Heterologous expression of Pleurotus eryngii peroxidase confirms its ability to oxidize Mn(2+) and different aromatic substrates.

A versatile ligninolytic peroxidase has been cloned from Pleurotus eryngii and its allelic variant MnPL2 expressed in Aspergillus nidulans, with properties similar to those of the mature enzyme from P. eryngii. These include the ability to oxidize Mn(2+) and aromatic substrates, confirming that this is a new peroxidase type sharing catalytic properties of lignin peroxidase and manganese peroxidase.     

Heterologous gene expression in continuous cultures of budding yeast

Summary In a previous paper we have studied the expression of -galactosidase from Escherichia coli, driven from the inducible GAL1-10/CYC1 hybrid promoter, in batch cultures of budding Saccharomyces cerevisiae and have described operating conditions for maximal productivity. In this paper we show that the plasmid instability in continuous cultures can be overcome by utilizing appropriate selection markers and a high copy number vector. The maximal level of expression is influenced by the dilution rate. Moreover, enzyme accumulation appears to depend also upon the degree of oxygenation. A possible explanation of these modulations is discussed, taking into account the interactions of the UAS-GAL and TATA-CYC1 elements. Offprint requests to: D. Porro     

Heterologous expression of functional G-protein-coupled receptors in Caenorhabditis elegans

New strategies for expression, purification, functional characterization, and structural determination of membrane-spanning G-protein-coupled receptors (GPCRs) are constantly being developed because of their importance to human health. Here, we report a Caenorhabditis elegans heterologous expression system able to produce milligram amounts of functional native and engineered GPCRs. Both bovine opsin [(b)opsin] and human adenosine A(2A) subtype receptor [(h)A(2A)R] expressed in neurons or muscles of C. elegans were localized to cell membranes. Worms expressing these GPCRs manifested changes in motor behavior in response to light and ligands, respectively. With a newly devised protocol, 0.6-1 mg of purified homogenous 9-cis-retinal-bound bovine isorhodopsin [(b)isoRho] and ligand-bound (h)A(2A)R were obtained from C. elegans from one 10-L fermentation at low cost. Purified recombinant (b)isoRho exhibited its signature absorbance spectrum and activated its cognate G-protein transducin in vitro at a rate similar to native rhodopsin (Rho) obtained from bovine retina. Generally high expression levels of 11 native and mutant GPCRs demonstrated the potential of this C. elegans system to produce milligram quantities of high-quality GPCRs and possibly other membrane proteins suitable for detailed characterization.     

Occurrence, regulation, and significance of progesterone receptors in human meningioma

The abundant expression of progesterone receptors (PR) in human meningiomas is well established. It is unknown, however, how PR expression is regulated, especially since estrogen receptors (ER) are virtually absent in these tumors. At the mRNA level, ER splice variants occur in meningioma but these appear not to be involved in the apparently autonomous PR expression. In an earlier study, because other ER-inducible proteins were either not expressed at all (pS2) or were expressed at a very low level compared to their expression in breast cancer (Cathepsin-D), the authors have postulated that the autonomous PR expression in meningioma is PR promoter-related rather than ER-related and have studied PR expression in cultured meningioma cells. PR levels appeared to decrease rapidly in vitro in monolayer as well as in three dimensional spheroid cultures. Culture conditions thus are not yet sufficient for the quantitative evaluation of PR expression. To evaluate whether PR deterioration is associated with cell turnover (meningiomas grow much faster in vitro than in vivo), the relationship between expression of the apoptotic proteins Bcl-2 and Bax and PR expression was investigated. Bcl-2 expression was found to be highest in meningioma with low PR levels, and in breast cancer tissue with high PR levels. Bax expression was not related to PR expression in any of the two tissues. Given the potential benefit of antiprogestin treatment and the occurrence in meningiomas of a protein capable of binding to the estrogen-responsive element, the expression of PR in meningioma remains a fascinating phenomenon which requires further investigation.     

Heterologous expression of a mammalian epithelial sodium channel in yeast

The alpha and beta subunits of the amiloride-sensitive rat epithelial sodium channel (alpha beta ENaC) were expressed in the yeast Saccharomyces cerevisiae. We used a combination of yeast strains, including a mutant in the secretory pathway (sec6), and Western blotting techniques, to show that alpha beta ENaC was synthesized and targeted through the secretory system to the plasma membrane. Yeasts expressing alpha beta ENaC were more sensitive to salt than the parent strain. In addition, amiloride, a specific blocker of ENaC, was found to suppress salt sensitivity in the yeast strain expressing alpha beta ENaC.     

Heterologous expression systems for P-glycoprotein:E. coli,yeast, and baculovirus

Chemotherapy, though it remains one of the front-line weapons used to treat human cancer, is often ineffective due to drug resistance mechanisms manifest in tumor cells. One common pattern of drug resistance, characterized by simultaneous resistance to multiple amphipathic, but otherwise structurally dissimilar anticancer drugs, is termed multidrug resistance. Multidrug resistance in various model systems, covering the phylogenetic range from bacteria to man, can be conferred by mammalian P-glycoproteins (PGPs), often termed multidrug transporters. PGPs are 170-kD polytopic membrane proteins, predicted to consist of two homologous halves, each with six membrane spanning regions and one ATP binding site. They are members of the ATP-binding cassette (ABC) superfamily of transporters, and are known to function biochemically as energy-dependent drug efflux pumps. However, much remains to be learned about PGP structure-function relationships, membrane topology, posttranslational regulation, and bioenergetics of drug transport. Much of the recent progress in the study of the human and mouse PGPs has come from heterologous expression systems which offer the benefits of ease of genetic selection and manipulation, and/or short generation times of the organism in which PGPs are expressed, and/or high-level expression of recombinant PGP. Here we review recent studies of PGP inE. coli, baculovirus, and yeast systems and evaluate their utility for the study of PGPs, as well as other higher eukaryotic membrane proteins.     

An assessment of the ability of yeast cells to incorporate photolabile fatty acids into their membrane phospholipids in vivo

The photolabile fatty acids 12-azidooleic, 12-(4-azido-2-nitrophenoxy)oleic, 12-azidolauric and 12-(4-azido-2-nitrophenoxy)lauric are readily taken up in vivo by an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. A low level of the two lauric acid derivatives and none of the two oleic acid derivatives are incorporated into membrane phospholipids. Under certain conditions of growth in the presence of 12-(4-azido-2-nitrophenoxy)oleic acid, the nitrophenylazide group is metabolized to a product that lacks the photolabile azido group.     

Heterologous expression, purification, and characterization of human triacylglycerol hydrolase.

Triacylglycerol hydrolase mobilizes stored triacylglycerol some of which is used for very-low-density lipoprotein assembly in the liver. A full-length cDNA coding for a human triacylglycerol hydrolase (hTGH) was isolated from a human liver cDNA library. The cDNA has an open reading frame of 576 amino acids with a cleavable 18-amino-acid signal sequence. The deduced amino acid sequence shows that the protein belongs to the carboxylesterase family. The hTGH was highly expressed in Escherichia coli as a 6xHis-tagged fusion protein, with the tag at the N-terminus in place of the signal peptide. However, the expressed protein was insoluble and inactive. Expression was confirmed by immunoblotting and N-terminal amino acid sequencing of the purified protein. Expression of hTGH with its native signal sequence and a C-terminal 6xHis-tag in Sf9 cells using the baculovirus expression system yielded active enzyme. N-terminal amino acid sequencing of the purified expressed protein showed correct processing of the signal peptide. The enzyme also undergoes glycosylation within the endoplasmic reticulum lumen. The results suggest that hTGH expressed in insect cells is properly folded. Therefore, baculovirus expression of hTGH and facile purification of the His-tagged enzyme will allow detailed characterization of the structure/activity relationship.     

Functional expression of parasite drug targets and their human orthologs in yeast

Background

The exacting nutritional requirements and complicated life cycles of parasites mean that they are not always amenable to high-throughput drug screening using automated procedures. Therefore, we have engineered the yeast Saccharomyces cerevisiae to act as a surrogate for expressing anti-parasitic targets from a range of biomedically important pathogens, to facilitate the rapid identification of new therapeutic agents.

Methodology/Principal Findings

Using pyrimethamine/dihydrofolate reductase (DHFR) as a model parasite drug/drug target system, we explore the potential of engineered yeast strains (expressing DHFR enzymes from Plasmodium falciparum, P. vivax, Homo sapiens, Schistosoma mansoni, Leishmania major, Trypanosoma brucei and T. cruzi) to exhibit appropriate differential sensitivity to pyrimethamine. Here, we demonstrate that yeast strains (lacking the major drug efflux pump, Pdr5p) expressing yeast (^ScDFR1), human (^HsDHFR), Schistosoma (^SmDHFR), and Trypanosoma (^TbDHFR and ^TcDHFR) DHFRs are insensitive to pyrimethamine treatment, whereas yeast strains producing Plasmodium (^PfDHFR and ^PvDHFR) DHFRs are hypersensitive. Reassuringly, yeast strains expressing field-verified, drug-resistant mutants of P. falciparum DHFR (^Pfdhfr ^51I,59R,108N) are completely insensitive to pyrimethamine, further validating our approach to drug screening. We further show the versatility of the approach by replacing yeast essential genes with other potential drug targets, namely phosphoglycerate kinases (PGKs) and N-myristoyl transferases (NMTs).

Conclusions/Significance

We have generated a number of yeast strains that can be successfully harnessed for the rapid and selective identification of urgently needed anti-parasitic agents.     

Heterologous expression and purification of active human phosphoribosylglycinamide formyltransferase as a single domain

We report here for the first time that the GART domain of the human trifunctional enzyme possessing GARS, AIRS, and GART activities can be expressed independently inEscherichia coli at high levels as a stable protein with enzymatic characteristics comparable to those of native trifunctional protein. Human trifunctional enzyme is involved inde novo purine biosynthesis, and has long been recognized as a target for antineoplastic intervention. The GART domain was expressed inE. coli under the control of bacteriophage T7 promotor and isolated by a three-step chromatographic procedure. Two residues, Asp 951 and His 915, were shown to be catalytically crucial by site-directed mutagenesis and subsequent characterization of purified mutant proteins. The active monofunctional GART protein produced inE. coli can serve as a valuable substitute of trifunctional enzyme for structural and functional studies which have been until now hindered because of insufficient quantity, instability, and size of the trifunctional GART protein.     

The structure and function of progesterone receptors in breast cancer

This paper is a review of the clinical role of progesterone receptors (PR) in the management of breast cancer, and the use of synthetic progestins in treatment of the metastatic disease. Also reviewed are our basic molecular studies dealing with the structure of human breast cancer PR, focusing on the two hormone binding proteins (the A- and B-receptors) and the role of phosphorylation. A model for the structure of PR and their subcellular compartmentalization following hormone treatment is presented.     

Heterologous expression,purification, and functional characterization of recombinant ovine angiotensinogen in the methylotrophic yeast Pichia pastoris

Angiotensinogen (AGT), a glycosylated plasma noninhibitory serpin, serves as a precursor for angiotensin peptides which regulate blood pressure and electrolyte balance. AGT is specifically cleaved by renin to produce angiotensin-I, the first product of the angiotensin-processing cascade. Ovine angiotensinogen (oAGT) is considered an effective substrate for human renin and consequently finds application in clinical renin assays. In this study, oAGT was cloned into the genome of Pichia pastoris and expressed under the control of alcohol oxidase (AOX1) promoter for high-level production. Compared to the shake flask study, the high cell density cultivation in bioreactor resulted in multifold increase in oAGT titer (420 ± 9.26 mg/L), which is its highest reported titer to date. We purified recombinant oAGT to homogeneity using two chromatography steps. The characterization studies revealed oAGT underwent a two-state transition during thermal denaturation process as assessed by differential scanning fluorimetry, and the melting temperature (T_m) of the purified oAGT from P. pastoris was 48.3°C. Renin reactivity with recombinant oAGT from P. pastoris (0.51 nM angiotensin-I/min) was slightly lower than the renin reactivity for recombinant oAGT from Escherichia coli (0.67 nM angiotensin-I/min), possibly because of its mannosylated N-glycan content. Enhanced production of functionally active recombinant oAGT using P. pastoris expression system reported in this study envisage the effective utilization of oAGT in clinical studies related to renin in near future.     

Functional characteristics of membrane progestin receptor alpha (mPRalpha) subtypes: a review with new data showing mPRalpha expression in seatrout sperm and its association with sperm motility

A novel cDNA with characteristics of the membrane progestin receptor regulating induction of oocyte maturation by a non-genomic mechanism, named st-mPRalpha, was recently discovered in seatrout. Subsequently, both recombinant and native mPRalphas have been localized to the plasma membrane in several vertebrate models where they have been shown to bind progestins specifically, resulting in activation of G proteins. Non-genomic actions of progestins to stimulate hypermotility and the acrosome reaction in sperm have been identified, but the receptors which mediate these processes are unknown. Here, we demonstrate progestin stimulation of sperm motility in seatrout and expression of st-mPRalpha mRNA and protein in sperm with the receptor localized on the plasma membrane. Immunocytochemical staining of non-permeabilized sperm shows st-mPRalpha is localized to the midpiece with an extracellular N-terminal region, indicating its likely role in progestin regulation of sperm motility. Moreover, the abundance of the st-mPRalpha protein on sperm membranes from seatrout donors with low motility was significantly reduced compared to that of normal motile sperm. Finally, progestin treatment of sperm membranes caused activation of G proteins. These results suggest that st-mPRalpha is an intermediary in progestin stimulation of sperm motility in seatrout by an unknown mechanism involving G protein activation.     

Synthesis and function of membrane phosphoinositides in budding yeast, Saccharomyces cerevisiae

It is now well appreciated that derivatives of phosphatidylinositol (PtdIns) are key regulators of many cellular processes in eukaryotes. Of particular interest are phosphoinositides (mono- and polyphosphorylated adducts to the inositol ring in PtdIns), which are located at the cytoplasmic face of cellular membranes. Phosphoinositides serve both a structural and a signaling role via their recruitment of proteins that contain phosphoinositide-binding domains. Phosphoinositides also have a role as precursors of several types of second messengers for certain intracellular signaling pathways. Realization of the importance of phosphoinositides has brought increased attention to characterization of the enzymes that regulate their synthesis, interconversion, and turnover. Here we review the current state of our knowledge about the properties and regulation of the ATP-dependent lipid kinases responsible for synthesis of phosphoinositides and also the additional temporal and spatial controls exerted by the phosphatases and a phospholipase that act on phosphoinositides in yeast.     

Fibronectin receptors of human keratinocytes and their expression during cell culture

Keratinocyte attachment to fibronectin (FN) substrata was inhibited by the peptide Gly-Arg-Gly-Asp-Ser-Pro-Cys, but not by the variant peptide Gly-Arg-Gly-Glu-Ser-Pro. The RGDS-containing peptide did not inhibit keratinocyte adhesion to collagen. Keratinocyte adhesion to FN substrata also was inhibited by polyclonal anti-FN receptor antibodies originally prepared against the 140-kD FN receptors of Chinese hamster ovary (CHO) cells. Anti-CHO FN receptor antibodies did not, however, inhibit keratinocyte adhesion to collagen substrata. A monoclonal antibody designated VM-1 that was prepared against human basal keratinocytes inhibited keratinocyte adhesion to collagen but not to FN. Based on these results, we conclude that keratinocytes have distinct FN and collagen receptors. Experiments were performed to compare the expression of FN receptors on keratinocytes freshly isolated from skin and keratinocytes harvested from cell cultures. Cells harvested from keratinocyte cultures were able to neutralize the inhibitory activity of anti-CHO FN receptor antibodies and were able to attach and spread on anti-CHO FN receptor-coated substrata. Cells freshly harvested from skin, however, did not neutralize the antibodies, nor did they attach and spread on antibody-coated substrata. To learn more about the biochemical nature of the keratinocyte FN receptors, we performed immunoaffinity chromatography and immunoprecipitation experiments using the anti-CHO FN receptor antibodies. Extracts from metabolically radiolabeled, 10-d cultured keratinocytes contained FN receptors that had a 135-kD component under reducing conditions and 115- and 155-kD components under nonreducing conditions. Similar components were observed in extracts from surface- radiolabeled cells indicating that the FN receptors were expressed on keratinocyte cell surfaces. On the other hand, extracts from metabolically radiolabeled, 1-d cultured keratinocytes lacked intact FN receptors but contained a component that migrated at 48 kD under reducing conditions and 50 kD under nonreducing conditions. Because this fragment was not detected in surface-radiolabeled keratinocytes that were freshly isolated from skin, it seems likely that the fragment was located inside the cells rather than on the cell surface. A 50-kD FN receptor fragment also was observed in extracts from 10-d cultured keratinocytes if leupeptin and pepstatin were omitted from the extraction buffer. The results suggested that human keratinocytes cultured for 10 d express the 140-kD class of FN receptors, but that these receptors are not expressed on the surfaces of keratinocytes freshly isolated from skin.(ABSTRACT TRUNCATED AT 400 WORDS)