PPase Activity Research Articles

Advanced electrochemical detection of pyrophosphatase activity in LNCaP cells utilizing in-situ redox of copper

The necessity for precise delineation and quantitation of pyrophosphatase (PPase) activities is underscored, especially in aggressive prostate cancer, driving the demand for more refined analytical techniques due to challenges associated with inadequate sensitivity and poor signal amplification. In response, a novel electrochemical method utilizing in-situ copper(II) (Cu(II))-based Fast Scan Cyclic Voltammetry (FSCV) is introduced for the advanced detection of PPase in LNCaP prostate cancer cells. This technique is characterized by the exploitation of competitive binding between Cu(II) and L-Phenylalanine (L-Phe) versus pyrophosphate (PPi), with FSCV technology enhanced by ohmic drop compensation to improve detection sensitivity and signal reliability. Through the application of the FSCV technique transitioned to Current(I)-Voltage(V) analysis, the signal generation is facilitated by an L-Phe-Cu(II) complex, driven by the redox reaction of in-situ Cu(II), specifically its rapid transition from the Cu(0) state back to the Cu(II) state. When this complex is anchored onto a gold substrate modified with cucurbit[7]uril (CB[7]) through supramolecular host–guest interactions, a significant FSCV signal is elicited. A detection limit of 0.05 mU/mL (S/N = 3) is subsequently achieved when the method is applied to the quantification of PPase concentration. Leveraging the FSCV method, we have effectively analyzed PPase activity in LNCaP cells, offering a fresh perspective on tumor marker analysis and fostering advancements in drug development.

BnVP1, a novel vacuolar H+ pyrophosphatase gene from Boehmeria nivea confers cadmium tolerance in transgenic Arabidopsis.

Plants have developed precise defense mechanisms against cadmium (Cd) stress, with vacuolar compartmentalization of Cd2+ being a crucial process in Cd detoxification. The transport of Cd into vacuoles by these cation / H+ antiporters is powered by the pH gradient created by proton pumps. In this study, the full-length cDNA of a vacuolar H+-pyrophosphatase (V-PPase) gene from Boehmeria nivea (ramie), BnVP1, was isolated using the rapid amplification of cDNA ends (RACE) method. The open reading frame (ORF) of BnVP1 is 2292 bp, encoding a 763 amino acid V-PPase protein with 15 predicted transmembrane domains. Sequence alignment and phylogenetic analysis revealed that BnVP1 belongs to the Type I V-PPase family. Quantitative RT-PCR assays demonstrated that BnVP1 expression was significantly higher in ramie roots than in shoots. Cd treatments markedly induced BnVP1 expression in both roots and leaves of ramie seedlings, with a more pronounced effect in roots. Additionally, BnVP1 expression was significantly upregulated by the plant hormone methyl jasmonate (MeJA). Heterologous expression of BnVP1 in transgenic Arabidopsis significantly enhanced V-PPase activity in the roots. The growth performance, root elongation, and total chlorophyll content of transgenic plants with high tonoplast H+-PPase (V-PPase) activity were superior to those of wild-type plants. Overexpression of BnVP1 reduced membrane lipid peroxidation and ion leakage, and significantly increased Cd accumulation in the roots of transgenic Arabidopsis seedlings. This study provides new genetic resources for the phytoremediation of Cd-contaminated farmland.

Enzymatic activity of bone markers on Lithobates catesbeianus (Shaw, 1802) growth during the ossification process.

In order to better understand the ossification processes in anurans our study was carried out on tadpoles and adults of Lithobates catesbeianus. In this sense, we characterized the kinetic properties of alkaline phosphatase with p-nitrophenylphosphatase (pNPP) and pyrophosphate (PPi) and evaluated the activities of tartrate-resistant acid phosphatase and acid phosphatase. The enzyme extracts were obtained from tadpoles and adult femurs, which were divided into epiphysis and diaphysis. After homogenization, the samples were submitted to differential centrifugation to obtain cell membranes and, further, to phospholipase C (PIPLC) treatment, to remove membrane-bound proteins anchored by phosphatidylinositol. The average of specific activity for pNPP hydrolysis (at pH 10.5) by alkaline phosphatase released by phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus cereus among different bone regions at different animal ages was 1,142.57 U.mg-1, while for PPi hydrolysis (at pH 8.0), it was 1,433.82 U.mg-1. Among the compounds tested for enzymatic activity, the one that influenced the most was EDTA, with approximately 67% of inhibition for pNPPase activity and 77% for PPase activity. In the case of kinetic parameters, the enzyme showed a "Michaelian" behavior for pNPP and PPi hydrolysis. The Km value was around 0.6mM for pNPPase activity and ranged from 0.01 to 0.11mM for PPase activity, indicating that the enzyme has a higher affinity for this substrate. The study of pNPP and PPi hydrolysis by the enzyme revealed that the optimum pH of actuation for pNPP was 10.5, while for PPi, which is considered the true substrate of alkaline phosphatase, was 8.0, close to the physiological value. The results show that regardless of the ossification type that occurs, the same enzyme or isoenzymes act on the different bone regions and different life stages of anurans. The similarity of the results of studies with other vertebrates shows that anurans can be considered excellent animal models for the study of biological calcification.

The Ingenious Multiple Efficacies of the Integrated Gene Fragment of Torque Teno Mini Virus in Orchestrating Promyelocytic Leukemogenesis Via Hijacking Rara

Introduction Torque teno mini virus (TTMV) is a ubiquitous virus which symbiotic with humans, but it has recently been reported to cause acute promyelocytic leukemia (APL) through a unique form of genome fragment integration and forming the TTMV::RARA fusion gene, with 4 cases reported till now. However, the leukemogenesis mechanism of TTMV::RARA-APL has not been explored. Methods We recruited transcriptome sequencing (RNA-seq) data of 7 TTMV::RARA-APL cases, 3 of them also with whole genome sequencing (WGS) data, to analyze the properties of the TTMV integrated sequences. Clinical information was analyzed. In vitro cell experiments were performed to verify the functions of the conserved regulatory sequences and the fusion protein. Results These cases were collected from 4 centers, 5 males and 2 females, aged 3 to 35 (median 9) years, with 6 pediatric cases. Of which 3 pediatric cases were identified from a continuous acute myeloid leukemia (AML) cohort. In this cohort, 3 TTMV::RARA-APL were identified in 205 AML cases under 14 years, but no cases in another 930 AML cases, showing a strong tendency towards pediatrics (p=0.00022). Moreover, we totally identified 85 PML::RARA-APL and 3 TTMV::RARA-APL, and no other X::RAR cases in this cohort, indicating TTMV::RARA is the most common X::RAR fusion besides PML::RARA. All cases manifested typical APL morphological features, and RNA-seq expression analysis showed clustering with PML::RARA-APL cases. Initial effect and remission can be achieved with chemotherapy regimens containing all-trans retinoic acid (ATRA), but early recurrence is prone to occur. RARA-I387T mutation was observed in case #TR4, who relapsed after treatment with ATRA, suggesting that the leukemic cells were responsive to ATRA. However, primary resistance to ATRA and extensive resistance to various agents were observed in case #TR5, who was initially diagnosed with myeloid sarcoma and transformed into APL at relapse. The TTMV integrated sequences of 3 cases with WGS data were 1164bp, 2113bp, and 2859bp (Fig. 1A), respectively, and the common feature was the involvement of the transcription regulatory region and the open reading frame 2 (ORF2). Alignment of TTMV transcription regulatory sequences (Fig. 1B) revealed a highly conserved basal transcription factor region, including the canonical TATA box and GC box. While in the proximal promoter region showing sequence diversity, all 3 cases carried 4 RUNX1 binding elements and 1 octamer motif. In vitro experiments (Fig. 1C) showed that TTMV basal transcription element can initiate basal expression in 293T cells, while RUNX1 can significantly enhance the expression in response to the RUNX1 binding elements. RUNX1 is an important transcription factor protein in the differentiation of myeloid cells, and the 4 RUNX1 binding elements might explain the myelotropic of TTMV and confer an important basis in promyelocytic leukemogenesis. The TTMV-ORF2 coding sequences of all 7 cases were analyzed, with the common region being the N-terminus 58 amino acids, of which 20 were highly conserved (Fig. 1D, 1E). Co-Immunoprecipitation analysis confirmed that the TTMV::RARA protein forms homopolymers and heteropolymers with RXRA (Fig.1F), which is the property shared by all X::RARA proteins. Serine/Threonine protein phosphatase (S/T PPase) assays showed that the fusion protein exhibits strong S/T PPase activity (Fig. 1G). UAS/GAL4 reporter experiment showed that TTMV::RARA protein exhibited a constrained but concentration-dependent response to ATRA compared to RARA (Fig. 1H). However, fusion proteins with ORF2-C32A/C34A mutation and impaired phosphatase activity were significantly more responsive to ATRA (Fig. 1H). This suggests that the S/T PPase activity of the ORF2 coding region plays a role in retarded ATRA response. Conclusions This is the first systematic report on this novelty disease. Data suggests that TTMV::RARA-APL may be more common in children, which may be related to the ubiquity of TTMV and its acquisition early in life. The clinical manifestations and treatment responses are diverse, with overall dismal prognosis. Molecular investigation indicated the multifunctional TTMV integrated molecular machinery in orchestrating RUNX1 enforced myelotropic, aberrant priming expression, homodimer formation, and S/T PPase activity, and confer promyelocytic leukemogenesis via hijacking RARA.

Click reaction-based single-particle dark-field microscopy: A visual platform for the highly sensitive and selective detection of pyrophosphatase

Inorganic pyrophosphatase (PPase) plays significant roles in diverse pathophysiological processes, therefore, developing a reliable method to measure PPase activity is crucial for the early diagnosis of the related diseases. In this work, a novel platform with single-particle dark-field microscopy (DFM), consisting of core gold nanoparticles (AuNPs), satellite AuNPs, pyrophosphate ions (PPi), copper ions (Cu2+), and sodium ascorbate (NaAsc), is designed for the selective and sensitive analysis of PPase according to a click reaction. After the introduction of PPase, the coordination between PPi and Cu2+ can be destroyed by catalyzing the hydrolysis of PPi to release free Cu2+ which can be reduced by NaAsc to produce cuprous ions (Cu+). Then the formed Cu+ promotes the click reaction between azide-modified core AuNPs and alkynyl-modified satellite AuNPs to cause the significant redshift of the localized surface plasmon resonance (LSPR) spectra of core AuNPs. Thus, PPase can be ultra-sensitively detected with a low detection limit of 0.003 U/L in a wide linear range from 0.01 to 1 U/L. This click reaction-based DFM technique can also be used to assess PPase inhibition, which suggests that it has a lot of potential for both the screening of PPase inhibitors and the diagnosis of PPase-related disorders.

Low soil available phosphorus level reduces cotton fiber length via osmoregulation.

Phosphorus (P) deficiency hinders cotton (Gossypium hirustum L.) growth and development, seriously affecting lint yield and fiber quality. However, it is still unclear how P fertilizer affects fiber length. Therefore, a two-year (2019-2020) pool-culture experiment was conducted using the split-plot design, with two cotton cultivars (CCRI-79; low-P tolerant and SCRC-28; low-P sensitive) as the main plot. Three soil available phosphorus (AP) contents (P0: 3 ± 0.5, P1: 6 ± 0.5, and P2 (control) with 15 ± 0.5 mg kg-1) were applied to the plots, as the subplot, to investigate the impact of soil AP content on cotton fiber elongation and length. Low soil AP (P0 and P1) decreased the contents of the osmotically active solutes in the cotton fibers, including potassium ions (K+), malate, soluble sugar, and sucrose, by 2.2-10.2%, 14.4-47.3%, 8.7-24.5%, and 10.1-23.4%, respectively, inhibiting the vacuoles from facilitating fiber elongation through osmoregulation. Moreover, soil AP deficiency also reduced the activities of enzymes participated in fiber elongation (plasma membrane H+-ATPase (PM-H+-ATPase), vacuole membrane H+-ATPase (V-H+-ATPase), vacuole membrane H+-translocating inorganic pyrophosphatase (V-H+-PPase), and phosphoenolpyruvate carboxylase (PEPC)). The PM-H+-ATPase, V-H+-ATPase, V-H+-PPase, and PEPC were reduced by 8.4-33.0%, 7.0-33.8%, 14.1-38.4%, and 16.9-40.2%, respectively, inhibiting the transmembrane transport of the osmotically active solutes and acidified conditions for fiber cell wall, thus limiting the fiber elongation. Similarly, soil AP deficiency reduced the fiber length by 0.6-3.0 mm, mainly due to the 3.8-16.3% reduction of the maximum velocity of fiber elongation (VLmax). Additionally, the upper fruiting branch positions (FB10-11) had higher VLmax and longer fiber lengths under low soil AP. Cotton fibers with higher malate content and V-H+-ATPase and V-H+-PPase activities yielded longer fibers. And the malate and soluble sugar contents and V-H+-ATPase and PEPC activities in the SCRC-28's fiber were more sensitive to soil AP deficiency in contrast to those of CCRI-79, possibly explaining the SCRC-28 fiber length sensitivity to low soil AP.

Assay of inorganic pyrophosphatase activity based on a fluorescence “turn–off” strategy using carbon quantum dots@Cu-MOF nanotubes

A highly sensitive and selective sensor for the quantitative assay of inorganic pyrophosphatase (PPase) activity was developed based on a fluorescence “turn–off” strategy. Carbon quantum dots@Cu(II)-based metal–organic framework nanotubes (CQDs@Cu-MOF) with length less than 300 nm and width less than 20 nm were synthesized. CQDs in the nanotubes exhibited weak fluorescence owing to static quenching. The coordination reaction between pyrophosphate ion (PPi) and Cu(II) decomposed CQDs@Cu-MOF and led to the release of CQDs, of which the fluorescence recovered. In the presence of PPase, the hydrolysis of PPi generated phosphate ion (Pi). CQDs@Cu-MOF remained their structural stability and the fluorescence turned off. The fluorescence intensity difference of the mixture of CQDs@Cu-MOF and PPi in the absence and presence of PPase (-ΔF) was proportional to the PPase concentration from 0.1 to 5 mU mL−1 and that from 5 to 50 mU mL−1, and a limit of detection at 0.03 mU mL−1 was obtained. PPase activity in human serum was analyzed using the proposed fluorescence sensor and the recovery values were found to vary from 95.0% to 104 %.

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%.

Ultraviolet radiation underlies metabolic energy reprograming in Coffea arabica and Coffea canephora genotypes

Depletion of the stratospheric ozone layer enhances UV exposure to living organisms. We hypothesized that the adaptative physiological and bioenergetic responses of modern genotypes of Coffea arabica cv. Catuaí Amarelo IAC 62, and C. canephora (Conilon LB1) to actual UV levels would generate additional costs to the detriment of biomass formation and partitioning. Coffee plants were cultivated for six months in a mini greenhouse under either near ambient (UVam) or reduced (UVre) UV conditions, after which leaf gas exchanges, chlorophyll a fluorescence parameters and proton pump phosphohydrolase activities were analyzed. Growth under UVam did not affect the photochemical efficiency and net CO2 assimilation in both Coffea species, but reduced stomatal conductance (gs) and increased intrinsic water use efficiency (iWUE), maximum photosynthetic O2 evolution, and apparent quantum yield. Coffee plants under UVam showed decreased P-type H+-ATPase activity, whilst H+-PPase activity increased, preserving metabolic energy. Coffee biomass accumulation decreases under UVam were more pronounced in C. canephora, which generally invested more in the root system than in shoot biomass as compared to C. arabica. Reduced biomass in C. canephora under UVam susuggested restrained C-sequestration in plant biomass, which might be partly associated with gs reduction, along with a equilibrium between the ATP- and PPi-driven electrochemical H+ gradients, providing a mechanism for energy buffering and cellular robustness. The stability of fluorescence parameters and net CO2 assimilation rate under UVam, reduced gs and increased iWUE, oxygen-evolving complex activity, and elevated SPAD values, supported photomorphogenetic responses acclimation to actual UV in two modern coffee genotypes, with detrimental impact on shoot biomass, especially in C. canephora.

A FRET ratiometric fluorescence biosensor for the selective determination of pyrophosphate ion and pyrophosphatase activity based on difunctional Cu-MOF nanozyme

A ratiometric fluorescent method based on the difunctional copper (II)-based metal-organic framework (Cu-MOF) for label-free monitoring pyrophosphatase (PPase) activity and pyrophosphate ion (PPi) was developed. Difunctional Cu-MOF possessed peroxidase mimetic activity and fluorescent emission property. The catalytic activity of Cu-MOF would be inhibited by the coordination reaction between Cu2+ and PPi because of the good affinity between Cu2+ and PPi. However, the PPi can be hydrolyzed by PPase catalyzation to Pi which can hardly affect the Cu-MOF nanozyme activity. Once the PPase participated, the catalytic activity of Cu-MOF was recovered and catalyzed the decomposition of hydrogen peroxide to oxidize o-phenylenediamine (OPD) to form a 2,3-diaminophenazine (DAP) solution and turns on the Förster resonance energy transfer (FRET) from Cu-MOF as a donor to DAP as an acceptor. The LODs of the fluorescence sensing platform were 0.30 mU mL−1 for PPase and 0.53 μmol L−1 for PPi, respectively. This method will broaden the applications of nanozyme and shows a great potential application in the diagnosis of PPi or PPase-related diseases.

DNAzyme-Amplified Label-Free Biosensor for the Simple and Sensitive Detection of Pyrophosphatase.

The level of pyrophosphatase (PPase) expression has been suggested as a potential biomarker of various cancers, and its prognostic value has been evaluated in patients suffering from lung cancer, colorectal cancer, and hyperthyroidism. However, the detection of PPase usually needs specific materials that require complicated, time-consuming reactions with restricted linear range and sensitivity, limiting their application in early clinical diagnosis. Herein, we developed a DNAzyme-based biosensor for the detection of PPase. In the presence of PPase, pyrophosphate (PPi) and Cu2+ ions released from the PPi–Cu2+–PPi complex induce the cleavage of the DNAzyme and the corresponding substrate. An apurinic/apyrimidinic (AP) site was elaborately designed within substrates that could encase the fluorophore 2-amino-5,6,7-trimethyl-1,8-naphthyridine (ATMND). The fluorescence of ATMND was initially quenched but restored when the DNAzyme/substrate complex was hydrolyzed with the release of ATMND. In this way, the PPase activity can be estimated by detecting the increased fluorescence of the released ATMND. Under optimized conditions, the activity of PPase could be analyzed at concentrations from 0.5 to 1000 mU, with the lowest detectable concentration being 0.5 mU. This work lays a foundation for developing a DNAzyme-amplified fluorescent biosensor with a high sensitivity, a wide linear range, and single-step operation for use as an easy diagnostic for PPase analysis.

Application of boron reduces vanadium toxicity by altering the subcellular distribution of vanadium, enhancing boron uptake and enhancing the antioxidant defense system of watermelon

Vanadium (V) is the fifth most abundant transition metal, elevated levels of V are hazardous to plants. Boron (B) is an essential micronutrient for plants and can mitigate heavy metal toxicity. However, the mechanism used by B to promote tolerance to vanadium is unknown. In this study, a combination of physiological and gene expression analysis was used to explain mechanism of B (75 µM) induced V (40 mg L-1) stress tolerance in watermelon. V stress severely reduced root and shoot growth and increased the accumulation of ROS. B application improved tolerance to V by enhancing the expression of B transporter genes (ClaNIP5;1–1, ClaNIP5;1–2, ClaBOR4) that facilitated B uptake and transport while restricting V transport in plant tissues. At cellular level, the higher V retention in leaves was achieved by cell wall chelation, whereas, the higher V exclusion in vacuole of root cell was driven by elevated vacuolar H+-ATPase, H+-PPase activities, and transcript level of ClaVHP1;1, ClaPDR12–1 and ClaPDR12–2 genes facilitated by B application. Moreover, B application reduced tissue ROS cascade by enhancing antioxidant enzymatic activity and expression of superoxide dismutase (ClaCSD1–1, ClaCSD1–2, ClaCSD3, ClaMSD1) and catalase (ClaCAT2–1, ClaCAT2–2) genes that enhanced the defense mechanism of the V treated plants, improved root and shoot growth and tolerance index of watermelon. In conclusion, we demonstrate that ameliorative effect of B in tolerance to V of watermelon was based on B homeostasis and improved antioxidant defense system. These findings might help to increase watermelon production in V polluted soils.

In situ Cu2+-triggered wavelength-tunable fluorescent sensor for highly sensitive sensing inorganic pyrophosphatase activity and its logic gate application

As inorganic pyrophosphatase (PPase) gets involved in the regulation of various pathophysiological procedures, rapid and effective detection of its activity is of great value for monitoring and managing PPase-related diseases. Herein, an in situ Cu2+-triggered wavelength-tunable fluorescent sensor for high sensitivity monitoring of PPase activity is constructed. The proposed sensor is based on the principle of Cu2+-triggered polymerization of dopamine (DA) and m-phenylenediamine (MPD), the stronger coupling effect of pyrophosphate ion (PPi) and Cu2+, and the effective hydrolysis of PPase to PPi. With the change of substituents of MPD, the wavelength of fluorescence polymer nanoparticles (FPNPs) exhibits tunable capacity from 535 nm to 565 nm. Compared with the traditional detection method, as developed sensor shows high sensitivity to PPase with a low detection limit of 0.333 mU/mL in green channel and 0.033 mU/mL in orange channel. Additionally, the sensor realizes PPase sensing in the diluted human serum with excellent recovery in both green and orange models. Furthermore, this sensor is also applicable for inhibition assay of PPase (NaF: IC50 of 134.7 μM) and logic gates construction. Summarily, as prepared sensor provides one better way for monitoring PPase in diagnostic as well as drug screening.

IGFBP-3 and TGF-β inhibit growth in epithelial cells by stimulating type V TGF-β receptor (TβR-V)-mediated tumor suppressor signaling.

The TGF‐β type V receptor (TβR‐V) mediates growth inhibition by IGFBP‐3 and TGF‐β in epithelial cells and loss of TβR‐V expression in these cells leads to development of carcinoma. The mechanisms by which TβR‐V mediates growth inhibition (tumor suppressor) signaling remain elusive. Previous studies revealed that IGFBP‐3 and TGF‐β inhibit growth in epithelial cells by stimulating TβR‐V‐mediated IRS‐1/2‐dependent activation and cytoplasm‐to‐nucleus translocation of IGFBP‐3‐ or TGF‐β‐stimulated protein phosphatase (PPase), resulting in dephosphorylation of pRb‐related proteins (p107, p130) or pRb, and growth arrest. To define the signaling, we characterized/identified the IGFBP‐3‐ and TGF‐β‐stimulated PPases in cell lysates and nucleus fractions in Mv1Lu cells treated with IGFBP‐3 and TGF‐β, using a cell‐free assay with 32P‐labeled casein as a substrate. Both IGFBP‐3‐ and TGF‐β‐stimulated PPase activities in cell lysates are abolished when cells are co‐treated with TGF‐β/IGFBP‐3 antagonist or RAP (LRP‐1/TβR‐V antagonist). However, the IGFBP‐3‐stimulated PPase activity, but not TGF‐β‐stimulated PPase activity, is sensitive to inhibition by okadaic acid (OA). In addition, OA or PP2Ac siRNA reverses IGFBP‐3 growth inhibition, but not TGF‐β growth inhibition, in Mv1Lu and 32D cells. These suggest that IGFBP‐3‐ and TGF‐β‐stimulated PPases are identical to PP2A and PP1, respectively. By Western blot/phosphorimager/immunofluorescence‐microscopy analyses, IGFBP‐3 and TGF‐β stimulate TβR‐V‐mediated IRS‐2‐dependent activation and cytoplasm‐to‐nucleus translocation of PP2Ac and PP1c, resulting in dephosphorylation of p130/p107 and pRb, respectively, and growth arrest. Small molecule TGF‐β enhancers, which potentiate TGF‐β growth inhibition by enhancing TβR‐I–TβR‐II‐mediated canonical signaling and thus activating TβR‐V‐mediated tumor suppressor signaling cascade (TβR‐V/IRS‐2/PP1/pRb), could be used to prevent and treat carcinoma.

Molecular characterization and transcriptional regulation of two types of H+-pyrophosphatases in the scuticociliate parasite Philasterides dicentrarchi

Proton-translocating inorganic pyrophosphatases (H+-PPases) are an ancient family of membrane bound enzymes that couple pyrophosphate (PPi) hydrolysis to H+ translocation across membranes. In this study, we conducted a molecular characterization of two isoenzymes (PdVP1 and PdVP2) located in respectively the alveolar sacs and in the membranes of the intracellular vacuoles of a scuticociliate parasite (Philasterides dicentrarchi) of farmed turbot. We analyzed the genetic expression of the isoenzymes after administration of antiparasitic drugs and after infection in the host. PdVP1 and PdVP2 are encoded by two genes of 2485 and 3069 bp, which respectively contain 3 and 11 exons and express proteins of 746 and 810 aa of molecular mass 78.9 and 87.6 kDa. Topological predictions from isoenzyme sequences indicate the formation of thirteen transmembrane regions (TMRs) for PdVP1 and seventeen TMRs for PdVP2. Protein structure modelling indicated that both isoenzymes are homodimeric, with three Mg2+ binding sites and an additional K+ binding site in PdVP2. The levels of identity and similarity between the isoenzyme sequences are respectively 33.5 and 51.2%. The molecular weights of the native proteins are 158 kDa (PdVP1) and 178 kDa (PdVP2). The isoenzyme sequences are derived from paralogous genes that form a monophyletic grouping with other ciliate species. Genetic expression of the isoenzymes is closely related to the acidification of alveolar sacs (PdVP1) and intracellular vacuoles (PdVP2): antiparasitic drugs inhibit transcription, while infection increases transcription of both isoenzymes. The study findings show that P. dicentrarchi possesses two isoenzymes with H+-PPase activity which are located in acidophilic cell compartment membranes and which are activated during infection in the host and are sensitive to antiparasitic drugs. The findings open the way to using molecular modelling to design drugs for the treatment of scuticociliatosis.

Good-Practice Non-Radioactive Assays of Inorganic Pyrophosphatase Activities.

Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PPi) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PPi as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PPi synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PPi hydrolysis, (b) continuous and fixed-time measurements of PPi synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium’s composition, whose components are involved in multiple equilibria.

Aggregation-Induced Emission of Au/Ag Alloy Nanoclusters for Fluorescence Detection of Inorganic Pyrophosphate and Pyrophosphatase Activity.

The development of sensitive and accurate detection of inorganic pyrophosphate (PPi) and pyrophosphatase activity (PPase) is important as they play vital roles in biological systems. However, it is still not satisfactory for most of the analytical methods for PPi and PPase because of their Cu2+-dependence and poor accuracy. Although the metal ion triggered aggregation-induced emission (AIE) of metal nanoclusters (NCs) offers a new approach to design a Cu2+-free strategy for the accurate determination of PPi and PPase recently, current methods are all focused on utilizing pure metal NCs. Alloy NCs incorporating the advantages of diverse metal usually can achieve improved behaviors in the application, such as enhanced sensitivity and stability. In this work, glutathione stabilized alloy Au/Ag NCs were synthesized via a simple method and used for the fluorescence detection of PPi and PPase based on a Zn2+-regulated AIE strategy. The controlled release of Zn2+ by PPi and PPase could regulate the AIE of Au/Ag NCs and be employed to response PPi concentration and PPase activity. This method processes simple procedure, high sensitivity and stability, and low toxicity. In addition, we also studied the AIE behaviors of this Au/Ag NCs and offer some fundamental understanding of the AIE properties of water-soluble alloy NCs. This study not only provides a straightforward and new approach for PPi and PPase determination but a basis for further study on the AIE properties of alloy NCs and their application.

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

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 (1mM) and nano-Fe2O3 (3mM) 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 12days 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.

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

BackgroundPrevious 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). MethodsHere, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure. ResultscPGI 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. ConclusionsThese 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 significanceThese 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.

The H+-Translocating Inorganic Pyrophosphatase From Arabidopsis thaliana Is More Sensitive to Sodium Than Its Na+-Translocating Counterpart From Methanosarcina mazei.

Overexpression of membrane-bound K+-dependent H+-translocating inorganic pyrophosphatases (H+-PPases) from higher plants has been widely used to alleviate the sensitivity toward NaCl in these organisms, a strategy that had been previously tested in Saccharomyces cerevisiae. On the other hand, H+-PPases have been reported to functionally complement the yeast cytosolic soluble pyrophosphatase (IPP1). Here, the efficiency of the K+-dependent Na+-PPase from the archaeon Methanosarcina mazei (MVP) to functionally complement IPP1 has been compared to that of its H+-pumping counterpart from Arabidopsis thaliana (AVP1). Both membrane-bound integral PPases (mPPases) supported yeast growth equally well under normal conditions, however, cells expressing MVP grew significantly better than those expressing AVP1 under salt stress. The subcellular distribution of the heterologously-expressed mPPases was crucial in order to observe the phenotypes associated with the complementation. In vitro studies showed that the PPase activity of MVP was less sensitive to Na+ than that of AVP1. Consistently, when yeast cells expressing MVP were grown in the presence of NaCl only a marginal increase in their internal PPi levels was observed with respect to control cells. By contrast, yeast cells that expressed AVP1 had significantly higher levels of this metabolite under the same conditions. The H+-pumping activity of AVP1 was also markedly inhibited by Na+. Our results suggest that mPPases primarily act by hydrolysing the PPi generated in the cytosol when expressed in yeast, and that AVP1 is more susceptible to Na+ inhibition than MVP both in vivo and in vitro. Based on this experimental evidence, we propose Na+-PPases as biotechnological tools to generate salt-tolerant plants.