Calcium-induced outgrowth of astrocytic peripheral processes requires actin binding by Profilin-1

Peripheral astrocytic processes (PAPs) are highly motile structures that are strategically positioned in close proximity to synapses. Long-lasting PAP retraction in hypothalamus is known to alter synaptic transmission [1]. The PAP motility is likely to be actin-based because they are known to contain actin-related proteins such as Ezrin [2]. However, the link between dynamic activity-dependent changes in astrocytic morphology and the synaptic function has not been established experimentally, presumably due to lack of appropriate tools. To selectively suppress activity-dependent morphological plasticity of astrocytes, we developed a bicistronic construct that allows simultaneous tracing and manipulating the morphology of PAPs. The construct is designed for co-expression of (i) the mutant actin binding protein Profilin-1 (abdProf-1) with a single amino acid substitution (H119E) that prevents its binding to actin monomers [3] with (ii) the membrane-targeted morphological tracer LckGFP [4]. Cultured cortical astrocytes transfected with this construct showed abdProf-1 overexpression at a 5-fold level compared to the endogenous Profilin-1. The cells also expressed LckGFP at a level sufficient for precise morphological tracing. We found that photolysis of caged Ca²⁺ induced a pronounced outgrowth of PAPs, which was suppressed by abdProf-1 overexpression in terms of PAP number, growth rate and maximal length. In contrast, the morphological complexity of astrocytes, basal motility of their PAPs and major cytoskeletal structures were not affected by abdProf-1 overexpression. In summary, we identified the actin binding by Profilin-1 as a pivotal mechanism in activity-dependent morphological plasticity of PAPs in cultured astrocytes.     

Multiple forms of poly(A) polymerases purified from HeLa cells function in specific mRNA 3''-end formation.

Poly(A) polymerases (PAPs) from HeLa cell cytoplasmic and nuclear fractions were extensively purified by using a combination of fast protein liquid chromatography and standard chromatographic methods. Several forms of the enzyme were identified, two from the nuclear fraction (NE PAPs I and II) and one from the cytoplasmic fraction (S100 PAP). NE PAP I had chromatographic properties similar to those of S100 PAP, and both enzymes displayed higher activities in the presence of Mn2+ than in the presence of Mg2+, whereas NE PAP II was chromatographically distinct and had approximately equal levels of activity in the presence of Mn2+ and Mg2+. Each of the enzymes, when mixed with other nuclear fractions containing cleavage or specificity factors, was able to reconstitute efficient cleavage and polyadenylation of pre-mRNAs containing an AAUAAA sequence element. The PAPs alone, however, showed no preference for precursors containing an intact AAUAAA sequence over a mutated one, providing further evidence that the PAPs have no intrinsic ability to recognize poly(A) addition sites. Two additional properties of the three enzymes suggest that they are related: sedimentation in glycerol density gradients indicated that the native size of each enzyme is approximately 50 to 60 kilodaltons, and antibodies against a rat hepatoma PAP inhibited the ability of each enzyme to function in AAUAAA-dependent polyadenylation.     

Poly(A) polymerase I of Escherichia coli: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation

Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) superfamily that includes the eukaryotic PAPs and all the known tRNA CCA-adding enzymes. Five highly conserved aspartic acids in the putative catalytic site of PAP I were changed to either alanine or proline, demonstrating their importance for polymerase activity. A glycine that is absolutely conserved in all Ntrs was also changed yielding a novel mutant protein in which ATP was wastefully hydrolysed in a primer-independent reaction. This is the first work to characterize the catalytic site of a eubacterial PAP and, despite the conservation of certain sequences, we predict that the overall architecture of the eukaryotic and eubacterial active sites is likely to be different. Binding sites for RNase E, a component of the RNA degradosome, and RNA were mapped by North-western and Far-western blotting using truncated forms of PAP I. Additional protein-protein interactions were detected between PAP I and CsdA, RhlE and SrmB, suggesting an unexpected connection between PAP I and these E. coli DEAD box RNA helicases. These results show that the functional organization of PAP I is similar to the eukaryotic PAPs with an N-terminal catalytic domain, a C-terminal RNA binding domain and sites for the interaction with other protein factors.     

Molecular characterization of the type 2 phosphatidic acid phosphatase.

Phosphatidic acid phosphatase (PAP) converts phosphatidic acid to diacylglycerol, thus regulating the de novo synthesis of glycerolipids and also signal transduction mediated by phospholipase D. We initially succeeded in the cDNA cloning of the mouse 35 kDa PAP bound to plasma membranes (type 2 enzyme). This work subsequently led us to the identification of two human PAP isozymes designated 2a and 2b. A third human PAP isozyme (2c) has also been described. The cloned enzymes are, in common, N-glycosylated and possess six transmembrane domains. The transmembrane dispositions of these enzymes are predicted and the catalytic sites are tentatively located in the 2nd and 3rd extracellular loops, thus suggesting that the type 2 PAPs may act as ecto-enzymes dephosphorylating exogenous substrates. Furthermore, the type 2 PAPs have been proposed to belong to a novel phosphatase superfamily consisting of a number of soluble and membrane-bound enzymes. In vitro enzyme assays show that the type 2 PAPs can dephosphorylate lyso-phosphatidate, ceramide-1-phosphate, sphingosine-1-phosphate and diacylglycerol pyrophosphate. Although the physiological implications of such a broad substrate specificity need to be further investigated, the type 2 PAPs appear to metabolize a wide range of lipid mediators derived from both glycero- and sphingolipids.     

Poly(A) polymerase (PAP) diversity in gene expression – Star-PAP vs canonical PAP

Almost all eukaryotic mRNAs acquire a poly(A) tail at the 3′-end by a concerted RNA processing event: cleavage and polyadenylation. The canonical PAP, PAPα, was considered the only nuclear PAP involved in general polyadenylation of mRNAs. A phosphoinositide-modulated nuclear PAP, Star-PAP, was then reported to regulate a select set of mRNAs in the cell. In addition, several non-canonical PAPs have been identified with diverse cellular functions. Further, canonical PAP itself exists in multiple isoforms thus illustrating the diversity of PAPs. In this review, we compare two nuclear PAPs, Star-PAP and PAPα with a general overview of PAP diversity in the cell. Emerging evidence suggests distinct niches of target pre-mRNAs for the two PAPs and that modulation of these PAPs regulates distinct cellular functions.     

Production of pokeweed antiviral proteins nontoxic to cells by Mutagenesis of PAP-cDNA

Pokeweed antiviral protein (PAP), one of ribosome inactivating proteins (RIPs), has very strong toxicity both to prokaryotic and eukaryotic cells. To produce mutant PAPs nontoxic to cells, the PAP-cDNA was inserted into a yeast-E.coli shuttle vector under the control of galactose promoter, mutagenized using hydroxylamine, and transformed into yeast cells. Transformed yeast cells were selected on the uracil-deficient plate containing glucose or raffinose, and the yeast cells producing mutant PAPs nontoxic to cells were then selected on the galactose plate. Eighteen mutants were obtained by immunoblot analyses of 1,000 transformants: among them, three, ten and five mutants produced unprocessed, mature and truncated PAPs, respectively. Fourteen PAP mutants among them did not inhibit the yeast cell growth, and showed no or less inhibition of protein synthesisin vitro. Six among fourteen mutants were able to protect TMV infection in coinoculation experiment. The mutant PAPs showing an antiviral activity either without or reduced RIP activity contain neither the active site mutation nor C-terminal deletion mutation. These results suggest that both the RIP activity and the antiviral activity will require other amino acid residue(s) besides the active site and that the antiviral acitivity of PAP can be dissociated from its toxicity.     

Mutational analysis of the interaction between active site residues and the loop region in mammalian purple acid phosphatases

Mammalian purple acid phosphatases (PAPs) can be divided into two groups, which exhibit distinct spectroscopic and kinetics properties: PAPs that consist of a single 36 kDa polypeptide, and PAPs that have undergone limited proteolysis to give two fragments with masses of 16 and 20 kDa, respectively. Proteolysis results in an increase in enzymatic activity, an increase in the optimal pH for activity, and a change in the g(z)() value of the characteristic EPR spectrum of the mixed-valence binuclear iron center. It has been proposed that these changes are due to the loss of interactions between Asp146 in an exposed loop region and active site residues upon proteolysis. In the present study, site-directed mutagenesis of Asp146 in recombinant rat bone PAP (recRPAP) has confirmed this hypothesis. Conversion of Asp146 into Ala, which eliminates the interaction of the side chain with the active site, resulted in an enzyme with properties typical of PAPs isolated in proteolytically cleaved forms. The Asp146Asn and Asp146Glu mutants were also prepared and examined to assess the effects of altered electrostatic interactions and side-chain length. Limited proteolysis of all three mutant enzymes with cathepsin L resulted in a significant increase in catalytic activity. Thus, although the interaction between Asp146 and (an) active site residue(s) is the major factor responsible for the low catalytic activity of uncleaved PAPs, other interactions are also important. Since both p-nitrophenyl phosphate and osteopontin, a potential in vivo substrate, show the same level of activation, the observed increase in catalytic activity upon proteolysis is likely to be due to electrostatic rather than steric effects. EPR spectra of FeZn-recRPAP before and after cleavage by cathepsin L suggest that cleavage primarily affects the divalent metal site. The observation that pK(es,1) is also sensitive to changes at the divalent site is consistent with the proposal that the nucleophilic hydroxide is that bridging the divalent and trivalent metals.     

Characterization of a novel plant poly(A) polymerase

We have purified and characterized poly(A) polymerases (PAPs) from Pisum sativum, Brassica juncea, and Zea mays. Through chromatography on DEAE-Sepharose and heparin-Sepharose, these PAPs copurified as a single enzyme along with RNPs that could provide RNA substrates for the enzyme. More extensive purification by chromatography on MonoQ resulted in the resolution of the PAPs into as many as three fractions. One of these (PAP-I) contained a 43-kDa polypeptide immunologically related to the yeast PAP, and two others (PAP-II and PAP-III) contained RNAs that could serve as substrates for polyadenylation. These fractions by themselves possessed little PAP activity, but mixtures containing combinations of these displayed substantial activity. Similar PAP factors (PAP-I and PAP-III) were identified after fractionation of extracts prepared from Brassica juncea and Zea mays. The factors from one plant were completely interchangeable with those from different plants. We conclude that the poly(A) polymerases present in vegetative plant tissues consist of more than one component. In this respect, they are substantially different from other reported plant, mammalian, and yeast PAPs.     

Novel function of the human presqualene diphosphate phosphatase as a type II phosphatidate phosphatase in phosphatidylcholine and triacylglyceride biosynthesis pathways

Phosphatidate phosphatases, PAPs, are key enzymes in lipid biosynthesis and signaling. Type I PAP enzymes participate in de-novo phospholipid biosynthesis, whereas type II PAP enzymes have an established role in lipid signaling. To identify novel human type II PAPs potentially involved in de-novo phospholipid synthesis we used bioinformatics to screen for enzymes with an active site exposed to the cytosolic side of membranes. Two related enzymes, a novel lipid phosphatase related protein (LPRP-A) and a presqualene diphosphate phosphatase (PA-PSP) met this criterion. PA-PSP and LPRP-A have differential tissue and subcellular distribution, and novel yet differential roles in lipid metabolism. Specifically, PA-PSP, but not LPRP-A, was a potent Mg(2+)-independent, NEM-insensitive type II PAP. Subcellular fractionation detection indicated that both proteins were associated with membranes, while immunofluorescent deconvolution imaging revealed that these membranes were exclusively from the nuclear envelope and the endoplasmic reticulum. PA-PSP overexpression, but not LPRP-A, accelerated the synthesis of phosphatidylcholine and caused accumulation of triacylglycerol with concomitant decrease in the rate of phosphatidylinositol synthesis. Coexpression of human CTP:phosphocholine cytidylyltransferase-alpha with PA-PSP enhanced the effect of PA-PSP on phosphatidylcholine levels, yet attenuated its effect on triacylglycerol. Taken together, our studies provide the first evidence that the eukaryotic, ER-resident PA-PSP is a bifunctional enzyme with specific type II PAP activity, and regulates, in addition to type I PAPs, the de-novo biosynthesis of phospholipids and triacylglycerols.     

Poly (A) polymerases in the nucleus and cytoplasm of frog oocytes: dynamic changes during oocyte maturation and early development.

Poly(A) can be added to mRNAs both in the nucleus and in the cytoplasm. During oocyte maturation and early embryonic development, cytoplasmic polyadenylation of preexisting mRNAs provides a common mechanism of translational control. In this report, to begin to understand the regulation of polyadenylation activities during early development, we analyze poly (A) polymerases (PAPs) in oocytes and early embryos of the frog, Xenopus laevis. We have cloned and sequenced a PAP cDNA that corresponds to a maternal mRNA present in frog oocytes. This PAP is similar in size and sequence to mammalian nuclear PAPs. By immunoblotting using monoclonal antibodies raised against human PAP, we demonstrate that oocytes contain multiple forms of PAP that display different electrophoretic mobilities. The oocyte nucleus contains primarily the slower migrating forms of PAP, whereas the cytoplasm contains primarily the faster migrating species. The nuclear forms of PAP are phosphorylated, accounting for their retarded mobility. During oocyte maturation and early postfertilization development, preexisting PAPs undergo regulated phosphorylation and dephosphorylation events. Using the cloned PAP cDNA, we demonstrate that the complex changes in PAP forms seen during oocyte maturation may be due to modifications of a single polypeptide. These results demonstrate that the oocyte contains a cytoplasmic polymerase closely related to the nuclear enzyme and suggest models for how its activity may be regulated during early development.     

cDNA cloning, expression pattern, genomic structure and chromosomal location of RAB6KIFL, a human kinesin-like gene

Purple acid phosphatases (PAPs) comprise a family of binuclear metal-containing hydrolases, members of which have been isolated from plants, mammals and fungi. Polypeptide chains differ in size (animal 35 kDa, plant 55 kDa) and exhibit low sequence homology between kingdoms but all residues involved in co-ordination of the metal ions are invariant. A search of genomic databases was undertaken using a sequence pattern which includes the conserved residues. Several novel potential PAP sequences were detected, including the first known examples from bacterial sources. Ten plant ESTs were also identified which, although possessing the conserved sequence pattern, were not homologous throughout their sequences to previously known plant PAPs. Based on these EST sequences, novel cDNAs from sweet potato, soybean, red kidney bean and Arabidopsis thaliana were cloned and sequenced. These sequences are more closely related to mammalian PAP than to previously characterized plant enzymes. Their predicted secondary structure is similar to that of the mammalian enzyme. A model of the sweet potato enzyme was generated based on the coordinates of pig PAP. These observations strongly suggest that the cloned cDNA sequences represent a second group of plant PAPs with properties more similar to the mammalian enzymes than to the high molecular weight plant enzymes.     

Poly(A) polymerase and the regulation of cytoplasmic polyadenylation

Translational activation in oocytes and embryos is often regulated via increases in poly(A) length. Cleavage and polyadenylation specificity factor (CPSF), cytoplasmic polyadenylation element binding protein (CPEB), and poly(A) polymerase (PAP) have each been implicated in cytoplasmic polyadenylation in Xenopus laevis oocytes. Cytoplasmic polyadenylation activity first appears in vertebrate oocytes during meiotic maturation. Data presented here shows that complexes containing both CPSF and CPEB are present in extracts of X. laevis oocytes prepared before or after meiotic maturation. Assessment of a variety of RNA sequences as polyadenylation substrates indicates that the sequence specificity of polyadenylation in egg extracts is comparable to that observed with highly purified mammalian CPSF and recombinant PAP. The two in vitro systems exhibit a sequence specificity that is similar, but not identical, to that observed in vivo, as assessed by injection of the same RNAs into the oocyte. These findings imply that CPSFs intrinsic RNA sequence preferences are sufficient to account for the specificity of cytoplasmic polyadenylation of some mRNAs. We discuss the hypothesis that CPSF is required for all polyadenylation reactions, but that the polyadenylation of some mRNAs may require additional factors such as CPEB. To test the consequences of PAP binding to mRNAs in vivo, PAP was tethered to a reporter mRNA in resting oocytes using MS2 coat protein. Tethered PAP catalyzed polyadenylation and stimulated translation approximately 40-fold; stimulation was exclusively cis-acting, but was independent of a CPE and AAUAAA. Both polyadenylation and translational stimulation required PAPs catalytic core, but did not require the putative CPSF interaction domain of PAP. These results demonstrate that premature recruitment of PAP can cause precocious polyadenylation and translational stimulation in the resting oocyte, and can be interpreted to suggest that the role of other factors is to deliver PAP to the mRNA.     

Plant purple acid phosphatases - genes, structures and biological function

The properties of plant purple acid phosphatases (PAPs), metallophosphoesterases present in some bacteria, plants and animals are reviewed. All members of this group contain a characteristic set of seven amino-acid residues involved in metal ligation. Animal PAPs contain a binuclear metallic center composed of two irons, whereas in plant PAPs one iron ion is joined by zinc or manganese ion. Among plant PAPs two groups can be distinguished: small PAPs, monomeric proteins with molecular mass around 35 kDa, structurally close to mammalian PAPs, and large PAPs, homodimeric proteins with a single polypeptide of about 55 kDa. Large plant PAPs exhibit two types of structural organization. One type comprises enzymes with subunits bound by a disulfide bridge formed by cysteines located in the C-terminal region around position 350. In the second type no cysteines are located in this position and no disulfide bridges are formed between subunits. Differences in structural organisation are reflected in substrate preferences. Recent data reveal in plants the occurrence of metallophosphoesterases structurally different from small or large PAPs but with metal-ligating sequences characteristic for PAPs and expressing pronounced specificity towards phytate or diphosphate nucleosides and inorganic pyrophosphate.     

Expression and distribution of tartrate-resistant purple acid phosphatase in the rat nervous system.

Tartrate-resistant purple acid phosphatase (TRAP) of osteoclasts and certain cells of the monocyte-macrophage lineage belongs to the family of purple acid phosphatases (PAPs). We provide here evidence for TRAP/PAP expression in the central and peripheral nervous systems in the rat. TRAP/PAP protein was partially purified and characterized from the trigeminal ganglion, brain, and spinal cord. The TRAP activity (U/mg tissue) in these tissues was about 10-20 times lower than in bone. Reducing agents, e.g. ascorbate and ferric iron, increased the TRAP activity from the neural tissues (nTRAP) and addition of oxidizing agents completely inactivated both bone and nTRAP. The IC(50) for three known oxyanion inhibitors of TRAP/PAP was similar for bone and nTRAP with the same rank order of potency (molybdate > tungstate > phosphate). This indicates that the redox-sensitive binuclear iron center characteristic of mammalian PAPs is present also in nTRAP. Western blots of partially purified nTRAP revealed a band with the expected size of 35 kD. The expression of TRAP in the trigeminal ganglion, brain, and spinal cord was confirmed at the mRNA level by RT-PCR. In situ hybridization histochemistry demonstrated TRAP mRNA expression in small ganglion cells of the trigeminal ganglion, in alpha-motor neurons of the ventral spinal cord, and in Purkinje cells of the cerebellum. TRAP-like immunoreactivity was encountered in the cytoplasm of neuronal cell bodies in specific areas of both the central and the peripheral nervous system. Together, the data demonstrate that active TRAP/PAP is expressed in certain parts of the rat nervous system.