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.

Application of Fenton chemistry in electrochemical determination of pyrophosphatase activity and fluoride.

Fenton chemistry has aroused widespread concern due to its application in the green oxidation and mineralization of organic wastes. Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of pyrophosphate ions (PPi) and provides a thermodynamic driving force for many biosynthetic reactions. Fluoride (F-) is widely applied to fight against tooth decay and reduce cavities. The electrochemical determination of PPase activity and F- was realized based on Fenton chemistry in this work. Glassy carbon electrode modified with poly (azure A) and acetylene black (GCE/PAA-AB) was fabricated. Hydroxyl radicals (∙OH) that were generated from a Cu2+-catalyzed Fenton-type reaction could oxidize PAA in the near-neutral medium, leading to a great increase of the cathodic peak current (Ipc). A coordination reaction between PPi and Cu2+ exerted a negative effect on Fenton reaction and hindered the Ipc enhancement. Cu2+-PPi complex was decomposed due to the hydrolysis of PPi induced by PPase, which caused the reappearance of the notably increased current response. F- could effectively inhibit PPase activity. As a result, the stable Cu2+-PPi complex remained and the high Ipc suffered from the decline again. The Ipc difference was used for the highly sensitive determination of PPase activity in the content range of 0.001-20 mU mL-1 with a detection of limit (LOD) at 0.6 µU mL-1 and that of F- in the concentration range of 0.01-100 µM with a LOD at 7 nM. The proposed PPase and F- sensor displayed a good selectivity, stability and reproducibility, and a high accuracy.

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.

GhVOZ1-AVP1 module positively regulates salt tolerance in upland cotton (Gossypium hirsutum L.).

Vascular Plant One­zinc Finger (VOZ) transcription factor can respond to a variety of abiotic stresses, however its function in cotton and the molecular mechanisms of response to salt tolerance remained unclear. In this study, we found that GhVOZ1 is highly expressed in stamen and stem of cotton under normal conditions. The expression of GhVOZ1 increased significantly after 3 h of salt treatment in three-leaf staged upland cotton. Overexpressed transgenic lines of GhVOZ1 in Arabidopsis and upland cotton were treated with salt stress and we found that GhVOZ1 could respond positively to salt stress. GhVOZ1 can regulate Arabidopsis Vacuolar Proton Pump Pyrophosphatase (H+-PPase) gene (AVP1) expression through specific binding to GCGTCTAAAGTACGC site on GhAVP1 promoter, which was examined through Dual-luciferase assay and Electrophoretic mobility shift assay (EMSA). AVP1 expression was significantly increased in Arabidopsis with GhVOZ1 overexpression, while GhAVP1 expression was decreased in virus induced gene silenced (VIGS) cotton plants of GhVOZ1. Knockdown of GhAVP1 expression in cotton plants by VIGS showed decreased superoxide dismutase (SOD) and peroxidase (POD) activities, whereas an increased malondialdehyde (MDA) content and ultimately decreased salt tolerance. The GhVOZ1-AVP1 module could maintain sodium ion homeostasis through cell ion transport and positively regulate the salt tolerance in cotton, providing new ideas and insights for the study of salt tolerance.

Exploration of the Catalytic Cycle Dynamics of Vigna Radiata H+-Translocating Pyrophosphatases Through Hydrogen-Deuterium Exchange Mass Spectrometry.

Vigna radiata H+-translocating pyrophosphatases (VrH+-PPases, EC 3.6.1.1) are present in various endomembranes of plants, bacteria, archaea, and certain protozoa. They transport H+ into the lumen by hydrolyzing pyrophosphate, which is a by-product of many essential anabolic reactions. Although the crystal structure of H+-PPases has been elucidated, the H+ translocation mechanism of H+-PPases in the solution state remains unclear. In this study, we used hydrogen-deuterium exchange (HDX) coupled with mass spectrometry (MS) to investigate the dynamics of H+-PPases between the previously proposed R state (resting state, Apo form), I state (intermediate state, bound to a substrate analog), and T state (transient state, bound to inorganic phosphate). When hydrogen was replaced by proteins in deuterium oxide solution, the backbone hydrogen atoms, which were exchanged with deuterium, were identified through MS. Accordingly, we used deuterium uptake to examine the structural dynamics and conformational changes of H+-PPases in solution. In the highly conserved substrate binding and proton exit regions, HDX-MS revealed the existence of a compact conformation with deuterium exchange when H+-PPases were bound with a substrate analog and product. Thus, a novel working model was developed to elucidate the in situ catalytic mechanism of pyrophosphate hydrolysis and proton transport. In this model, a proton is released in the I state, and the TM5 inner wall serves as a proton piston.

Only one beer can be mortal: a case report of two sisters with cardiac arrest due to a homozygous mutation in PPA2 gene.

We report the long way to the correct diagnosis in two teenage sisters who developed a cardiac arrest after consuming minimal amounts of alcohol. The older girl dramatically survived two cardiac arrests at the age of 14 and 15 years. She underwent an extensive examination that revealed isolated cardiac abnormalities including fibrosis, dilated cardiomyopathy and inflammation. The younger girl also had a cardiac arrest at the age of 15 and died suddenly after consuming 1-2 beers, 3 years after her sister´s first incident. Autopsy of the heart revealed acute myocarditis without structural alterations. Multigene panel analysis (not including PPA2) showed SCN5A and CACNA1D variants in both sisters and their healthy mother. Six years later duo exome allowed the diagnosis of an autosomal recessive PPA2-related mitochondriopathy. We discuss the molecular results and clinical picture of our patients compared to other PPA2-related cases. We highlight the diagnostic contribution of multigene panels and exome analysis. The genetic diagnosis is important for medical care and for everyday life, specifically because alcohol intake can result in cardiac arrest and should be strictly avoided.   Conclusion: Duo exome sequencing clarified the diagnosis of PPA2-related mitochondriopathy in two sisters with isolated cardiac features and sudden cardiac arrest triggered by minimal amounts of alcohol. What is Known: • Multigene-Panel or exome analysis is a valuable tool to identify genetic causes of hereditary cardiac arrhythmias. • Variants of unknown significance can lead to misinterpretation. PPA2-related mitochondriopathy is a very rare autosomal recessive condition that is normally fatal in infancy. What is New: • Duo exome analysis in two teeenage sisters with cardiac arrest revealed a homozygous mild PPA2 mutation as the underlying pathology restricted to the heart muscle.

Acetylation of inorganic pyrophosphatase by S-RNase signaling induces pollen tube tip swelling by repressing pectin methylesterase.

Self-incompatibility (SI) is a widespread genetically determined system in flowering plants that prevents self-fertilization to promote gene flow and limit inbreeding. S-RNase-based SI is characterized by the arrest of pollen tube growth through the pistil. Arrested pollen tubes show disrupted polarized growth and swollen tips, but the underlying molecular mechanism is largely unknown. Here, we demonstrate that the swelling at the tips of incompatible pollen tubes in pear (Pyrus bretschneideri [Pbr]) is mediated by the SI-induced acetylation of the soluble inorganic pyrophosphatase (PPA) PbrPPA5. Acetylation at Lys-42 of PbrPPA5 by the acetyltransferase GCN5-related N-acetyltransferase 1 (GNAT1) drives accumulation of PbrPPA5 in the nucleus, where it binds to the transcription factor PbrbZIP77, forming a transcriptional repression complex that inhibits the expression of the pectin methylesterase (PME) gene PbrPME44. The function of PbrPPA5 as a transcriptional repressor does not require its PPA activity. Downregulating PbrPME44 resulted in increased levels of methyl-esterified pectins in growing pollen tubes, leading to swelling at their tips. These observations suggest a mechanism for PbrPPA5-driven swelling at the tips of pollen tubes during the SI response. The targets of PbrPPA5 include genes encoding cell wall-modifying enzymes, which are essential for building a continuous sustainable mechanical structure for pollen tube growth.

Targeted mutagenesis of the vacuolar H+ translocating pyrophosphatase gene reduces grain chalkiness in rice.

Grain chalkiness is a major concern in rice production because it impacts milling yield and cooking quality, eventually reducing market value of the rice. A gene encoding vacuolar H+ translocating pyrophosphatase (V-PPase) is a major quantitative trait locus in indica rice, controlling grain chalkiness. Higher transcriptional activity of this gene is associated with increased chalk content. However, whether the suppression of V-PPase could reduce chalkiness is not clear. Furthermore, natural variation in the chalkiness of japonica rice has not been linked with V-PPase. Here, we describe promoter targeting of the japonica V-PPase allele that led to reduced grain chalkiness and the development of more translucent grains. Disruption of a putative GATA element by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 suppressed V-PPase activity, reduced grain chalkiness and impacted post-germination growth that could be rescued by the exogenous supply of sucrose. The mature grains of the targeted lines showed a much lower percentage of large or medium chalk. Interestingly, the targeted lines developed a significantly lower chalk under heat stress, a major inducer of grain chalk. Metabolomic analysis showed that pathways related to starch and sugar metabolism were affected in the developing grains of the targeted lines that correlated with higher inorganic pyrophosphate and starch contents and upregulation of starch biosynthesis genes. In summary, we show a biotechnology approach of reducing grain chalkiness in rice by downregulating the transcriptional activity of V-PPase that presumably leads to altered metabolic rates, including starch biosynthesis, resulting in more compact packing of starch granules and formation of translucent rice grains.

Alcohol-Induced Sudden Cardiac Death in a Teenager With PPA2 Gene Mutations.

ABSTRACT: The PPA2 gene encodes a mitochondrial pyrophosphatase protein. Mutations in the gene are inherited in an autosomal recessive fashion and, when mutated, function to induce mitochondrial ATP production failure resulting in increased stress on the heart and sudden cardiac death, especially when combined with alcohol. Herein, we describe a case of a 19-year-old female patient with a history of "alcohol intolerance" who was found unexpectedly deceased after consuming a minimal amount of alcohol. Histological examination of her heart revealed widespread fibrosis of the left ventricle and the interventricular septum. Other findings include hypertrophied myocytes, including some with pleomorphic nuclei. Genetic studies were performed on postmortem blood, revealing heterozygous PPA2 gene mutations, the pathogenic variant c.683C>T (p.Pro228Leu), and the other variant c.814C>T (p.His272Tyr), a novel variant of undetermined significance. We propose that the variant of undetermined significance is likely a pathogenic mutation due to the decedent's phenotype.

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.

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 %.

Thermophilic Inorganic Pyrophosphatase Ton1914 from Thermococcus onnurineus NA1 Removes the Inhibitory Effect of Pyrophosphate.

Pyrophosphate (PPi) is a byproduct of over 120 biosynthetic reactions, and an overabundance of PPi can inhibit industrial synthesis. Pyrophosphatases (PPases) can effectively hydrolyze pyrophosphate to remove the inhibitory effect of pyrophosphate. In the present work, a thermophilic alkaline inorganic pyrophosphatase from Thermococcus onnurineus NA1 was studied. The optimum pH and temperature of Ton1914 were 9.0 and 80 °C, respectively, and the half-life was 52 h at 70 °C and 2.5 h at 90 °C. Ton1914 showed excellent thermal stability, and its relative enzyme activity, when incubated in Tris-HCl 9.0 containing 1.6 mM Mg2+ at 90 °C for 5 h, was still 100%, which was much higher than the control, whose relative activity was only 37%. Real-time quantitative PCR (qPCR) results showed that the promotion of Ton1914 on long-chain DNA was more efficient than that on short-chain DNA when the same concentration of templates was supplemented. The yield of long-chain products was increased by 32-41%, while that of short-chain DNA was only improved by 9.5-15%. Ton1914 also increased the yields of UDP-glucose and UDP-galactose enzymatic synthesis from 40.1% to 84.8% and 20.9% to 35.4%, respectively. These findings suggested that Ton1914 has considerable potential for industrial applications.

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 Mechanism of Energy Coupling in H+/Na+-Pumping Membrane Pyrophosphatase-Possibilities and Probabilities.

Membrane pyrophosphatases (mPPases) found in plant vacuoles and some prokaryotes and protists are ancient cation pumps that couple pyrophosphate hydrolysis with the H+ and/or Na+ transport out of the cytoplasm. Because this function is reversible, mPPases play a role in maintaining the level of cytoplasmic pyrophosphate, a known regulator of numerous metabolic reactions. mPPases arouse interest because they are among the simplest membrane transporters and have no homologs among known ion pumps. Detailed phylogenetic studies have revealed various subtypes of mPPases and suggested their roles in the evolution of the "sodium" and "proton" bioenergetics. This treatise focuses on the mechanistic aspects of the transport reaction, namely, the coupling step, the role of the chemically produced proton, subunit cooperation, and the relationship between the proton and sodium ion transport. The available data identify H+-PPases as the first non-oxidoreductase pump with a "direct-coupling" mechanism, i.e., the transported proton is produced in the coupled chemical reaction. They also support a "billiard" hypothesis, which unifies the H+ and Na+ transport mechanisms in mPPase and, probably, other transporters.

The Xanthomonas type III effector XopAP prevents stomatal closure by interfering with vacuolar acidification.

Plant stomata close rapidly in response to a rise in the plant hormone abscisic acid (ABA) or salicylic acid (SA) and after recognition of pathogen-associated molecular patterns (PAMPs). Stomatal closure is the result of vacuolar convolution, ion efflux, and changes in turgor pressure in guard cells. Phytopathogenic bacteria secrete type III effectors (T3Es) that interfere with plant defense mechanisms, causing severe plant disease symptoms. Here, we show that the virulence and infection of Xanthomonas oryzae pv. oryzicola (Xoc), which is the causal agent of rice bacterial leaf streak disease, drastically increased in transgenic rice (Oryza sativa L.) plants overexpressing the Xoc T3E gene XopAP, which encodes a protein annotated as a lipase. We discovered that XopAP binds to phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ), a membrane phospholipid that functions in pH control in lysosomes, membrane dynamics, and protein trafficking. XopAP inhibited the acidification of vacuoles by competing with vacuolar H+ -pyrophosphatase (V-PPase) for binding to PtdIns(3,5)P2 , leading to stomatal opening. Transgenic rice overexpressing XopAP also showed inhibition of stomatal closure when challenged by Xoc infection and treatment with the PAMP flg22. Moreover, XopAP suppressed flg22-induced gene expression, reactive oxygen species burst and callose deposition in host plants, demonstrating that XopAP subverts PAMP-triggered immunity during Xoc infection. Taken together, these findings demonstrate that XopAP overcomes stomatal immunity in plants by binding to lipids.

Purification and characterization of inorganic pyrophosphatase for in vitro RNA transcription.

Inorganic pyrophosphatase (iPPase) is an enzyme that cleaves pyrophosphate into two phosphate molecules. This enzyme is an essential component of in vitro transcription (IVT) reactions for RNA preparation as it prevents pyrophosphate from precipitating with magnesium, ultimately increasing the rate of the IVT reaction. Large-scale RNA production is often required for biochemical and biophysical characterization studies of RNA, therefore requiring large amounts of IVT reagents. Commercially purchased iPPase is often the most expensive component of any IVT reaction. In this paper, we demonstrate that iPPase can be produced in large quantities and high quality using a reasonably generic laboratory facility and that laboratory-purified iPPase is as effective as commercially available iPPase. Furthermore, using size exclusion chromatography coupled with multi-angle light scattering and dynamic light scattering, analytical ultracentrifugation, and small-angle X-ray scattering, we demonstrate that yeast iPPase can form tetramers and hexamers in solution as well as the enzymatically active dimer. Our work provides a robust protocol for laboratories involved with RNA in vitro transcription to efficiently produce active iPPase, significantly reducing the financial strain of large-scale RNA production.

The His23 and Lys79 pair determines the high catalytic efficiency of the inorganic pyrophosphatase of the haloacid dehalogenase superfamily.

Haloacid dehalogenase (HAD) superfamily members are mainly phosphomonoesterases, while BT2127 from Bacteroides thetaiotaomicron of the HAD superfamily is identified as an inorganic pyrophosphatase. In this study, to explore the roles of the Lys79 and His23 pair in the hydrolysis reaction of inorganic pyrophosphate (PPi) catalyzed by BT2127, a series of models were designed. Calculations were performed by using the density functional theory (DFT) method with the dispersion energy D3-B3LYP. The His23 and Lys79 pair plays a key role in the high catalytic efficiency of BT2127 with PPi. First, the His23 and Lys79 pair prompts Asp13 to easily provide a proton to the leaving group, which remarkably reduces the energy barrier of the phospho-transfer step; then, Lys79 provides a proton to the first leaving phosphate group via His23, produces a more electrically stabilized phosphate (H3PO4), makes this step exothermal, and further promotes the subsequent phospho-enzyme intermediate hydrolysis. The results suggest that the Lys79-His23 pair helps BT2127 reach high catalytic efficiency by strengthening the acid catalysis. Our study provides detailed chemical insights into the evolution of the inorganic pyrophosphatase function of BT2127 from the phosphomonoesterase of the HAD superfamily and the biomimetic enzyme design.