and chronic allergic inflammation. However, it remains unclear how the chronic allergic inflammation is regulated. Here, we showed that ecto-nucleotide pyrophosphatase-phosphodiesterase 3 (E-NPP3), also known as CD203c, rapidly induced by FcεRI crosslinking, negatively regulated chronic allergic inflammation. Basophil and mast cell numbers increased in Enpp3(-/-) mice with augmented serum ATP concentrations. Enpp3(-/-) mice were highly sensitive to chronic allergic pathologies, which was reduced by ATP blockade. FcεRI crosslinking induced ATP secretion from basophils and mast cells, and ATP activated both cells. ATP clearance was impaired in Enpp3(-/-) cells. Enpp3(-/-)P2rx7(-/-) mice showed decreased responses to FcεRI crosslinking. Thus, ATP released by FcεRI crosslinking stimulates basophils and mast cells for further activation causing allergic inflammation. E-NPP3 decreases ATP concentration and suppresses basophil and mast cell activity.

Regulation of ENPP5, a senescence-associated secretory phenotype factor, prevents skin aging.

Aging negatively affects the appearance and texture of the skin owing to the accumulation of senescent fibroblasts within the dermis. Senescent cells undergo abnormal remodeling of collagen and the extracellular matrix through an inflammatory histolytic senescence-associated secretory phenotype (SASP). Therefore, suppression of SASP in senescent cells is essential for the development of effective skin anti-aging therapies. Ectonucleotide pyrophosphatase/phosphodiesterase family member 5 (ENPP5), an extracellular signaling molecule, has been implicated in vascular aging and apoptosis; however, its role in SASP remains unclear. Therefore, this study aimed to investigate the role of ENPP5 in SASP and skin aging using molecular techniques. We investigated the effects of siRNA-mediated ENPP5 knockdown, human recombinant ENPP5 (rENPP5) treatment, and lentiviral overexpression of ENPP5 on SASP and aging in human skin fibroblasts. Additionally, we investigated the effect of siRNA-mediated ENPP5 knockdown on the skin of C57BL/6 mice. We found that ENPP5 was significantly expressed in replication-aged and otherwise DNA-damaged human skin fibroblasts and that treatment with human rENPP5 and lentiviral overexpression of ENPP5 promoted SASP and senescence. By contrast, siRNA-mediated knockdown of ENPP5 suppressed SASP and the expression of skin aging-related factors. Additionally, ENPP5 knockdown in mouse skin ameliorated the age-related reduction of subcutaneous adipose tissue, the panniculus carnosus muscle layer, and thinning of collagen fibers. Conclusively, these findings suggest that age-related changes may be prevented through the regulation of ENPP5 expression to suppress SASP in aging cells, contributing to the development of anti-aging treatments for the skin.

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.

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.

Disease-associated inosine misincorporation into RNA hinders translation.

Failure to prevent accumulation of the non-canonical nucleotide inosine triphosphate (ITP) by inosine triphosphate pyrophosphatase (ITPase) during nucleotide synthesis results in misincorporation of inosine into RNA and can cause severe and fatal developmental anomalies in humans. While the biochemical activity of ITPase is well understood, the pathogenic basis of ITPase deficiency and the molecular and cellular consequences of ITP misincorporation into RNA remain cryptic. Here, we demonstrate that excess ITP in the nucleotide pool during in vitro transcription results in T7 polymerase-mediated inosine misincorporation in luciferase RNA. In vitro translation of inosine-containing luciferase RNA reduces resulting luciferase activity, which is only partly explained by reduced abundance of the luciferase protein produced. Using Oxford Nanopore Direct RNA sequencing, we reveal inosine misincorporation to be stochastic but biased largely towards misincorporation in place of guanosine, with evidence for misincorporation also in place of cytidine, adenosine and uridine. Inosine misincorporation into RNA is also detected in Itpa-null mouse embryonic heart tissue as an increase in relative variants compared with the wild type using Illumina RNA sequencing. By generating CRISPR/Cas9 rat H9c2 Itpa-null cardiomyoblast cells, we validate a translation defect in cells that accumulate inosine within endogenous RNA. Furthermore, we observe hindered cellular translation of transfected luciferase RNA containing misincorporated inosine in both wild-type and Itpa-null cells. We therefore conclude that inosine misincorporation into RNA perturbs translation, thus providing mechanistic insight linking ITPase deficiency, inosine accumulation and pathogenesis.