Pro-phenol oxidase activating proteinase from an insect, Manduca sexta: A bacteria-inducible protein similar to Drosophila easter

Activation of pro-phenol oxidase (proPO) in insects and crustaceans is important in defense against wounding and infection. The proPO zymogen is activated by a specific proteolytic cleavage. PO oxidizes phenolic compounds to produce quinones, which may help to kill pathogens and can also be used for synthesis of melanin to seal wounds and encapsulate parasites. We have isolated from the tobacco hornworm, Manduca sexta, a serine proteinase that activates proPO, and have cloned its cDNA. The isolated proPO activating proteinase (PAP) hydrolyzed artificial substrates but required other protein factors for proPO activation, suggesting that proPO-activating enzyme may exist as a protein complex, one component of which is PAP. PAP (44 kDa) is composed of two disulfide-linked polypeptide chains (31 kDa and 13 kDa). A cDNA for PAP was isolated from a hemocyte library, by using a PCR-generated probe based on the amino-terminal amino acid sequence of the 31-kDa catalytic domain. PAP belongs to a family of arthropod serine proteinases containing a carboxyl-terminal proteinase domain and an amino-terminal “clip” domain. The member of this family most similar in sequence to PAP is the product of the easter gene from Drosophila melanogaster. PAP mRNA was present at a low level in larval hemocytes and fat body, but became much more abundant in fat body after insects were injected with Escherichia coli. Sequence data and 3H-diisopropyl fluorphosphate labeling results suggest that the same PAP exists in hemolymph and cuticle.

Development of an in vitro mRNA decay system for Escherichia coli: Poly(A) polymerase I is necessary to trigger degradation

Using a novel Escherichia coli in vitro decay system in which polysomes are the source of both enzymes and mRNA, we demonstrate a requirement for poly(A) polymerase I (PAP I) in mRNA turnover. The in vitro decay of two different mRNAs (trxA and lpp) is triggered by the addition of ATP only when polysomes are prepared from a strain carrying the wild-type gene for PAP I (pcnB+). The relative decay rates of these two messages are similar in vitro and in vivo. Poly(A) tails are formed on both mRNAs, but no poly(A) tails are detected on the 3′ end of mature 23S rRNA. The size distribution of poly(A) tails generated in vitro, averaging 50 nt in length, is comparable to that previously reported in vivo. PAP I activity is associated exclusively with the polysomes. Exogenously added PAP I does not restore mRNA decay to PAP I− polysomes, suggesting that, in vivo, PAP I may be part of a multiprotein complex. The potential of this in vitro system for analyzing mRNA decay in E. coli is discussed.

Sulfate reduction in higher plants: Molecular evidence for a novel 5′-adenylylsulfate reductase

Sulfate-assimilating organisms reduce inorganic sulfate for Cys biosynthesis. There are two leading hypotheses for the mechanism of sulfate reduction in higher plants. In one, adenosine 5′-phosphosulfate (APS) (5′-adenylylsulfate) sulfotransferase carries out reductive transfer of sulfate from APS to reduced glutathione. Alternatively, the mechanism may be similar to that in bacteria in which the enzyme, 3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase, catalyzes thioredoxin (Trx)-dependent reduction of PAPS. Three classes of cDNA were cloned from Arabidopsis thaliana termed APR1, -2, and -3, that functionally complement a cysH, PAPS reductase mutant strain of Escherichia coli. The coding sequence of the APR clones is homologous with PAPS reductases from microorganisms. In addition, a carboxyl-terminal domain is homologous with members of the Trx superfamily. Further genetic analysis showed that the APR clones can functionally complement a mutant strain of E. coli lacking Trx, and an APS kinase, cysC. mutant. These results suggest that the APR enzyme may be a Trx-independent APS reductase. Cell extracts of E. coli expressing APR showed Trx-independent sulfonucleotide reductase activity with a preference for APS over PAPS as a substrate. APR-mediated APS reduction is dependent on dithiothreitol, has a pH optimum of 8.5, is stimulated by high ionic strength, and is sensitive to inactivation by 5′-adenosinemonophosphate (5′-AMP). 2′-AMP, or 3′-phosphoadenosine-5′-phosphate (PAP), a competitive inhibitor of PAPS reductase, do not affect activity. The APR enzymes may be localized in different cellular compartments as evidenced by the presence of an amino-terminal transit peptide for plastid localization in APR1 and APR3 but not APR2. Southern blot analysis confirmed that the APR clones are members of a small gene family, possibly consisting of three members.

Improved cervical smear assessment using antibodies against proteins that regulate DNA replication

Carcinoma of the cervix is one of the most common malignancies. Papanicolaou (Pap) smear tests have reduced mortality by up to 70%. Nevertheless their interpretation is notoriously difficult with high false-negative rates and frequently fatal consequences. We have addressed this problem by using affinity-purified antibodies against human proteins that regulate DNA replication, namely Cdc6 and Mcm5. These antibodies were applied to sections and smears of normal and diseased uterine cervix by using immunoperoxidase or immunofluorescence to detect abnormal precursor malignant cells. Antibodies against Cdc6 and Mcm5 stain abnormal cells in cervical smears and sections with remarkably high specificity and sensitivity. Proliferation markers Ki-67 and proliferating cell nuclear antigen are much less effective. The majority of abnormal precursor malignant cells are stained in both low-grade and high-grade squamous intraepithelial lesions. Immunostaining of cervical smears can be combined with the conventional Pap stain so that all the morphological information from the conventional method is conserved. Thus antibodies against proteins that regulate DNA replication can reduce the high false-negative rate of the Pap smear test and may facilitate mass automated screening.

Binding of factor VIIa to tissue factor induces alterations in gene expression in human fibroblast cells: Up-regulation of poly(A) polymerase

Tissue factor (TF) is the cellular receptor for an activated form of clotting factor VII (VIIa) and the binding of factor VII(a) to TF initiates the coagulation cascade. Sequence and structural patterns extracted from a global alignment of TF confers homology with interferon receptors of the cytokine receptor super family. Several recent studies suggested that TF could function as a genuine signal transducing receptor. However, it is unknown which biological function(s) of cells are altered upon the ligand, VIIa, binding to TF. In the present study, we examined the effect of VIIa binding to cell surface TF on cellular gene expression in fibroblasts. Differential mRNA display PCR technique was used to identify transcriptional changes in fibroblasts upon VIIa binding to TF. The display showed that VIIa binding to TF either up or down-regulated several mRNA species. The differential expression of one such transcript, VIIa-induced up-regulation, was confirmed by Northern blot analysis. Isolation of a full-length cDNA corresponding to the differentially expressed transcript revealed that VIIa-up-regulated gene was poly(A) polymerase. Northern blot analysis of various carcinomas and normal human tissues revealed an over expression of PAP in cancer tissues. Enhanced expression of PAP upon VIIa binding to tumor cell TF may potentially play an important role in tumor metastasis.

Sequential Actions of Phospholipase D and Phosphatidic Acid Phosphohydrolase 2b Generate Diglyceride in Mammalian Cells

Phosphatidylcholine (PC) is a major source of lipid-derived second messenger molecules that function as both intracellular and extracellular signals. PC-specific phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) are two pivotal enzymes in this signaling system, and they act in series to generate the biologically active lipids phosphatidic acid (PA) and diglyceride. The identity of the PAP enzyme involved in PLD-mediated signal transduction is unclear. We provide the first evidence for a functional role of a type 2 PAP, PAP2b, in the metabolism of PLD-generated PA. Our data indicate that PAP2b localizes to regions of the cell in which PC hydrolysis by PLD is taking place. Using a newly developed PAP2b-specific antibody, we have characterized the expression, posttranslational modification, and localization of endogenous PAP2b. Glycosylation and localization of PAP2b appear to be cell type and tissue specific. Biochemical fractionation and immunoprecipitation analyses revealed that PAP2b and PLD2 activities are present in caveolin-1–enriched detergent-resistant membrane microdomains. We found that PLD2 and PAP2b act sequentially to generate diglyceride within this specialized membrane compartment. The unique lipid composition of these membranes may provide a selective environment for the regulation and actions of enzymes involved in signaling through PC hydrolysis.

The Glycosylphosphatidylinositol-Anchored Phosphatase from Spirodela oligorrhiza Is a Purple Acid Phosphatase1

We recently presented clear evidence that the major low-phosphate-inducible phosphatase of the duckweed Spirodela oligorrhiza is a glycosylphosphatidylinositol (GPI)-anchored protein, and, to our knowledge, is the first described from higher plants (N. Morita, H. Nakazato, H. Okuyama, Y. Kim, G.A. Thompson, Jr. [1996] Biochim Biophys Acta 1290: 53–62). In this report the purified 57-kD phosphatase is shown to be a purple metalloenzyme containing Fe and Mn atoms and having an absorption maximum at 556 nm. The phosphatase activity was only slightly inhibited by tartrate, as expected for a purple acid phosphatase (PAP). Furthermore, the protein cross-reacted with an anti-Arabidopsis PAP antibody on immunoblots. The N-terminal amino acid sequence of the phosphatase was very similar to those of Arabidopsis, red kidney bean (Phaseolus vulgaris), and soybean (Glycine max) PAP. Extracts of S. oligorrhiza plants incubated with the GPI-specific precursor [3H]ethanolamine were treated with antibodies raised against the purified S. oligorrhiza phosphatase. Radioactivity from the resulting immunoprecipitates was specifically associated with a 57-kD band on sodium dodecyl sulfate-polyacrylamide gels. These results, together with previous findings, strongly indicate that the GPI-anchored phosphatase of S. oligorrhiza is a PAP.

Identification of the coding region for a second poly(A) polymerase in Escherichia coli.

We had earlier identified the pcnB locus as the gene for the major Escherichia coli poly(A) polymerase (PAP I). In this report, we describe the disruption and identification of a candidate gene for a second poly(A) polymerase (PAP II) by an experimental strategy which was based on the assumption that the viability of E. coli depends on the presence of either PAP I or PAP II. The coding region thus identified is the open reading frame f310, located at about 87 min on the E. coli chromosome. The following lines of evidence support f310 as the gene for PAP II: (i) the deduced peptide encoded by f310 has a molecular weight of 36,300, similar to the molecular weight of 35,000 estimated by gel filtration of PAP II; (ii) the deduced f310 product is a relatively hydrophobic polypeptide with a pI of 9.4, consistent with the properties of partially purified PAP II; (iii) overexpression of f310 leads to the formation of inclusion bodies whose solubilization and renaturation yields poly(A) polymerase activity that corresponds to a 35-kDa protein as shown by enzyme blotting; and (iv) expression of a f310 fusion construct with hexahistidine at the N-terminus of the coding region allowed purification of a poly(A) polymerase fraction whose major component is a 36-kDa protein. E. coli PAP II has no significant sequence homology either to PAP I or to the viral and eukaryotic poly(A) polymerases, suggesting that the bacterial poly(A) polymerases have evolved independently. An interesting feature of the PAP II sequence is the presence of sets of two paired cysteine and histidine residues that resemble the RNA binding motifs seen in some other proteins.

Isolation and characterization of pokeweed antiviral protein mutations in Saccharomyces cerevisiae: identification of residues important for toxicity.

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP. The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast. The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.