Upregulation of LHPP by saRNA inhibited hepatocellular cancer cell proliferation and xenograft tumor growth.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer worldwide and no pharmacological treatment is available that can achieve complete remission of HCC. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a recently identified HCC tumor suppressor gene which plays an important role in the development of HCC and its inactivation and reactivation has been shown to result in respectively HCC tumorigenesis and suppression. Small activating RNAs (saRNAs) have been used to achieve targeted activation of therapeutic genes for the restoration of their encoded protein through the RNAa mechanism. Here we designed and validated saRNAs that could activate LHPP expression at both the mRNA and protein levels in HCC cells. Activation of LHPP by its saRNAs led to the suppression of HCC proliferation, migration and the inhibition of Akt phosphorylation. When combined with targeted anticancer drugs (e.g., regorafenib), LHPP saRNA exhibited synergistic effect in inhibiting in vitro HCC proliferation and in vivo antitumor growth in a xenograft HCC model. Findings from this study provides further evidence for a tumor suppressor role of LHPP and potential therapeutic value of restoring the expression of LHPP by saRNA for the treatment of HCC.

Ultrasensitive NIR fluorometric assay for inorganic pyrophosphatase detection via Cu2+-PPi interaction using bimetallic Au-Ag nanoclusters.

BACKGROUND: Inorganic pyrophosphatase (PPase) is key enzyme playing a key role in biochemical transformations such as biosynthesis of DNA and RNA, bone formation, metabolic pathways associated with lipid, carbohydrate and phosphorous. It has been reported that lung adenocarcinomas, colorectal cancer, and hyperthyroidism disorders can result from abnormal level of PPase. Therefore, it is of notable significance to develop simple and effective real time assay for PPase enzyme activity monitoring for screening of many metabolic pathways as well as for early disease diagnosis. RESULT: The fluorometric detection of PPase enzyme in near infrared region-1 (NIR-1) has been carried out using bimetallic nanoclusters (LA@AuAg NCs). The developed sensing strategy was based on quenching of fluorescence intensity of LA@AuAg NCs upon interaction with copper (Cu2+) ions. The off state of LA@AuAg_Cu2+ ensemble was turned on upon addition of pyrophosphate anion (PPi) due to strong binding interaction between PPi and Cu2+. The catalytic conversion of PPi into phosphate anion (Pi) in the presence of PPase led to liberation of Cu2+ ions, and again quenched off state was retrieved due to interaction of free Cu2+ with LA@AuAg NCs. The ultrasensitive detection of PPase was observed in the linear range of 0.06-250 mU/mL with LOD as 0.0025 mU/mL. The designed scheme showed good selectivity towards PPase enzyme in comparison to other bio-substrates, along with good percentage recovery for PPase detection in real human serum samples. SIGNIFICANCE: The developed NIR based assay is ultrasensitive, highly selective and robust for PPase enzyme and can be safely employed for other enzymes detection. This highly sensitive nature of biosensor was result of involvement of fluorescence-based technique and synergistic effect of dual metal in NIR based bimetallic NCs. Moreover, owing to the emission in NIR domain, in future, these nanoclusters can be safely employed for many biomedical applications for In vivo studies.

Inorganic pyrophosphatase 1 activates the phosphatidylinositol 3-kinase/Akt signaling to promote tumorigenicity and stemness properties in colorectal cancer.

Colorectal cancer (CRC) is a common malignant tumor of the human digestive tract. Inorganic pyrophosphatase 1 (PPA1) plays an imperative role in the advancement of malignant tumors, but its function in CRC is ill-defined. In this study, we inspected the functions of PPA1 in CRC. The abundance of PPA1 in CRC tissues was analyzed by utilizing publicly available data from the The Cancer Genome Atlas and Human Protein Atlas project. Cell counting kit-8 assay and 5-ethynyl-2'-deoxyuridine assay were used to evaluate the viability and proliferation of CRC cells. Bioinformatics analysis was used to forecast the PPA1 related genes and signal pathways in CRC. The protein expression was examined by western blot. The xenograft model was implemented to determine the influence of PPA1 in CRC in vivo. Proliferating cell nuclear antigen, CD133, and CD44 contents in xenograft tumors were evaluated by immunohistochemistry. In the present study, we found that the PPA1 content was heightened in CRC, and the diagnostic value of PPA1 in CRC was enormous. Overexpression of PPA1 enhanced cell proliferation and stemness properties in CRC cells, while downregulation of PPA1 had the opposite effects. PPA1 promoted the activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Activation of the PI3K/Akt signaling reversed the effect of PPA1 silencing on cell proliferation and stemness properties in CRC cells. Silencing of PPA1 reduced xenograft tumor growth via modulating the PI3K/Akt signaling pathway in vivo. In conclusion, PPA1 promoted cell proliferation and stemness properties in CRC by activating the PI3K/Akt signaling pathway.

HDAC4 mediated LHPP deacetylation enhances its destabilization and promotes the proliferation and metastasis of nasopharyngeal carcinoma.

Studies have shown that acetylation modification plays an important role in tumor proliferation and metastasis. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is downregulated in certain tumors, as a tumor suppressor role. However, the regulation of LHPP expression and its function in nasopharyngeal carcinoma (NPC) remain unclear. In the present study, we found that LHPP was downregulated in NPC, and overexpression of LHPP inhibited the proliferation and invasion of NPC cells. Mechanistically, HDAC4 deacetylated LHPP at K6 and promoted the degradation of LHPP through TRIM21 mediated K48-linked ubiquitination. HDAC4 was confirmed to be highly expressed in NPC cells and promoted the proliferation and invasion of NPC cells through LHPP. Further research found that LHPP could inhibit the phosphorylation of tyrosine kinase TYK2, thereby inhibiting the activity of STAT1. In vivo, knockdown of HDAC4 or treatment with small molecule inhibitor Tasquinimod targeting HDAC4 could significantly inhibit the proliferation and metastasis of NPC by upregulating LHPP. In conclusion, our finding demonstrated that HDAC4/LHPP signal axis promotes the proliferation and metastasis of NPC through upregulating TYK2-STAT1 phosphorylation activation. This research will provide novel evidence and intervention targets for NPC metastasis.

LHPP promotes the intracellular reactive oxygen species accumulation and sensitivity of gastric cancer to cisplatin via JNK and p38 MAPK pathways.

BACKGROUND: Cisplatin is the first-line chemotherapy drug for the treatment of gastric cancer (GC) patients. However, GC patients who are resistant to cisplatin often do not benefit from it. Therefore, finding a key molecule that affects cisplatin sensitivity is expected to enhance the efficacy of cisplatin in GC treatment. METHODS: The human GC cell lines SGC-7901 and BGC-823 were used. The protein chip array was used to screen the cisplatin-resistance genes from the complete response and non-complete response GC patients' tissues, then, the differential gene expression analysis, GO function annotation analysis, and KEGG pathway enrichment analysis were performed. The GC tissue chip in the GEO database was analyzed to screen the target gene. Flow cytometry, Hoechst 33342 staining assay, Western Blot, MTT, tumor sphere formation, cell cycle, and apoptosis assays were performed to explore the effect of Phospholysine Phosphohistidine Inorganic Pyrophosphate Phosphatase (LHPP) on the apoptosis, stemness, and reactive oxygen species (ROS) accumulation of cisplatin-resistant GC cells treated with cisplatin. In vivo, the cisplatin-resistant GC cell lines transfected with pcDNA-LHPP or si-LHPP were injected subcutaneously into mice to construct GC subcutaneous xenograft GC models. RESULTS: Based on protein chip array and bioinformatics analysis, it was found that LHPP is the core molecule in the cisplatin resistance regulatory network in GC, and its expression is down-regulated in GC cisplatin-resistant tissues and cells. In vitro and in vivo experimental results show that the up-regulated expression of LHPP is closely related to the increase in sensitivity of GC to cisplatin. Mechanically, we found that overexpression of LHPP may inhibit the activation of the JNK and p38 MAPK pathways, promote cisplatin-induced ROS accumulation, suppress stemness, and enhance the sensitivity of GC to cisplatin. CONCLUSIONS: Up-regulation of LHPP may inhibit the activation of the JNK and p38 MAPK pathways, attenuate stemness, and enhance the accumulation of intracellular ROS, thereby promoting cisplatin-mediated GC cell apoptosis and enhancing cisplatin sensitivity.

Washing-Free and Label-Free Onsite Assay for Inorganic Pyrophosphatase Activity Using a Personal Glucose Meter.

In this study, we demonstrated a personal glucose meter-based method for washing-free and label-free inorganic pyrophosphatase (PPase) detection, which relies on the cascade enzymatic reaction (CER) promoted by hexokinase and pyruvate kinase. In principle, the absence of target PPase enables adenosine triphosphate sulfurylase to catalyze the conversion of pyrophosphate (PPi) to ATP, a substrate of CER, which results in the significant reduction of glucose levels by the effective CER process. In contrast, the PPi cleavage activity works in the presence of target PPase by decomposing PPi to orthophosphate (Pi). Therefore, the CER process cannot be effectively executed, leading to the maintenance of the initial high glucose level that may be measured by a portable personal glucose meter. Based on this novel strategy, a quantitative evaluation of the PPase activity may be achieved in a dynamic linear range of 1.5-25 mU/mL with a detection limit of 1.18 mU/mL. Compared with the previous PPase detection methods, this method eliminates the demand for expensive and bulky analysis equipment as well as a complex washing step. More importantly, the diagnostic capability of this method was also successfully verified by reliably detecting PPase present in an undiluted human serum sample with an excellent recovery ratio of 100 ± 2%.

The histidine phosphatase LHPP: an emerging player in cancer.

Cancers continue to have high incidence and mortality rates worldwide. Therefore, cancer control remains the main public health goal. Growing research evidence suggests that phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) plays an important role in inhibiting tumor cell progression. It has been reported in the literature that LHPP is expressed at low levels in tumor tissues and cells and that patients with low LHPP expression have a poorer prognosis. Functional studies have shown that LHPP can inhibit tumor cell proliferation, metastasis, and apoptosis by affecting different target genes. In addition, researchers have used iDPP nanoparticles to deliver LHPP plasmids to treat tumors, demonstrating the great potential of LHPP plasmids for cancer therapy. In our review, we highlight the biological functions and important downstream target genes of LHPP in tumors, providing a theoretical basis for the treatment of human cancers. Although not thoroughly studied in terms of tumor mechanisms, LHPP still represents a promising and effective anticancer drug target.

Circ_0067934 reduces JNK phosphorylation through a microRNA-545-3p/PPA1 axis to enhance tumorigenesis and cisplatin resistance in ovarian cancer.

BACKGROUND: Circular RNA 0067934 (circ_0067934) has been revealed as a cancer driver in multiple human malignancies, whereas its action in the pathogenesis of ovarian cancer (OC) remains unclear. This study focuses on the function of circ_0067934 in tumorigenesis and cisplatin (DDP) resistance in OC and the molecular mechanism. METHODS: Expression of circ_0067934 in OC tissues and cells was examined, and its correlation with the clinical characteristics of patients was analyzed. Candidate targets of circ_0067934 were predicted using bioinformatics systems. Binding relationships between circ_0067934 and microRNA (miR)-545-3p and between miR-545-3p and inorganic pyrophosphatase 1 (PPA1) were validated via luciferase assays. Gain- and loss-of functions of circ_0067934, miR-545-3p and PPA1 were performed to determine their functions in proliferation, invasion, apoptosis and DDP resistance of OC cells in vitro and in vivo. RESULTS: Circ_0067934 was overexpressed in OC samples and associated with advanced tumor staging and lymph node metastasis. Downregulation of circ_0067934 reduced DDP resistance of the DDP-resistant A2780/DDP cell line and reduced cell proliferation and invasion, but the malignant behaviors of OC cells were restored after further miR-545-3p downregulation. Circ_0067934 served as a sponge for miR-545-3p and diminished its suppressive effect on PPA1 translation. Artificial upregulation of PPA1 enhanced proliferation, invasion and DDP resistance of A2780/DDP cells, and it reduced phosphorylation of the pro-apoptotic JNK signaling. Similar results were found in vivo. CONCLUSION: This study suggests that circ_0067934 sequesters miR-545-3p and enhances PPA1 expression to promote tumorigenesis and DDP resistance in OC. This study may provide novel approaches in the management of OC.

As a Novel Tumor Suppressor, LHPP Promotes Apoptosis by Inhibiting the PI3K/AKT Signaling Pathway in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) refers to the malignant tumor of the head and neck with a highest morbidity. It exhibits a poor prognosis and unsatisfactory treatment partially attributed to delayed diagnosis. As indicated from existing reports, the protein histidine phosphatase LHPP acts as a vital factor in tumorigenesis in liver, lung, bladder, breast and pancreatic tumor tissues. Thus far, the functional mechanism of LHPP in OSCC remains unclear. DGE analysis, OSCC cell lines and OSCC cases were found that LHPP was down-regulated in OSCC tissues and cells compared with that in normal oral mucosa tissues and cells, and was closely related to OSCC differentiation. Cell counting Kit 8 test, EdU proliferation test, scratches test, invasion test, monoclonal formation test, mouse xenograft tumor model, HE staining and immunohistochemistry showed that LHPP inhibited OSCC growth, proliferation and migration in vivo and in vitro. GO and KEGG enrichment analysis, LHPP transcription factor analysis and flow cytometry found that LHPP promotes the apoptosis of OSCC by decreasing the transcriptional activity of p-PI3K and p-Akt. Finally, our results suggested that LHPP inhibited the progression of OSCC through the PI3K/AKT signaling pathway, indicating that LHPP may be a new target for the treatment of OSCC.

Functional Analysis of H+-Pumping Membrane-Bound Pyrophosphatase, ADP-Glucose Synthase, and Pyruvate Phosphate Dikinase as Pyrophosphate Sources in Clostridium thermocellum.

The atypical glycolysis of Clostridium thermocellum is characterized by the use of pyrophosphate (PPi) as a phosphoryl donor for phosphofructokinase (Pfk) and pyruvate phosphate dikinase (Ppdk) reactions. Previously, biosynthetic PPi was calculated to be stoichiometrically insufficient to drive glycolysis. This study investigates the role of a H+-pumping membrane-bound pyrophosphatase, glycogen cycling, a predicted Ppdk-malate shunt cycle, and acetate cycling in generating PPi. Knockout studies and enzyme assays confirmed that clo1313_0823 encodes a membrane-bound pyrophosphatase. Additionally, clo1313_0717-0718 was confirmed to encode ADP-glucose synthase by knockouts, glycogen measurements in C. thermocellum, and heterologous expression in Escherichia coli. Unexpectedly, individually targeted gene deletions of the four putative PPi sources did not have a significant phenotypic effect. Although combinatorial deletion of all four putative PPi sources reduced the growth rate by 22% (0.30 ± 0.01 h-1) and the biomass yield by 38% (0.18 ± 0.00 gbiomass gsubstrate-1), this change was much smaller than what would be expected for stoichiometrically essential PPi-supplying mechanisms. Growth-arrested cells of the quadruple knockout readily fermented cellobiose, indicating that the unknown PPi-supplying mechanisms are independent of biosynthesis. An alternative hypothesis that ATP-dependent Pfk activity circumvents a need for PPi altogether was falsified by enzyme assays, heterologous expression of candidate genes, and whole-genome sequencing. As a secondary outcome, enzymatic assays confirmed functional annotation of clo1313_1832 as ATP- and GTP-dependent fructokinase. These results indicate that the four investigated PPi sources individually and combined play no significant PPi-supplying role, and the true source(s) of PPi, or alternative phosphorylating mechanisms, that drive(s) glycolysis in C. thermocellum remain(s) elusive. IMPORTANCE Increased understanding of the central metabolism of C. thermocellum is important from a fundamental as well as from a sustainability and industrial perspective. In addition to showing that H+-pumping membrane-bound PPase, glycogen cycling, a Ppdk-malate shunt cycle, and acetate cycling are not significant sources of PPi supply, this study adds functional annotation of four genes and availability of an updated PPi stoichiometry from biosynthesis to the scientific domain. Together, this aids future metabolic engineering attempts aimed to improve C. thermocellum as a cell factory for sustainable and efficient production of ethanol from lignocellulosic material through consolidated bioprocessing with minimal pretreatment. Getting closer to elucidating the elusive source of PPi, or alternative phosphorylating mechanisms, for the atypical glycolysis is itself of fundamental importance. Additionally, the findings of this study directly contribute to investigations into trade-offs between thermodynamic driving force versus energy yield of PPi- and ATP-dependent glycolysis.

Three LHPP gene-targeting co-expressed microRNAs (microRNA-765, microRNA-21, and microRNA-144) promote proliferation, epithelial-mesenchymal transition, invasion, and are independent prognostic biomarkers in renal cell carcinomas patients.

BACKGROUND: Renal cell carcinoma (RCC) is one of the highly malignant tumors in the world. Global Cancer Statistics 2020 estimated that there were 179,368 deaths from kidney tumors. Therefore, exploring the prognostic biomarkers of RCC is of great significance for RCC patients. This study aims to explore the potential mechanism and prognostic value of phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) gene-targeting co-expression microRNAs in RCC patients. METHODS: A total of 60 RCC patients were included. Quantitative real-time PCR (qRT-PCR), western blotting, and immunohistochemistry were used for LHPP, microRNA-765, microRNA-21, and microRNA-144 levels evaluation. Cell Counting Kit-8 assay, dual-luciferase reporter gene assay, invasion assay, and RNA fluorescence in situ hybridization were used for functional analyses. RESULTS: Compared with adjacent tissues, LHPP levels in cancer tissues were significantly increased (p < .001). Herein, we confirmed that microRNA-765, microRNA-21, and microRNA-144 were direct biological targets of LHPP. MicroRNA-765 (r = -0.570, p < 0.001), microRNA-21 (r = -0.495, p < .001), and microRNA-144 (r = -0.463, p < .001) expression levels were negatively correlated with LHPP expression levels. The high expression levels of microRNA-765, microRNA-21, and microRNA-144 in RCC tissues were associated with poor differentiation, recurrence, and poor prognosis (p < .05). In vitro, microRNA-765, microRNA-21, and microRNA-144 act as oncogenes to promote proliferation, invasion, and epithelial-mesenchymal transition (EMT) through targeting LHPP. CONCLUSIONS: MicroRNA-765, microRNA-21, and microRNA-144 are independent risk biomarkers for RCC patients. Inhibiting the expression levels of microRNA-765, microRNA-21, and microRNA-144 can reduce the proliferation, EMT, and invasion of RCC cells. Therefore, the above three microRNAs are expected to become molecular biomarkers for RCC therapy.

Downregulation of LHPP Expression Associated with AFP Acts as a Good Prognostic Factor in Human Hepatocellular Carcinoma.

BACKGROUND: Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) serves as a tumor suppressor in hepatocellular carcinoma (HCC), but the correlation between the expression of LHPP and the clinical parameters of oncogenic progression is still not well defined. This study is to reveal the correlation between the expression of LHPP in HCC and their clinical parameters. METHODS: Immunohistochemical analysis was used to assess the correlation between the expression of LHPP and the clinical parameters of HCC. Expressions of LHPP in HCC tissues and cultured HCC cells were detected by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). LHPP, gamma-glutamyl transferase (GGT), and α-fetoprotein (AFP) expression levels in blood or HCC tissues were detected by enzyme-linked immunosorbent assay (ELISA). The Spearman rank correlation coefficient was used to evaluate the correlation of the expression of LHPP and the clinical index of HCC. Correlation of survival and expression of LHPP were analyzed using the Kaplan-Meier method and the log-rank test. RESULTS: Expressions of LHPP in HCC tissues were significantly downregulated than their paired adjacent normal tissues. A significant positive correlation was found between the cytoplasm and nuclear expression of LHPP in both HCC and their paired adjacent normal tissues. The expression of LHPP negatively correlated with the levels of GGT in the cytoplasm of adjacent tissues and with the AFP level in the nucleus of HCC cells. Relative levels of LHPP in HCC tissues were markedly lower than those of the paired adjacent normal tissues. Relative levels of LHPP in LO-2 cells were higher than those of HepG2, BEL-7404, and SMMC-7721 cell lines. The overall survival and DSF survival of patients with the high expression of LHPP were much higher than those with the low expression of LHPP in paired adjacent normal tissue. CONCLUSIONS: LHPP is associated with the AFP level and acts as a good prognostic factor in HCC.

Prognostic Correlation of Glycolysis-Related Gene Signature in Patients with Laryngeal Cancer.

BACKGROUND: Aerobic glycolysis is one of the metabolic characteristics of tumor cells, which is regulated by many genes. The aim of our study was to construct glycolysis-related gene signature to accurately predict the prognosis of laryngeal cancer (LC) patients. METHODS: We analyzed the mRNA expression profiles of LC patients from The Cancer Genome Atlas (TCGA). Eleven glycolysis-related gene sets were analyzed by gene set enrichment analysis (GSEA). In order to acquire the gene signature related to prognosis, we used univariate and multivariate Cox regression analysis. RESULTS: We confirmed that a gene signature composed of two genes (STC2, LHPP) can predict the overall survival (OS) of patients with LC. Based on each patient's risk score, we found that the survival results of patients in the high-risk group were significantly lower than those in the low-risk group (log-rank test P-value=0.002). Multivariate Cox regression analysis confirmed that gene signature could independently predict OS in LC patients (HR = 1.981, 95% CI 1.446-2.714 P<0.001). In addition, a nomogram including the age, sex, grade and risk score was constructed. The nomogram demonstrated good accuracy for OS prediction, with a C-index of 0.752. CONCLUSION: The glycolysis-related two-gene risk score model could be used as a biomarker for LC prognosis.

Crystallographic and modeling study of the human inorganic pyrophosphatase 1: A potential anti-cancer drug target.

Inorganic pyrophosphatases (PPases) catalyze the hydrolysis of pyrophosphate to phosphates. PPases play essential roles in growth and development, and are found in all kingdoms of life. Human possess two PPases, PPA1 and PPA2. PPA1 is present in all tissues, acting largely as a housekeeping enzyme. Besides pyrophosphate hydrolysis, PPA1 can also directly dephosphorylate phosphorylated c-Jun N-terminal kinases 1 (JNK1). Upregulated expression of PPA1 has been linked to many human malignant tumors. PPA1 knockdown induces apoptosis and decreases proliferation. PPA1 is emerging as a potential prognostic biomarker and target for anti-cancer drug development. In spite of the biological and physiopathological importance of PPA1, there is no detailed study on the structure and catalytic mechanisms of mammalian origin PPases. Here we report the crystal structure of human PPA1 at a resolution of 2.4 Å. We also carried out modeling studies of PPA1 in complex with JNK1 derived phosphor-peptides. The monomeric protein fold of PPA1 is similar to those found in other family I PPases. PPA1 forms a dimeric structure that should be conserved in animal and fungal PPases. Analysis of the PPA1 structure and comparison with available structures of PPases from lower organisms suggest that PPA1 has a largely pre-organized and relatively rigid active site for pyrophosphate hydrolysis. Results from the modeling study indicate the active site of PPA1 has the potential to accommodate double-phosphorylated peptides from JNK1. In short, results from the study provides new insights into the mechanisms of human PPA1 and basis for structure-based anti-cancer drug developments using PPA1 as the target.

LHPP suppresses tumorigenesis of intrahepatic cholangiocarcinoma by inhibiting the TGFß/smad signaling pathway.

Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP), a histidine phosphatase, plays an important role in tumor progression and metastasis as a tumor suppressor. Here, we investigate the effect of LHPP in intrahepatic cholangiocarcinoma (ICC). We discovered that LHPP was downregulated in tumor tissues and low levels of LHPP predicted poor survival. LHPP inhibited ICC cell growth, cell invasion and epithelial-mesenchymal transition (EMT) in vitro and in vivo. Mechanically, LHPP deactivated transforming growth factor­beta (TGFß) signaling pathway, and low level LHPP upregulated the expression of TGFß and phosphorylation of smad2/3. Moreover, inhibition of this pathway reversed the biofunction of LHPP. In summary, these findings demonstrated that LHPP suppressed ICC through inhibiting the activation of TGFß/smad signaling. Our results indicated that LHPP is a potential therapeutic target in ICC.

Improving ATPase and PPase activities, nutrient uptake and growth of salt stressed ajowan plants by salicylic acid and iron-oxide nanoparticles.

KEY MESSAGE: Salicylic acid and iron-oxide nanoparticles alleviated salt toxicity and improved plant growth by stimulating the activities of H+-ATPase and H+-PPase and preventing nutrient imbalance. Two factorial experiments were undertaken in a greenhouse during 2018 and 2019, to evaluate the impacts of SA (1 mM) and nano-Fe2O3 (3 mM) sprays at 7 leaves and flowering stages on vacuolar H+-pumps, growth and essential oil of salt-subjected (0, 4, 8 and 12 dS m-1 NaCl) ajowan plants. Measurements of plant traits were started at about 12 days after the last foliar spray and continued up to maturity. The H+-ATPase and H+-PPase activities and root ATP content were enhanced under low salinity, but higher salinities reduced these parameters. Rising salinity enhanced Na uptake and translocation, endogenous SA and DPPH activity, while reduced K+/Na+ ratio and nutrients uptake, leading to a reduction in plant biomass. Treatment with SA, nano-Fe2O3 and their combination improved H+-pumps activities and ATP content in roots and leaves. The SA-related treatments caused the highest activities of H+-pumps in roots, but Fe-related treatments resulted in the highest activities of these pumps in leaves. Increasing H+-pumps activities reduced sodium uptake and translocation and enhanced nutrients uptake. Foliar treatments, especially SA + nano-Fe2O3 augmented endogenous SA, DPPH activity, and plant growth in salt-stressed plants. Essential oil contents of vegetative and inflorescence organs under severe salinity and seeds under moderate and severe salinities were enhanced. Maximum essential oil was obtained from seeds of SA + nano-Fe2O3-treated plants, which was strongly correlated with endogenous SA and DPPH. Nevertheless, the SA + nano-Fe2O3 was the best treatment for diminishing salt toxicity and improving ajowan plant growth and essential oil production.

LHPP exerts a tumor-inhibiting role in glioblastoma via the downregulation of Akt and Wnt/ß-catenin signaling.

Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) has been recently identified as a novel inhibitor of multiple tumors; however, its role in glioblastoma (GBM) has not been investigated. This study aimed to evaluate whether LHPP exerts a potential tumor-inhibiting role in GBM. Compared with that in normal tissues, LHPP expression was lower in GBM tissues and various GBM cell lines. LHPP up-regulation in GBM cells markedly reduced their proliferation and invasion, and its knockdown had an oncogenic effect on these cells. Further studies revealed that overexpressed LHPP decreased the levels of Akt and glycogen synthase-3ß phosphorylation and down-regulated Wnt/ß-catenin signaling. By contrast, LHPP knockdown produced opposite effects. Akt suppression markedly abrogated the activation of Wnt/ß-catenin signaling induced by LHPP knockdown. The reactivation of Wnt/ß-catenin signaling partially reversed the inhibition of tumor growth in GBM mediated by LHPP overexpression. In addition, LHPP overexpression markedly retarded the tumorigenesis of GBM cells in vivo. These findings revealed that LHPP acts a potential inhibitor of tumor growth in GBM, and its overexpression represses GBM proliferation and invasion by down-regulating Akt and Wnt/ß-catenin signaling. This work highlights the crucial role of LHPP in GBM progression and suggests its potential as an anticancer target for the treatment of this disease.

Purpurin binding interacts with LHPP protein that inhibits PI3K/AKT phosphorylation and induces apoptosis in colon cancer cells HCT-116.

Colorectal cancer (CRC) is the leading type of diagnosed cancer; globally, it resides in the fourth-leading origin of cancer-interrelated mortality in the globe. The treatment strategies were chemotherapy and potent radiotherapy. Although chemotherapy treatment can eliminate tumor cells, it remains with unnecessary toxic effects in cancer patients. Therefore, the identification of natural-based compounds, which have selectively inhibiting target proteins with limited toxicity that can facilitate the therapeutic approaches against CRC. In this existing approach, which highlights the binding efficacy of our anthraquinone compound, purpurin against phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) protein restrains the CRC cell growth by inhibiting phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT), cell proliferation, and inducing apoptosis signaling. Primarily, purpurin (36 µM) exposed to HCT-116 cells and incubated for 24 and 48 h could induce reactive oxygen species production, subsequently alter mitochondrion membrane, and increase the apoptotic cells in HCT-116. LHPP, a kind of histidine phosphatase protein, has been considered as a tumor suppressor in numerous carcinomas. However, purpurin-mediated LHPP proteins and its associated molecular events in CRC remain unclear. In our docking studies revealed that purpurin has been strongly interacts with LHPP via hydrophobic and hydrophilic binding interaction. Western blot results confirmed that purpurin enhances the expression of LHPP protein, thereby inhibits the expression of phosphorylated-PI3K/AKT, EGFR, cyclin-D1, PCNA in HCT-116 cells. Moreover, purpurin induces messenger RNA expression of apoptotic genes (Bax, CASP-9, and CASP-3) in HCT-116 cells. Thus, we conclude that purpurin could be a natural and useful compound, which inhibits the growth of CRC cells through the activation of LHPP proteins.

Constitutive inorganic pyrophosphatase as a reciprocal regulator of three inducible enzymes in Escherichia coli.

BACKGROUND: Previous studies have demonstrated the formation of stable complexes between inorganic pyrophosphatase (PPase) and three other Escherichia coli enzymes - cupin-type phosphoglucose isomerase (cPGI), class I fructose-1,6-bisphosphate aldolase (FbaB) and l-glutamate decarboxylase (GadA). METHODS: Here, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure. RESULTS: cPGI activity was modulated by all partner proteins, but none was reciprocally affected by cPGI. PPase activity was down-regulated upon complex formation, whereas all other enzymes were up-regulated. For cPGI, the activation was partially counteracted by a shift in dimer ⇆ hexamer equilibrium to inactive hexamer. Complex stoichiometry appeared to be 1:1 in most cases, but FbaB formed both 1:1 and 1:2 complexes with both GadA and PPase, FbaB activation was only observed in the 1:2 complexes. FbaB and GadA induced functional asymmetry (negative kinetic cooperativity) in hexameric PPase, presumably by favoring partial dissociation to trimers. CONCLUSIONS: These four enzymes form all six possible binary complexes in vitro, resulting in modulated activity of at least one of the constituent enzymes. In five complexes, the effects on activity were unidirectional, and in one complex (FbaB⋅PPase), the effects were reciprocal. The effects of potential physiological significance include inhibition of PPase by FbaB and GadA and activation of FbaB and cPGI by PPase. Together, they provide a mechanism for feedback regulation of FbaB and GadA biosynthesis. GENERAL SIGNIFICANCE: These findings indicate the complexity of functionally significant interactions between cellular enzymes, which classical enzymology treats as individual entities, and demonstrate their moonlighting activities as regulators.

Development of a chemical probe against NUDT15.

The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.