Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a dual specificity NUDT enzyme that attacks diadenosine polyphosphates and diphosphoinositol polyphosphates

Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5',5"'-P(1),P(6)-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate attached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP(5)) and bis-diphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)). Guided by multiple sequence alignments, we used site-directed mutagenesis to obtain new information concerning catalytically essential amino acid residues in DIPP. Mutagenesis of either of two conserved glutamate residues (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catalytic motif impaired hydrolysis of Ap(6)A, PP-InsP(5), and (PP)(2)-InsP(4) >95%; thus, all three substrates are hydrolyzed at the same active site. Two Gly-rich domains (glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with potential sites for cation coordination and substrate binding. GR1 comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX(2)GX(6)G) that is conserved in yeast homologues of DIPP. Mutagenesis of any of these Gly residues in GR1 and GR2 reduced catalytic activity toward all three substrates by up to 95%. More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap(6)A and (PP)(2)-InsP(4) metabolism (by 71 and 96%), yet PP-InsP(5) hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His(91) and Phe(84). This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.     

The g5R (D250) gene of African swine fever virus encodes a Nudix hydrolase that preferentially degrades diphosphoinositol polyphosphates.

The African swine fever virus (ASFV) g5R gene encodes a protein containing a Nudix hydrolase motif which in terms of sequence appears most closely related to the mammalian diadenosine tetraphosphate (Ap4A) hydrolases. However, purified recombinant g5R protein (g5Rp) showed a much wider range of nucleotide substrate specificity compared to eukaryotic Ap4A hydrolases, having highest activity with GTP, followed by adenosine 5'-pentaphosphate (p5A) and dGTP. Diadenosine and diguanosine nucleotides were substrates, but the enzyme showed no activity with cap analogues such as 7mGp3A. In common with eukaryotic diadenosine hexaphosphate (Ap6A) hydrolases, which prefer higher-order polyphosphates as substrates, g5Rp also hydrolyzes the diphosphoinositol polyphosphates PP-InsP5 and [PP]2-InsP4. A comparison of the kinetics of substrate utilization showed that the k(cat)/K(m) ratio for PP-InsP5 is 60-fold higher than that for GTP, which allows classification of g5R as a novel diphosphoinositol polyphosphate phosphohydrolase (DIPP). Unlike mammalian DIPP, g5Rp appeared to preferentially remove the 5-beta-phosphate from both PP-InsP5 and [PP]2-InsP4. ASFV infection led to a reduction in the levels of PP-InsP5, ATP and GTP by ca. 50% at late times postinfection. The measured intracellular concentrations of these compounds were comparable to the respective K(m) values of g5Rp, suggesting that one or all of these may be substrates for g5Rp during ASFV infection. Transfection of ASFV-infected Vero cells with a plasmid encoding epitope-tagged g5Rp suggested localization of this protein in the rough endoplasmic reticulum. These results suggest a possible role for g5Rp in regulating a stage of viral morphogenesis involving diphosphoinositol polyphosphate-mediated membrane trafficking.     

Synthesis and biological actions of diphosphoinositol phosphates (inositol pyrophosphates), regulators of cell homeostasis

Abstract Diphosphoinositol phosphates are a subclass of inositol phosphates possessing one or two high energy diphosphate groups instead of phosphoester substituents of the myo-inositol. Here we describe the enzymes responsible for their synthesis and degradation and how these may be regulated. Formation of diphosphoinositol phosphates in yeast and mammals is driven by an increase of the cellular energy charge, a lack of inorganic phosphate, and in mammals by osmotic or heat stress and in some cases by receptor mediated signaling. Known cellular actions are an improvement of the cell homeostasis by a reduction of the energy charge, increased phosphate uptake, improvement of mitochondrial performance, and an increase of insulin secretion in mammals. The underlying molecular mechanisms of action are far from being clarified but an increasing body of knowledge about molecular details has highlighted their complex participation in many cellular systems and metabolic processes.     

Nudix hydrolases that degrade dinucleoside and diphosphoinositol polyphosphates also have 5-phosphoribosyl 1-pyrophosphate (PRPP) pyrophosphatase activity that generates the glycolytic activator ribose 1,5-bisphosphate

A total of 17 Nudix hydrolases were tested for their ability to hydrolyze 5-phosphoribosyl 1-pyrophosphate (PRPP). All 11 enzymes that were active toward dinucleoside polyphosphates with 4 or more phosphate groups as substrates were also able to hydrolyze PRPP, whereas the 6 that could not and that have coenzyme A, NDP-sugars, or pyridine nucleotides as preferred substrates did not degrade PRPP. The products of hydrolysis were ribose 1,5-bisphosphate and P(i). Active PRPP pyrophosphatases included the diphosphoinositol polyphosphate phosphohydrolase (DIPP) subfamily of Nudix hydrolases, which also degrade the non-nucleotide diphosphoinositol polyphosphates. K(m) and k(cat) values for PRPP hydrolysis for the Deinococcus radiodurans DR2356 (di)nucleoside polyphosphate hydrolase, the human diadenosine tetraphosphate hydrolase, and human DIPP-1 (diadenosine hexaphosphate and diphosphoinositol polyphosphate hydrolase) were 1 mm and 1.5 s(-1), 0.13 mm and 0.057 s(-1), and 0.38 mm and 1.0 s(-1), respectively. Active site mutants of the Caenorhabditis elegans diadenosine tetraphosphate hydrolase had no activity, confirming that the same active site is responsible for nucleotide and PRPP hydrolysis. Comparison of the specificity constants for nucleotide, diphosphoinositol polyphosphate, and PRPP hydrolysis suggests that PRPP is a significant substrate for the D. radiodurans DR2356 enzyme and for the DIPP subfamily. In the latter case, generation of the glycolytic activator ribose 1,5-bisphosphate may be a new function for these enzymes.     

Synthesis of (di)adenosine polyphosphates by non-ribosomal peptide synthetases (NRPS)

In response to nutritional stress conditions, Bacillus brevis produces the cyclodecapeptide antibiotic tyrocidine via tyrocidine synthetase, a multifunctional non-ribosomal peptide synthetase. The apo-form of tyrocidine synthetase 1 forms adenosine (5')tetraphospho(5')adenosine, when incubated with MgATP(2-), amino acid and inorganic pyrophosphatase. The synthesis is an intrinsic property of the adenylation domain, is strictly dependent upon the amino acid, and proceeds from a reverse reaction of adenylate formation involving a second ATP molecule. In the presence of tri- or tetrapolyphosphate preferential synthesis of adenosine 5'-tetraphosphate and adenosine 5'-pentaphosphate occurs, respectively. A potential involvement of adenosine (5')-n-phospho(5')adenosine in the regulation of the biosynthetic process has been suggested.     

忍冬属植物花药药隔的特化结构及其生物学意义

花药药隔的特化结构通常具有重要的生物学意义。目前,有关药隔特化出贮存酚类物质的异细胞群的研究报道甚少,该特化结构有何生物学意义则未见报道。本研究运用半薄切片技术、冰冻切片技术、组织化学和细胞化学等方法对忍冬属Lonicera植物特化药隔异细胞群的主要内含物进行定性分析,实验证明忍冬属特化药隔异细胞群内含丰富的酚类物质。另外通过对忍冬属华南忍冬L.confusa药隔薄壁组织的发育及其与药隔维管束的关系分析,推测忍冬属特化药隔可能一方面对发育中的花药维管束起保护作用,另一方面又可作为酚类物质的临时贮存场所。同时研究发现忍冬属植物特化药隔异细胞群的形成及其分布特点可能具有重要的系统学意义。     

Synthesis of (di)nucleoside polyphosphates by the ubiquitin activating enzyme E1

Previous work from this laboratory had shown that ligases may catalyze the synthesis of (di)nucleoside polyphosphates. Here, we show that one of the enzymes of the proteasome system (E1 or the ubiquitin (Ub) activating enzyme, EC 6.3.2.19) catalyzes very effectively (k(cat) = 0.29+/-0.05 s(-1)) the transfer of AMP from the E-AMP-ubiquitin complex to tripolyphosphate or tetrapolyphosphate with formation of adenosine tetra- or pentaphosphate (p4A or p5A), respectively. Whereas the concomitant formation of AMP is stimulated by the presence of dithiothreitol in a concentration dependent manner, the synthesis of p4A is only slightly inhibited by this compound. Previous treatment of the enzyme (E1) with iodoacetamide inhibited only partially the synthesis of p4A. p4A can substitute for ATP as substrate of the reaction to generate the ubiquityl adenylate complex. A small amount of diadenosine pentaphosphate (Ap5A) was also synthesized in the presence of p4A.     

On the occurrence of diphosphoinositol in the lipids of liver and pancreas

• 1.1. The lipids of liver were reexamined for polyphosphoinositide, previously reported absent. The presence of a diphosphoinositide (DPI) was revealed by the isolation of inositol diphosphate (IP₂) from an acid hydrolysate, and its glyceryl derivative from an alkaline hydrolysate of the concentrated phospholipid fraction.
• 2.2. Starting with lipids extracted from liver and pancreas by azeotropic dehydration with 1,2-dichloroethane, materials soluble in acetone and alcohol were removed. After partition of the alcohol-insoluble phospholipids in CCl₄-light petroleum-methanol-H₂O (31:31:35:3, V/V), the non-polar fraction was assayed for inositol polyphosphate by chromatographic fractionation of the acid hydrolysate on Dowex-2 X8 Cl⁻ A diphosphate mixture eluted by NaCl or LiCl was separated by refractionation with a formate system yielding IP₂ and glycerol diphosphate.
• 3.3. The mixed glyceryl derivatives of IP₂ and glycerol diphosphate were secured by chromatographic fractionation of the alkaline hydrolysate. Based on the recovery of IP₂ and its glyceryl derivative, the content of DPI in the original pork liver lipid is 0.28 mmoles/kg.
• 4.4. DPI could not be extracted by ether from acetone-dehydrated fresh beef liver but was extracted with neutral chloroform-methanol (2:1, V/V).
• 5.5. Most of the IP₂ found in pacreatic lipid occured in material insoluble in CHCl₃ and in the CCl₄-light petroleum solvent.
     

Selectivity of the peripheral auditory system of spadefoot toads (Scaphiopus couchi) for sounds of biological significance

Summary Scaphiopus couchi is a primitive anuran whose vocal repertoire consists of a mating call and a release call. The two calls are distinct and differ in trill rate. Reception of airborne sound is achieved by means of a poorly differentiated region of skin on the head which serves as an eardrum.Whereas more modern anurans possessthree distinct types of auditory nerve fibers, spadefoot toads possess onlytwo types: a low-frequency-sensitive group which exhibits tone-on-tone inhibition and a high-frequency-sensitive group which is not inhibitable. The sharpness of frequency tuning of primary fibers in each group is comparable to more advanced vertebrate species. While the response properties of auditory fibers in the high-frequency-sensitive group are well matched to the spectral and temporal features in the spadefoot's mating call and release call, the low-frequency-sensitive fibers do not respond to these calls. Instead they may be involved in detection of bodily transmitted sounds during clasping, as well as other low-frequency sounds in the environment. The two groups of auditory fibers probably derive from separate auditory organs within the inner ear. Thresholds of auditory nerve fibers in spadefoot toads are relatively poorer than in more advanced anurans, which likely is due to their less developed eardrum. The role of tone-on-tone inhibition in the peripheral auditory system is questioned with regard to its significance in processing sounds of biological value.We wish to dedicate this paper to Jasper J. Loftus-Hills who was killed in a tragic accident near Austin, Texas on June 11, 1974. His post-doctoral appointment in our laboratory and his assistance in collecting spadefoot toads in the field recall fond memories.We also wish to thank R. Sage for helping us collect animals and W. F. Blair for supplying tape recordings ofScaphiopus mating calls. The assistance of J. Paton in photographing the animal in Fig. 1 is gratefully appreciated. This research was supported by the U.S. Public Health Service (NIH Research Grant NS-09244) and the National Science Foundation (Grant GB-18836); travel expenses involved in collecting animals were supported by a Cornell University Research Grant.     

Unraveling the biological significance of nitric oxide

Independent investigations into the biochemical changes and cytostatic properties induced in immunostimulated macrophages and studies involving the identity and mechanism of action of endothelium-derived relaxing factor led to the finding of a new metabolic pathway which converts L-arginine to nitric oxide and citrulline. The pathway has since been reported in a number of additional cell types including cells in the central nervous system (CNS). In the endothelium and CNS nitric oxide is acting as a signaling agent with the evidence supporting activation of the enzyme guanylate cyclase in the target cell. Nitric oxide is toxic and evidence supports a cytostatic/cytotoxic function as the primary action of macrophage-derived nitric oxide.     

Dinucleoside polyphosphates stimulate the primer independent synthesis of poly(A) catalyzed by yeast poly(A) polymerase.

Novel properties of the primer independent synthesis of poly(A), catalyzed by the yeast poly(A) polymerase are presented. The commercial enzyme from yeast, in contrast to the enzyme from Escherichia coli, is unable to adenylate the 3'-OH end of nucleosides, nucleotides or dinucleoside polyphosphates (NpnN). In the presence of 0.05 mm ATP, dinucleotides (at 0.01 mm) activated the enzyme velocity in the following decreasing order: Gp4G, 100; Gp3G, 82; Ap6A, 61; Gp2G, 52; Ap4A, 51; Ap2A, 41; Gp5G, 36; Ap5A, 27; Ap3A, 20, where 100 represents a 10-fold activation in relation to a control without effector. The velocity of the enzyme towards its substrate ATP displayed sigmoidal kinetics with a Hill coefficient (nH) of 1.6 and a Km(S0.5) value of 0.308 +/- 0.120 mm. Dinucleoside polyphosphates did not affect the maximum velocity (Vmax) of the reaction, but did alter its nH and Km(S0.5) values. In the presence of 0.01 mm Gp4G or Ap4A the nH and Km(S0.5) values were (1.0 and 0.063 +/- 0.012 mm) and (0.8 and 0.170 +/- 0.025 mm), respectively. With these kinetic properties, a dinucleoside polyphosphate concentration as low as 1 micro m may have a noticeable activating effect on the synthesis of poly(A) by the enzyme. These findings together with previous publications from this laboratory point to a potential relationship between dinucleoside polyphosphates and enzymes catalyzing the synthesis and/or modification of DNA or RNA.     

Understanding cultural significance, the edible mushrooms case

Background

This article analyzes whether the distribution or area of use of 18 medicinal plants is influenced by ecological and cultural factors which might account for their traditional use and/or phytonymy in Navarra. This discussion may be helpful for comparative studies, touching as it does on other ethnopharmacological issues: a) which cultural and ecological factors affect the selection of medicinal plants; b) substitutions of medicinal plants in popular medicine; c) the relation between local nomenclature and uses. To analyze these questions, this paper presents an example of a species used for digestive disorders (tea and camomile: Jasonia glutinosa, J. tuberosa, Sideritis hyssopifolia, Bidens aurea, Chamaemelum nobile, Santolina chamaecyparissus...), high blood pressure (Rhamnus alaternus, Olea europaea...) or skin diseases (Hylotelephium maximum, H. telephium, Anagallis arvensis, A. foemina).

Methods

Fieldwork began on January 2004 and continued until December 2006. During that time we interviewed 505 informants in 218 locations in Navarra. Information was collected using semi-structured ethnobotanical interviews, and we subsequently made maps using Arc-View 8.0 program to determine the area of use of each taxon. Each map was then compared with the bioclimatic and linguistic map of Navarra, using the soil and ethnographic data for the region, and with other ethnobotanical and ethnopharmacological studies carried out in Europe.

Results

The results clearly show that ecological and cultural factors influence the selection of medicinal plants in this region. Climate and substrate are the most important ecological factors that influence the distribution and abundance of plants, which are the biological factors that affect medicinal plant selection.

Conclusion

The study of edaphological and climatological factors, on the one hand, and culture, on the other, can help us to understand why a plant is replaced by another one for the same purposes, either in the same or in a different area. In many cases, the cultural factor means that the use of a species is more widespread than its ecological distribution. This may also explain the presence of synonyms and polysemies which are useful for discussing ethnopharmacological data.     

Hypothetical double-helical poly(A) formation in a cell and its possible biological significance

Arguments are presented in favor of capability of poly(A)-tracts of cellular RNA to form double helices in vivo. It is suggested that formation of the double helix in the mRNA poly(A) tall provides the basis for such processes as polyadenylation termination, PAB I synthesis autoregulation, and stabilization of ARE-containing mRNA by ELAV-like proteins.     

Structural analysis and detection of biological inositol pyrophosphates reveal that the family of VIP/diphosphoinositol pentakisphosphate kinases are 1/3-kinases

We have characterized the positional specificity of the mammalian and yeast VIP/diphosphoinositol pentakisphosphate kinase (PPIP5K) family of inositol phosphate kinases. We deployed a microscale metal dye detection protocol coupled to a high performance liquid chromatography system that was calibrated with synthetic and biologically synthesized standards of inositol pyrophosphates. In addition, we have directly analyzed the structures of biological inositol pyrophosphates using two-dimensional 1H-1H and 1H-31P nuclear magnetic resonance spectroscopy. Using these tools, we have determined that the mammalian and yeast VIP/PPIP5K family phosphorylates the 1/3-position of the inositol ring in vitro and in vivo. For example, the VIP/PPIP5K enzymes convert inositol hexakisphosphate to 1/3-diphosphoinositol pentakisphosphate. The latter compound has not previously been identified in any organism. We have also unequivocally determined that 1/3,5-(PP)2-IP4 is the isomeric structure of the bis-diphosphoinositol tetrakisphosphate that is synthesized by yeasts and mammals, through a collaboration between the inositol hexakisphosphate kinase and VIP/PPIP5K enzymes. These data uncover phylogenetic variability within the crown taxa in the structures of inositol pyrophosphates. For example, in the Dictyostelids, the major bis-diphosphoinositol tetrakisphosphate is 5,6-(PP)2-IP4 ( Laussmann, T., Eujen, R., Weisshuhn, C. M., Thiel, U., Falck, J. R., and Vogel, G. (1996) Biochem. J. 315, 715-725 ). Our study brings us closer to the goal of understanding the structure/function relationships that control specificity in the synthesis and biological actions of inositol pyrophosphates.