PPA1 Regulates Systemic Insulin Sensitivity by Maintaining Adipocyte Mitochondria Function as a Novel PPARγ Target Gene.

Downregulation of mitochondrial function in adipose tissue is considered as one important driver for the development of obesity-associated metabolic disorders. Inorganic pyrophosphatase 1 (PPA1) is an enzyme that catalyzes the hydrolysis of inorganic pyrophosphate to inorganic phosphate and is required for anabolism to take place in cells. Although alteration of PPA1 has been related to some diseases, the importance of PPA1 in metabolic syndromes has never been discussed. In this study, we found that global PPA1 knockout mice (PPA1+/-) showed impaired glucose tolerance and severe insulin resistance under high-fat-diet feeding. In addition, impaired adipose tissue development and ectopic lipid accumulation were observed. Conversely, overexpression of PPA1 in adipose tissue by adeno-associated virus injection can partly reverse the metabolic disorders in PPA1+/- mice, suggesting that impaired adipose tissue function is responsible for the metabolic disorders observed in PPA1+/- mice. Mechanistic studies revealed that PPA1 acted as a PPARγ target gene to maintain mitochondrial function in adipocytes. Furthermore, specific knockdown of PPA1 in fat body of Drosophila led to impaired mitochondria morphology, decreased lipid storage, and made Drosophila more sensitive to starvation. In conclusion, for the first time, our findings demonstrate the importance of PPA1 in maintaining adipose tissue function and whole-body metabolic homeostasis.

Biological properties and roles of a Trichinella spiralis inorganic pyrophosphatase in molting and developmental process of intestinal larval stages.

Inorganic pyrophosphatase (PPase) participates in energy cycle and plays a vital role in hydrolysis of inorganic pyrophosphate (PPi) into inorganic phosphate (Pi). The aim of this study was to investigate the biological properties of a Trichinella spiralis PPase (TsPPase) and its role in larval molting and developmental process. The predicted TsPPase consisted of 367 amino acids with a molecular mass of 41.48 kDa and a pI of 5.76. Amino acid sequence alignment and phylogenetic analysis showed that the TsPPase gene encodes a functional family I soluble PPase with the same characteristics as prokaryotic, plant and animal/fungal soluble PPase. The rTsPPase was expressed and purified, it has the activity to catalyze the hydrolysis of PPi to Pi, and the activity was dependent on Mg2+, pH and temperature. The enzymatic activity of rTsPPase was significantly inhibited after its metal binding sites mutation. TsPPase was transcribed and expressed in all T. spiralis phases, especially in muscle larvae (ML) and intestinal infective larvae (IIL). Immunofluorescence assay (IFA) revealed that TsPPase was mainly located in cuticle and stichosome. When the ML and IIL were treated with TsPPase-specific siRNA-279, TsPPase expression and enzymatic activity were obviously reduced, the larval molting and development were also impeded. Intestinal IIL as well as AW burden, IIL molting rates from mice infected with siRNA-treated ML were obviously suppressed. The results indicated that rTsPPase possesses the enzymatic activity of native inorganic pyrophosphatase, and TsPPase plays an important role in development and molting process of intestinal T. spiralis larval stages.

Improving drought-, salinity-, and heat-tolerance in transgenic plants by co-overexpressing Arabidopsis vacuolar pyrophosphatase gene AVP1 and Larrea Rubisco activase gene RCA.

The severity and frequency of many abiotic stresses such as drought, salinity and heat, cause substantial crop losses worldwide, which poses a serious challenge in food security. To increase crop production, new approaches are needed. Previous research has shown that overexpression of the tonoplast H+ pyrophosphatase gene AVP1 leads to improved drought and salt tolerance in transgenic plants. Other research showed that overexpression of thermotolerant ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase gene could maintain photosynthesis at higher temperatures, which contributes to higher heat tolerance in transgenic plants. In nature, abiotic stresses rarely come alone, instead these stresses often occur in various combinations. Therefore, it is desirable to make crops more tolerant to multiple stresses, which will likely lead to higher crop yield under various stress conditions. It is shown here that co-overexpression of the Arabidopsis gene AVP1 and the Larrea Rubisco activase gene RCA significantly increases drought, salinity and heat tolerance, resulting in higher biomass and seed yield than wild-type plants. AVP1/RCA co-overexpressing plants are as more drought- and salt-tolerant as AVP1-overexpressing plants, and as more heat-tolerant as RCA-overexpressing plants. More importantly, they produce higher seed yields than AVP1-overexpressing, RCA-overexpressing, and wild-type plants under combined drought and heat conditions.

Do metabolic HAD phosphatases moonlight as protein phosphatases?

Mammalian haloacid dehalogenase (HAD)-type phosphatases have evolved to dephosphorylate a wide range of small metabolites, but can also target macromolecules such as serine/threonine, tyrosine-, and histidine-phosphorylated proteins. To accomplish these tasks, HAD phosphatases are equipped with cap domains that control access to the active site and provide substrate specificity determinants. A number of capped HAD phosphatases impact protein phosphorylation, although structural data are consistent with small metabolite substrates rather than protein substrates. This review discusses the structures, functions and disease implications of the three closely related, capped HAD phosphatases pyridoxal phosphatase (PDXP or chronophin), phosphoglycolate phosphatase (PGP, also termed AUM or glycerol phosphatase) and phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP or HDHD2B). Evidence in support of small metabolite and protein phosphatase activity is discussed in the context of the diversity of their biological functions.

Molecular Cloning, Expression Analysis, and Functional Characterization of the H(+)-Pyrophosphatase from Jatropha curcas.

H(+)-pyrophosphatase (H(+)-PPase) is a primary pyrophosphate (PPi)-energized proton pump to generate electrochemical H(+) gradient for ATP production and substance translocations across membranes. It plays an important role in stress adaptation that was intensively substantiated by numerous transgenic plants overexpressing H(+)-PPases yet devoid of any correlated studies pointing to the elite energy plant, Jatropha curcas. Herein, we cloned the full length of J. curcas H(+)-PPase (JcVP1) complementary DNA (cDNA) by reverse transcription PCR, based on the assembled sequence of its ESTs highly matched to Hevea brasiliensis H(+)-PPase. This gene encodes a polypeptide of 765 amino acids that was predicted as a K(+)-dependent H(+)-PPase evolutionarily closest to those of other Euphorbiaceae plants. Many cis-regulatory elements relevant to environmental stresses, molecular signals, or tissue-specificity were identified by promoter prediction within the 1.5-kb region upstream of JcVP1 coding sequence. Meanwhile, the responses of JcVP1 expression to several common abiotic stresses (salt, drought, heat, cold) were characterized with a considerable accordance with the inherent stress tolerance of J. curcas. Moreover, we found that the heterologous expression of JcVP1 could significantly improve the salt tolerance in both recombinant Escherichia coli and Saccharomyces cerevisiae, and this effect could be further fortified in yeast by N-terminal addition of a vacuole-targeting signal peptide from the H(+)-PPase of Trypanosoma cruzi.

H(+) -pyrophosphatase from Salicornia europaea confers tolerance to simultaneously occurring salt stress and nitrogen deficiency in Arabidopsis and wheat.

High salinity and nitrogen (N) deficiency in soil are two key factors limiting crop productivity, and they usually occur simultaneously. Here we firstly found that H(+) -PPase is involved in salt-stimulated NO3 (-) uptake in the euhalophyte Salicornia europaea. Then, two genes (named SeVP1 and SeVP2) encoding H(+) -PPase from S. europaea were characterized. The expression of SeVP1 and SeVP2 was induced by salt stress and N starvation. Both SeVP1 or SeVP2 transgenic Arabidopsis and wheat plants outperformed the wild types (WTs) when high salt and low N occur simultaneously. The transgenic Arabidopsis plants maintained higher K(+) /Na(+) ratio in leaves and exhibited increased NO3 (-) uptake, inorganic pyrophosphate-dependent vacuolar nitrate efflux and assimilation capacity under this double stresses. Furthermore, they had more soluble sugars in shoots and roots and less starch accumulation in shoots than WT. These performances can be explained by the up-regulated expression of ion, nitrate and sugar transporter genes in transgenic plants. Taken together, our results suggest that up-regulation of H(+) -PPase favours the transport of photosynthates to root, which could promote root growth and integrate N and carbon metabolism in plant. This work provides potential strategies for improving crop yields challenged by increasing soil salinization and shrinking farmland.

A vacuolar H(+)-pyrophosphatase differential activation and energy coupling integrate the responses of weeds and crops to drought stress.

BACKGROUND: Cyperus rotundus L. is a C4 weed of large vegetative and reproductive vigor endowed with competitive advantages over most crop species mainly under adverse environmental conditions. Vacuole functions are critical for the mechanisms of drought resistance, and here the modulation of the primary system of vacuolar ion transport is investigated during a transient water stress imposed to this weed and to C4 crop species (Zea mays L.). METHODS: The vacuolar H(+) pumps, the H(+)-ATPase and H(+)-PPiase, expression, activities and the energy coupling were spectrophotometrically investigated as key elements in the differential drought-resistance mechanisms developed by weeds and crops. RESULTS: In C. rotundus tonoplasts, ATP hydrolysis was more sensitive to drought than its coupled H(+) transport, which was in turn at least 3-folds faster than that mediated by the H(+)-PPiase. Its PPi hydrolysis was only slightly affected by severe water deficit, contrasting with the disruption induced in the PPi-dependent H(+)-gradient. This effect was antagonized by plant rehydration as the H(+)-PPiase activity was highly stimulated, reassuming a coupled PPi-driven H(+) pumping. Maize tonoplasts exhibited 2-4 times lower hydrolytic activities than that of C. rotundus, but were able to overactivate specifically PPi-dependent H(+) pumping in response to stress relief, resulting in an enhanced H(+)-pumps coupling efficiency. CONCLUSION: These results together with immunoanalysis revealed profiles consistent with pre- and post-translational changes occurring on the tonoplast H(+)-pumps, which differ between weeds and crops upon water deficit. GENERAL SIGNIFICANCE: The evidences highlight an unusual modulation of the H(+)-PPiase energy coupling as a key biochemical change related to environmental stresses adaptive capacity of plants.

Improved growth, drought tolerance, and ultrastructural evidence of increased turgidity in tobacco plants overexpressing Arabidopsis vacuolar pyrophosphatase (AVP1).

An increasing volume of evidence indicating the mechanisms of drought tolerance of AVP1-overexpressing transgenic plants has been reported. In the present study, we are reporting the experiments conducted for the drought tolerance of AVP1 overexpressing plants and WT tobacco plants in three water regimes named as "fully watered," "less-watered," and "desiccated". Results suggest that AVP1 plants exhibited greater vigor and drought tolerance in quantitative terms i.e., increase in size and weight of shoots and capsules. AVP1 plants produced more seeds than WT across all three water regimes. The less-watered regime was found to produce the greatest contrast. AVP1 overexpression enhanced solute accumulation in vacuoles resulting in an increase in water retention and turgor of the cell. The ultrastructure study of AVP1 overexpressing cells and WT leaf cells revealed that AVP1 plants displayed more turgid and hyperosmotic cells than WT. Moreover, guard cells in the AVP1 plants exhibited thick cell walls, few vacuoles, and deep and close stomata, whereas WT plants showed larger vacuoles and relatively open stomata aperture with no significant difference in size and number of the cells per unit area.

Identification of direct forkhead box O1 targets involved in palmitate-induced apoptosis in clonal insulin-secreting cells using chromatin immunoprecipitation coupled to DNA selection and ligation.

AIMS/HYPOTHESIS: The transcription factor, forkhead box (FOX)O1, is involved in fatty acid-induced apoptosis in pancreatic beta cells, but the precise mechanism is poorly understood. We aimed to identify which direct downstream targets of FOXO1 are involved in palmitate-induced apoptosis in the pancreatic beta cell line MIN6. METHODS: Chromatin immunoprecipitation (ChIP) coupled to a DNA selection and ligation technique (ChIP-DSL) was used to identify the direct targets of FOXO1. The mRNA level was examined by real-time PCR assay. The ChIP-DSL results were verified using ChIP-PCR and luciferase assay, respectively. The cell apoptosis rate was determined by TUNEL assay and by scoring cells with pycnotic nuclei. RESULTS: We identified 189 target genes and selected 106 targets for expression analysis in MIN6 cells treated with palmitate. The results showed that six genes were significantly upregulated and four were downregulated. Binding of FOXO1 to the promoters was determined by ChIP-PCR and confirmed by luciferase assay. Among the ten up- and downregulated genes, mRNA expression of A930038C07Rik was significantly decreased and that of Ppa1 was increased in 8-week-old db/db mice. The apoptosis assay showed that overproduction of the protein 'RIKEN cDNA A930038C07' (A930038C07Rik) drastically enhanced palmitate-induced apoptosis, while pyrophosphatase (inorganic) 1 (PPA1) partially protected the cells from apoptosis. Knockdown of PPA1, moreover, significantly increased apoptosis. CONCLUSIONS/INTERPRETATION: We identified for the first time FOXO1 targets in MIN6 cells treated with palmitate, thus revealing the important roles of A930038C07Rik and PPA1 in palmitate-induced cell apoptosis. These results shed light on the mechanisms of palmitate-induced apoptosis in pancreatic beta cells.

Expression of an Arabidopsis vacuolar H+-pyrophosphatase gene (AVP1) in cotton improves drought- and salt tolerance and increases fibre yield in the field conditions.

The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase that functions as a proton pump on the vacuolar membrane. Overexpression of AVP1 in Arabidopsis, tomato and rice enhances plant performance under salt and drought stress conditions, because up-regulation of the type I H+-PPase from Arabidopsis may result in a higher proton electrochemical gradient, which facilitates enhanced sequestering of ions and sugars into the vacuole, reducing water potential and resulting in increased drought- and salt tolerance when compared to wild-type plants. Furthermore, overexpression of AVP1 stimulates auxin transport in the root system and leads to larger root systems, which helps transgenic plants absorb water more efficiently under drought conditions. Using the same approach, AVP1-expressing cotton plants were created and tested for their performance under high-salt and reduced irrigation conditions. The AVP1-expressing cotton plants showed more vigorous growth than wild-type plants in the presence of 200 mM NaCl under hydroponic growth conditions. The soil-grown AVP1-expressing cotton plants also displayed significantly improved tolerance to both drought and salt stresses in greenhouse conditions. Furthermore, the fibre yield of AVP1-expressing cotton plants is at least 20% higher than that of wild-type plants under dry-land conditions in the field. This research indicates that AVP1 has the potential to be used for improving crop's drought- and salt tolerance in areas where water and salinity are limiting factors for agricultural productivity.

Distance variations between active sites of H(+)-pyrophosphatase determined by fluorescence resonance energy transfer.

Homodimeric H(+)-pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H(+)-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PP(i)), for H(+) translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PP(i) binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H(+)-PPase are 49.3 + or - 4.0 and 67.2 + or - 5.7 A, respectively. Furthermore, putative PP(i) binding motifs on individual subunits are found to be relatively far away from each other (70.8 + or - 4.8 A), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H(+)-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H(+)-PPase upon substrate binding.

Molecular cloning and characterization of a novel H+-translocating pyrophosphatase gene in Zea mays.

A cDNA encoding a putative H+-translocating pyrophosphatase (H+-PPase) has been cloned from Zea mays by suppression subtractive hybridization (SSH) coupled with in silico cloning approach. The isolated 2974 bp full-length cDNA named ZmGPP contains a single 2400 bp open reading frame encoding a putative protein of 799 amino acids. The predicted protein has 16 transmembrane domains and is significantly similar to Golgi apparatus resident type-II H+-PPase from Arabidopsis thaliana. DNA gel blotting analysis shows that ZmGPP is a low-copy gene. Organ expression pattern analysis reveals that ZmGPPexpressed highly in leaf and tassel, followed by in stem, root, and ear. The Real-time RT-PCR assays showed that the expression of ZmGPP was up-regulated both in shoots and roots of maize seedlings under dehydration, cold and high salt stresses. Those results suggest that the ZmGPP product may play an important role in abiotic stress tolerance of Z. mays.

H+-PPases: yesterday, today and tomorrow.

Suggestions by Calvin about a role of inorganic pyrophosphate (PPi) in early photosynthesis and by Lipmann that PPi may have been the original energy-rich phosphate donor in biological energy conversion, were followed in the mid-1960s by experimental results with isolated chromatophore membranes from the purple photosynthetic bacterium Rhodospirillum rubrum. PPi was shown to be hydrolysed in an uncoupler stimulated reaction by a membrane-bound inorganic pyrophosphatase (PPase), to be formed at the expense of light energy in photophosphorylation and to be utilized as an energy donor for various energy-requiring reactions, as a first known alternative to ATP. This direct link between PPi and photosynthesis led to increasing attention concerning the role of PPi in both early and present biological energy transfer. In the 1970s, the PPase was shown to be a proton pump and to be present also in higher plants. In the 1990s, sequences of H(+)-PPase genes were obtained from plants, protists, bacteria and archaea and two classes of H(+)-PPases differing in K(+) sensitivity were established. Over 200 H(+)-PPase sequences have now been determined. Recent biochemical and biophysical results have led to new progress and questions regarding the H(+)-PPase family, as well as the families of soluble PPases and the inorganic polyphosphatases, which hydrolyse inorganic linear high-molecular-weight polyphosphates (HMW-polyP). Here we will focus attention on the H(+)-PPases, their evolution and putative active site motifs, response to monovalent cations, genetic regulation and some very recent results, based on new methods for obtaining large quantities of purified protein, about their tertiary and quaternary structures.

Roles of basic residues and salt-bridge interaction in a vacuolar H+-pumping pyrophosphatase (AVP1) from Arabidopsis thaliana.

To investigate the possible role of basic residues in H+ translocation through vacuolar-type H+-pumping pyrophosphatases (V-PPases), conserved arginine and lysine residues predicted to reside within or close to transmembrane domains of an Arabidopsis thaliana V-PPase (AVP1) were subjected to site-directed mutagenesis. One of these mutants (K461A) exhibited a "decoupled" phenotype in which proton-pumping but not hydrolysis was inhibited. Similar results were reported previously for an E427Q mutant, resulting in the proposal that E427 might be involved in proton translocation. However, the double mutant E427K/K461E has a wild type phenotype, suggesting that E427 and K461 form a stabilising salt bridge, but that neither residue plays a critical role in proton translocation.

Crystallization and preliminary crystallographic analysis of two Streptococcus agalactiae proteins: the family II inorganic pyrophosphatase and the serine/threonine phosphatase.

Streptococcus agalactiae, which infects human neonates and causes sepsis and meningitis, has recently been shown to possess a eukaryotic-like serine/threonine protein phosphorylation signalling cascade. Through their target proteins, the S. agalactiae Ser/Thr kinase and Ser/Thr phosphatase together control the growth as well as the morphology and virulence of this organism. One of the targets is the S. agalactiae family II inorganic pyrophosphatase. The inorganic pyrophosphatase and the serine/threonine phosphatase have therefore been purified and crystallized and diffraction data have been collected from their crystals. The data were processed using XDS. The inorganic pyrosphosphatase crystals diffracted to 2.80 A and the Ser/Thr phosphatase crystals to 2.65 A. Initial structure-solution experiments indicate that structure solution will be successful in both cases. Solving the structure of the proteins involved in this cascade is the first step towards understanding this phenomenon in atomic detail.

Cloning of an H+-PPase gene from Thellungiella halophila and its heterologous expression to improve tobacco salt tolerance.

An H(+)-pyrophosphatase (PPase) gene named TsVP involved in basic biochemical and physiological mechanisms was cloned from Thellungiella halophila. The deduced translation product has similar characteristics to H(+)-PPases from other species, such as Arabidopsis and rice, in terms of bioinformation. The heterologous expression of TsVP in the yeast mutant ena1 suppressed Na(+) hypersensitivity and demonstrated the function of TsVP as an H(+)-PPase. Transgenic tobacco overexpressing TsVP had 60% greater dry weight than wild-type tobacco at 300 mM NaCl and higher viability of mesophyll protoplasts under salt shock stress conditions. TsVP and AVP1, another H(+)-PPase from Arabidopsis, were heterologously expressed separately in both the yeast mutant ena1 and tobacco. The salt tolerance of TsVP or AVP1 yeast transformants and transgenic tobacco were improved to almost the same level. The TsVP transgenic tobacco lines TL3 and TL5 with the highest H(+)-PPase hydrolytic activity were studied further. These transgenic tobacco plants accumulated 25% more solutes than wild-type plants without NaCl stress and 20-32% more Na(+) under salt stress conditions. Although transgenic tobacco lines TL3 and TL5 accumulated more Na(+) in leaf tissues, the malondialdehyde content and cell membrane damage were less than those of the wild type under salt stress conditions. Presumably, compartmentalization of Na(+) in vacuoles reduces its toxic effects on plant cells. This result supports the hypothesis that overexpression of H(+)-PPase causes the accumulation of Na(+) in vacuoles instead of in the cytoplasm and avoids the toxicity of excessive Na(+) in plant cells.

Importance of Rhodospirillum rubrum H(+)-pyrophosphatase under low-energy conditions.

The physiological role of the membrane-bound pyrophosphatase of Rhodospirillum rubrum was investigated by the characterization of a mutant strain. Comparisons of growth levels between the wild type and the mutant under different low-potential conditions and during transitions between different metabolisms indicate that this enzyme provides R. rubrum with an alternative energy source that is important for growth in low-energy states.

Vacuolar proton pumps in malaria parasite cells.

The malaria parasite is a unicellular protozoan parasite of the genus Plasmodium that causes one of the most serious infectious diseases for human beings. Like other protozoa, the malaria parasite possesses acidic organelles, which may play an essential role(s) in energy acquisition, resistance to antimalarial agents, and vesicular trafficking. Recent evidence has indicated that two types of vacuolar proton pumps, vacuolar H+-ATPase and vacuolar H+-pyrophosphatase, are responsible for their acidification. In this mini-review, we discuss the recent progress on vacuolar proton pumps in the malaria parasite.