The 3′-Phosphoadenosine 5′-Phosphosulfate Transporters,PAPST1 and 2, Contribute to the Maintenance and Differentiation of Mouse Embryonic Stem Cells

Recently, we have identified two 3′-phosphoadenosine 5′-phosphosulfate (PAPS) transporters (PAPST1 and PAPST2), which contribute to PAPS transport into the Golgi, in both human and Drosophila. Mutation and RNA interference (RNAi) of the Drosophila PAPST have shown the importance of PAPST-dependent sulfation of carbohydrates and proteins during development. However, the functional roles of PAPST in mammals are largely unknown. Here, we investigated whether PAPST-dependent sulfation is involved in regulating signaling pathways required for the maintenance of mouse embryonic stem cells (mESCs), differentiation into the three germ layers, and neurogenesis. By using a yeast expression system, mouse PAPST1 and PAPST2 proteins were shown to have PAPS transport activity with an apparent K_m value of 1.54 µM or 1.49 µM, respectively. RNAi-mediated knockdown of each PAPST induced the reduction of chondroitin sulfate (CS) chain sulfation as well as heparan sulfate (HS) chain sulfation, and inhibited mESC self-renewal due to defects in several signaling pathways. However, we suggest that these effects were due to reduced HS, not CS, chain sulfation, because knockdown of mouse N-deacetylase/N-sulfotransferase, which catalyzes the first step of HS sulfation, in mESCs gave similar results to those observed in PAPST-knockdown mESCs, but depletion of CS chains did not. On the other hand, during embryoid body formation, PAPST-knockdown mESCs exhibited abnormal differentiation, in particular neurogenesis was promoted, presumably due to the observed defects in BMP, FGF and Wnt signaling. The latter were reduced as a result of the reduction in both HS and CS chain sulfation. We propose that PAPST-dependent sulfation of HS or CS chains, which is regulated developmentally, regulates the extrinsic signaling required for the maintenance and normal differentiation of mESCs.     

Heterodimerization of the Entamoeba histolytica EhCPADH virulence complex through molecular dynamics and protein–protein docking

EhCPADH is a protein complex involved in the virulence of Entamoeba histolytica, the protozoan responsible for human amebiasis. It is formed by the EhCP112 cysteine protease and the EhADH adhesin. To explore the molecular basis of the complex formation, three-dimensional models were built for both proteins and molecular dynamics simulations (MDS) and docking calculations were performed. Results predicted that the pEhCP112 proenzyme and the mEhCP112 mature enzyme were globular and peripheral membrane proteins. Interestingly, in pEhCP112, the propeptide appeared hiding the catalytic site (C167, H329, N348); while in mEhCP112, this site was exposed and its residues were found structurally closer than in pEhCP112. EhADH emerged as an extended peripheral membrane protein with high fluctuation in Bro1 and V shape domains. 500 ns-long MDS and protein–protein docking predictions evidenced different heterodimeric complexes with the lowest free energy. pEhCP112 interacted with EhADH by the propeptide and C-terminal regions and mEhCP112 by the C-terminal through hydrogen bonds. In contrast, EhADH bound to mEhCP112 by 442–479 residues, adjacent to the target cell-adherence region (480–600 residues), and by the Bro1 domain (9–349 residues). Calculations of the effective binding free energy and per residue free energy decomposition showed that EhADH binds to mEhCP112 with a higher binding energy than to pEhCP112, mainly through van der Waals interactions and the nonpolar part of solvation energy. The EhADH and EhCP112 structural relationship was validated in trophozoites by immunofluorescence, TEM, and immunoprecipitation assays. Experimental findings fair agreed with in silico results.     

Two Golgi-resident 3′-Phosphoadenosine 5′-Phosphosulfate Transporters Play Distinct Roles in Heparan Sulfate Modifications and Embryonic and Larval Development in Caenorhabditis elegans

Synthesis of extracellular sulfated molecules requires active 3′-phosphoadenosine 5′-phosphosulfate (PAPS). For sulfation to occur, PAPS must pass through the Golgi membrane, which is facilitated by Golgi-resident PAPS transporters. Caenorhabditis elegans PAPS transporters are encoded by two genes, pst-1 and pst-2. Using the yeast heterologous expression system, we characterized PST-1 and PST-2 as PAPS transporters. We created deletion mutants to study the importance of PAPS transporter activity. The pst-1 deletion mutant exhibited defects in cuticle formation, post-embryonic seam cell development, vulval morphogenesis, cell migration, and embryogenesis. The pst-2 mutant exhibited a wild-type phenotype. The defects observed in the pst-1 mutant could be rescued by transgenic expression of pst-1 and hPAPST1 but not pst-2 or hPAPST2. Moreover, the phenotype of a pst-1;pst-2 double mutant were similar to those of the pst-1 single mutant, except that larval cuticle formation was more severely defected. Disaccharide analysis revealed that heparan sulfate from these mutants was undersulfated. Gene expression reporter analysis revealed that these PAPS transporters exhibited different tissue distributions and subcellular localizations. These data suggest that pst-1 and pst-2 play different physiological roles in heparan sulfate modification and development.     

Preparation of 3′-Phosphoadenosine 5′-Phospho35S-sulfate Using ATP Sulfurylase and APS Kinase from Bacillus stearothermophilus: Enzymatic Synthesis and Purification

Radioactive PAPS was synthesized with a predetermined specific activity using enzymes purified from Bacillus stearothermophilus. The reaction system, incorporating 1.5mCi of ³⁵S-H₂SO₄ and 500 nmol of ATP, produced 582 μCi of ³⁵S-PAPS. The synthesized ³⁵S-PAPS was purified by ECTEOLA cellulose chromatography, followed by desalting with DOWEX 50W-X8. The final yield was 206 μmol of ³⁵S-PAPS with a radioactivity of 411 μCi.     

Evolution of the 2′-5′-Oligoadenylate Synthetase Family in Eukaryotes and Bacteria

The 2′-5′-oligoadenylate synthetase (OAS) belongs to a nucleotidyl transferase family that includes poly(A) polymerases and CCA-adding enzymes. In mammals and birds, the OAS functions in the interferon system but it is also present in an active form in sponges, which are devoid of the interferon system. In view of these observations, we have pursued the idea that OAS genes could be present in other metazoans and in unicellular organisms as well. We have identified a number of OAS1 genes in annelids, mollusks, a cnidarian, chordates, and unicellular eukaryotes and also found a family of proteins in bacteria that contains the five OAS-specific motifs. This indicates a specific relationship to OAS. The wide distribution of the OAS genes has made it possible to suggest how the OAS1 gene could have evolved from a common ancestor to choanoflagellates and metazoans. Furthermore, we suggest that the OASL may have evolved from an ancestor of cartilaginous fishes, and that the OAS2 and the OAS3 genes evolved from a mammalian ancestor. OAS proteins function in the interferon system in mammals. This system is only found in jawed vertebrates. We therefore suggest that the original function of OAS may differ from its function in the interferon system, and that this original function of OAS is preserved even in OAS genes that code for proteins, which do not have 2′-5′-oligoadenylate synthetase activity.     

Substrate Specificities of Mitochondrial Thymidine Kinase and Cytosolic Deoxycytidine Kinase Against 5-Aryl Substituted Pyrimidine-2′-deoxyribose Analogues

Abstract

Some 5-aryl-2′-deoxyuridine and -deoxycytidine analogues, many with known antiviral activity, were evaluated as substrates for pure deoxycytidine kinase (dCK) and pure mitochondrial thymidine kinase (TK2). Some of the deoxyuridine compounds were also tested with pure cytosolic thymidine kinase (TK1). TK2 showed the highest activity with this type of analogues.     

Cloning, Functional Identification and Sequence Analysis of Flavonoid 3′-hydroxylase and Flavonoid 3′,5′-hydroxylase cDNAs Reveals Independent Evolution of Flavonoid 3′,5′-hydroxylase in the Asteraceae Family

Flavonoids are ubiquitous secondary plant metabolites which function as protectants against UV light and pathogens and are involved in the attraction of pollinators as well as seed and fruit dispersers. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of the vast and versatile cytochrome P450 protein (P450) family, the flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H). Phylogenetic analysis of known sequences of F3′H and F3′5′H indicated that F3′5′H was recruited from F3′H before the divergence of angiosperms and gymnosperms. Seven cDNAs were isolated from species of the Asteraceae family, all of which were predicted to code for F3′Hs based on their sequences. The recombinant proteins of four of the heterologously in yeast expressed cDNAs exhibited the expected F3′H activity but surprisingly, three recombinant proteins showed F3′5′H activity. Phylogenetic analyses indicated the independent evolution of an Asteraceae-specific F3′5′H. Furthermore, sequence analysis of these unusual F3′5′H cDNAs revealed an elevated rate of nonsynonymous substitutions as typically found for duplicated genes acquiring new functions. Since F3′5′H is necessary for the synthesis of 3′,4′,5′-hydroxylated delphinidin-derivatives, which normally provide the basis for purple to blue flower colours, the evolution of an Asteraceae-specific F3′5′H probably reflects the adaptive value of efficient attraction of insect pollinators.     

Evidence for the presence of 3′, 5′-cyclic AMP in plant tissues

3′, 5′-cyclic AMP has been highly purified by chromatography from sterile higher plant tissues and assayed by bioluminescence as well as by activation of protein kinase. Both methods give comparable results: the amount of cyclic AMP was found to vary between 30 and 200 pmoles per mg of protein nitrogen.     

Synthesis and Properties of the Oligonucleotide N3′ →P5′ Phosphoramidates

Abstract

Uniformly modified oligonucleotide N3′ → P5′ phosphoramidates were synthesized. The prepared N3′ → P5′ phosphoramidates form extremely stable duplexes and triplexes with complementary nucleic acids. Moreover, these compounds are highly resistant to enzymatic hydrolysis by snake venom phosphodiesterase and cellular nucleases and they show high antisense activity in vitro and in vivo.     

Purification and properties of the ATP: adenylylsulphate 3′-phosphotransferase from Chlamydomonas reinhardii

APS-kinase (ATP: adenylylsulphate 3-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg²⁺ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.The purified enzyme attained a V _max of 360 pkat under optimal reaction conditions declining to v _limit of 260 pkat in the presence of excess substrate APS. This sensitivity towards changes in substrate concentrations was parallelled by a high affinity and specificity: apparent K _m APS: 2 · 10^-6 mol · l^-1, and K _m ATP: 7 · 10^-6 mol · l^-1. The enzyme was found specific for ATP, d-ATP and CTP, while UTP, ITP and GTP showed marginal activity. The Hill coefficients suggested 4 binding sites for APS and 1 for ATP. Excessive APS resulted in a negative slope indicating 3 inhibiting sites of the substrate.Abbreviations APS Adenosine 5-phosphosulphate - dATP 2-deoxyadenosine 5-triphosphate - p-CMB p-chloromercuribenzoate - DTE dithioerythritol - DTT dithiothreitol - -MSH -mercaptoethanol - PAPS 3-phosphoadenosine 5-phosphosulphate - PAP 3-phosphoadenosine 5-phosphate - SDS sodium dodecyl sulphate This work is part of a dissertation submitted by H. G. J., Bochum 1982     

Evidence for the Stereoelectronic Control of the Acid Hydrolysis of Adenosine Cyclic 3′,5′-Phosphoramidate Diastereoisomers

Abstract

The substitution by a methyl group of the axial 5′hydrogen atom of adenosine cyclic 3′,5′-phosphoramidate diastereoisomers significantly increases the P-N bond breaking for the R^?p diastereoisomer, but does not change that for the S^?p diastereoisomer as anticipated on the basis of ground state stereoelectronic effects.     

Stimulation of ion transport by ascorbic acid through inhibition of 3′:5′-cyclic-AMP phosphodiesterase in the corneal epithelium and other tissues

Ascorbic acid stimulates active transport of Cl^? by the isolated intact corneas. The effect is not present in corneas previously stimulated by theophylline, an inhibitor of 3′: 5′-cyclic-AMP phosphodiesterase (EC 3.1.4.17), and vice versa, theophylline has no action after stimulation with ascorbic acid. This indicated inhibition of 3′: 5′-cyclic-AMP phosphodiesterase by ascorbic acid. Assay of phosphodiesterase using ³H-labeled cyclid AMP of frog and rabbit corneal epithelial homogenates showed an inhibitory effect of ascorbic acid. Concentration of 5 mM produced 16% inhibition with 20 mM producing 46 %. This compares with 58 % inhibition by theophylline at 5 mM. Phosphodiesterase activity is mostly soluble in frog corneal epithelium but in rabbit 45 % is particulate. Soluble and particulate fractions are inhibited by ascorbate, but in rabbits greater inhibition (50 %) was observed in the particulate fraction than in the soluble fraction. Other tissues showed inhibition also: frog retina 12 %, rat brain (caudate nucleus) 48 %, rabbit brain 14 %, rabbit liver 16 %. It is concluded that ascorbate produces an increase in cyclic AMP content of corneal epithelium and other tissues by inhibition of 3′: 5′-cyclic-AMP phosphodiesterase. This action may be one of the main functions of the high ascorbic acid content of ocular tissues and explain some of the effects of high dosis of ascorbate in other systems.     

Effects of 3′-C-Methylation on the Hydrolytic Stability and Hydroxyl pK a Values of Dinucleoside 2′,5′- and 3′,5′-Monophosphates

Abstract

The first-order rate constants for hydrolysis of 3′-C-methyluridylyl(2′,5′)- and -(3′,5′)adenosine and the corresponding native dinucleoside monophosphates (2′,5′- and 3′,5′-UpA) have been determined as a function of hydroxide-ion concentration (0.025 - 7 M) at 25°C. In addition to the effects on the hydrolytic stability of the compounds, the effects of the 3′-C-methyl substitution on the kinetically determined pK _a values for the sugar hydroxyls of the undine moiety are discussed.     

The occurrence of the syn-C3′ endo conformation and the distorted backbone conformations for C4′-C5′ and P-O5′ in oligo and polynucleotides

Abstract

The nucleoside constituents of nucleic acids prefer the anti conformation (1). When the sugar pucker is taken into account the nucleosides prefer the C2′endo-anti conformation. Of the nearly 300 nucleosides known, about 250 are in the anti conformation and 50 are in the syn-conformation, i.e., anti to syn conformation is 5:1. The nucleotide building blocks of nucleic acids show the same trend as nucleosides. Both the deoxy-guanosine and ribo- guanosine residues in nucleosides and nucleotides prefer the syn-C2′endo conformation with an intra-molecular hydrogen bond (for nucleosides) between the O5′- H and the N3 of the base and, a few syn-C3′endo conformations are also observed. Evidence is presented for the occurrence of the C3′endo-syn conformation for guanines in mis-paired double helical right-handed structures with the distorted sugar phosphate C4′-C5′ and P-O5′ bonds respectively, from g+ (gg) and g- to trans. Evidence is also provided for guanosine nucleotides in left-handed double-helical (Z-DNA) oligo and polynucleotides which has the same syn-C3′endo conformation and the distorted backbone sugar-phosphate bonds (C4′-C5′ and P- O5′) as in the earlier right-handed case.     

The NLRP3 Inflammasome Is a Pathogen Sensor for Invasive Entamoeba histolytica via Activation of α5β1 Integrin at the Macrophage-Amebae Intercellular Junction

Entamoeba histolytica (Eh) is an extracellular protozoan parasite of humans that invades the colon to cause life-threatening intestinal and extra-intestinal amebiasis. Colonized Eh is asymptomatic, however, when trophozoites adhere to host cells there is a considerable inflammatory response that is critical in the pathogenesis of amebiasis. The host and/or parasite factors that trigger the inflammatory response to invading Eh are not well understood. We recently identified that Eh adherence to macrophages induces inflammasome activation and in the present study we sought to determine the molecular events upon contact that coordinates this response. Here we report that Eh contact-dependent activation of α₅β₁ integrin is critical for activation of the NLRP3 inflammasome. Eh-macrophage contact triggered recruitment of α₅β₁ integrin and NLRP3 into the intercellular junction, where α₅β₁ integrin underwent activation by an integrin-binding cysteine protease on the parasite surface, termed EhCP5. As a result of its activation, α₅β₁ integrin induced ATP release into the extracellular space through opening of pannexin-1 channels that signalled through P2X₇ receptors to deliver a critical co-stimulatory signal that activated the NLRP3 inflammasome. Both the cysteine protease activity and integrin-binding domain of EhCP5 were required to trigger α₅β₁ integrin that led to ATP release and NLRP3 inflammasome activation. These findings reveal engagement of α₅β₁ integrin across the parasite-host junction is a key regulatory step that initiates robust inflammatory responses to Eh. We propose that α₅β₁ integrin distinguishes Eh direct contact and functions with NLRP3 as pathogenicity sensor for invasive Eh infection.