Cell-Free Protein Synthesis by Diversifying Bacterial Transcription Machinery.

We have evaluated several approaches to increase protein synthesis in a cell-free coupled bacterial transcription and translation system. A strong pargC promoter, originally isolated from a moderate thermophilic bacterium Geobacillus stearothermophilus, was used to improve the performance of a cell-free system in extracts of Escherichia coli BL21 (DE3). A stimulating effect on protein synthesis was detected with extracts prepared from recombinant cells, in which the E. coli RNA polymerase subunits α, ß, ß' and ω are simultaneously coexpressed. Appending a 3' UTR genomic sequence and a T7 transcription terminator to the protein-coding region also improves the synthetic activity of some genes from linear DNA. The E. coli BL21 (DE3) rna::Tn10 mutant deficient in a periplasmic RNase I was constructed. The mutant cell-free extract increases by up to four-fold the expression of bacterial and human genes mediated from both bacterial pargC and phage pT7 promoters. By contrast, the RNase E deficiency does not affect the cell-free expression of the same genes. The regulatory proteins of the extremophilic bacterium Thermotoga, synthesized in a cell-free system, can provide the binding capacity to target DNA regions. The advantageous characteristics of cell-free systems described open attractive opportunities for high-throughput screening assays.

Rational Engineering of the Substrate Specificity of a Thermostable D-Hydantoinase (Dihydropyrimidinase).

D-hydantoinases catalyze an enantioselective opening of 5- and 6-membered cyclic structures and therefore can be used for the production of optically pure precursors for biomedical applications. The thermostable D-hydantoinase from Geobacillus stearothermophilus ATCC 31783 is a manganese-dependent enzyme and exhibits low activity towards bulky hydantoin derivatives. Homology modeling with a known 3D structure (PDB code: 1K1D) allowed us to identify the amino acids to be mutated at the substrate binding site and in its immediate vicinity to modulate the substrate specificity. Both single and double substituted mutants were generated by site-directed mutagenesis at appropriate sites located inside and outside of the stereochemistry gate loops (SGL) involved in the substrate binding. Substrate specificity and kinetic constant data demonstrate that the replacement of Phe159 and Trp287 with alanine leads to an increase in the enzyme activity towards D,L-5-benzyl and D,L-5-indolylmethyl hydantoins. The length of the side chain and the hydrophobicity of substrates are essential parameters to consider when designing the substrate binding pocket for bulky hydantoins. Our data highlight that D-hydantoinase is the authentic dihydropyrimidinase involved in the pyrimidine reductive catabolic pathway in moderate thermophiles.

Screening and discovery of nitro-benzoxadiazole compounds activating epidermal growth factor receptor (EGFR) in cancer cells.

Peptide ligand-induced dimerization of the extracellular region of the epidermal growth factor receptor (sEGFR) is central to the signal transduction of many cellular processes. A small molecule microarray screen has been developed to search for non-peptide compounds able to bind to sEGFR. We describe the discovery of nitro-benzoxadiazole (NBD) compounds that enhance tyrosine phosphorylation of EGFR and thereby trigger downstream signaling pathways and other receptor tyrosine kinases in cancer cells. The protein phosphorylation profile in cells exposed to NBD compounds is to some extent reminiscent of the profile induced by the cognate ligand. Experimental studies indicate that the small compounds bind to the dimerization domain of sEGFR, and generate stable dimers providing allosteric activation of the receptor. Moreover, receptor phosphorylation is associated with inhibition of PTP-1B phosphatase. Our data offer a promising paradigm for investigating new aspects of signal transduction mediated by EGFR in cancer cells exposed to electrophilic NBD compounds.

Acid phosphatase 5 is responsible for removing the mannose 6-phosphate recognition marker from lysosomal proteins.

Most newly synthesized proteins destined for the lysosome reach this location via a specific intracellular pathway. In the Golgi, a phosphotransferase specifically labels lysosomal proteins with mannose 6-phosphate (Man-6-P). This modification is recognized by receptors that target the lysosomal proteins to the lysosome where, in most cell types, the Man-6-P recognition marker is rapidly removed. Despite extensive characterization of this pathway, the enzyme responsible for the removal of the targeting modification has remained elusive. In this study, we have identified this activity. Preliminary investigations using a cell-based bioassay were used to follow a dephosphorylation activity that was associated with the lysosomal fraction. This activity was high in the liver, where endogenous lysosomal proteins are efficiently dephosphorylated, but present at a much lower level in the brain, where the modification persists. This observation, combined with an analysis of the expression of lysosomal proteins in different tissues, led us to identify acid phosphatase 5 (ACP5) as a candidate for the enzyme that removes Man-6-P. Expression of ACP5 in N1E-115 neuroblastoma cells, which do not efficiently dephosphorylate lysosomal proteins, significantly decreased the steady state levels of Man6-P glycoproteins. Analysis of ACP5-deficient mice revealed that levels of Man-6-P glycoproteins were highly elevated in tissues that normally express ACP5, and this resulted from a failure to dephosphorylate lysosomal proteins. These results indicate a central role for ACP5 in removal of the Man-6-P recognition marker and open up new avenues to investigate the importance of this process in cell biology and medicine.

Competition on nitrocellulose-immobilized antibody arrays: from bacterial protein binding assay to protein profiling in breast cancer cells.

Large scale comparative evaluation of protein expression requires miniaturized techniques to provide sensitive and accurate measurements of the abundance of molecules present as individual and/or assembled protein complexes in cells. The principle of competition between target molecules for binding to arrayed antibodies has recently been proposed to assess differential expression of numerous proteins with one-color or two-color fluorescence detection methods. To establish the limiting factors and to validate the use of alternative detection for protein profiling, we performed competitive binding assays under different conditions. A model experimental protocol was developed whereby the competitive displacement of multi-subunit bacterial RNA polymerase and/or its subunits was evaluated through binding to subunit-specific immobilized monoclonal antibodies. We show that the difference in physico-chemical properties of unlabeled and labeled molecules significantly affects the performance of one-color detection, whereas epitope inaccessibility in the protein complex can prohibit the assessment of competition by both detection methods. Our data also demonstrate that antibody cross-reactivity, target protein truncation and abundance, as well as the cellular compartment of origin are major factors that affect protein profiling on antibody arrays. The experimental conditions established for prokaryotic proteins were adopted to compare protein profiles in the breast tumor-derived cell lines MDA MB-231 and SKBR3. Competitive displacement was detected and confirmed for a number of proteins using both detection methods; however, we show that overall the two-color method is better suited for accurate expression profile evaluation of a large, complex set of proteins. Antibody array data confirm the functional linkage between the ErbB2 receptor and AP-2 transcription factors in these cell lines and highlight unexpected differences in G1 cyclin expression.

Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection.

BACKGROUND: The mechanism of gene transfer into hepatocytes by the hydrodynamics-based transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the transfection. METHODS: We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with (35)S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA. RESULTS: When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of (35)S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time. CONCLUSIONS: Our results show that the efficiency of hydrodynamics-based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure.

Dissecting DNA-protein and protein-protein interactions involved in bacterial transcriptional regulation by a sensitive protein array method combining a near-infrared fluorescence detection.

The protein array methodology is used to study DNA-protein and protein-protein interactions governing gene expression from the Bacillus stearothermophilus PargCo promoter-operator region. Using probes labelled with near-infrared fluorescence dyes with exitation characteristics close to 700 or 800 nm, it is possible to detect signals from proteins (purified or non-purified in Escherichia coli cell extracts) immobilised on a nitrocellulose membrane with a high sensitivity (almost 12 amol of a spotted protein for protein-DNA interactions). Protein array data are confirmed by other methods indicating that molecular interactions of the order 10(-7) M can be monitored with the proposed protein array approach. We show that the PargCo region is a target for binding at least three types of regulatory proteins, ArgR repressors from thermophilic bacteria, the E. coli RNA polymerase alpha subunit and cyclic AMP binding protein CRP. We also demonstrate that the high strength of the PargC promoter is related to an upstream element that binds to the E. coli RNA polymerase alpha subunit.

Uptake by mouse liver and intracellular fate of plasmid DNA after a rapid tail vein injection of a small or a large volume.

BACKGROUND: An efficient gene transfer can be achieved in mouse liver by a rapid tail vein injection of a large volume of plasmid DNA solution (hydrodynamics-based transfection). The mechanism of gene transfer by this procedure is not known. It must be related to the uptake and intracellular fate of DNA. METHODS: We have investigated the problem by following the uptake by mouse liver and the intracellular distribution of DNA after a rapid tail vein injection of a large (2.0 ml) or a small (0.2 ml) volume of (35)S-DNA solution. Total and acid-soluble radioactivity were measured in liver homogenates at increasing times after injection, and their subcellular distributions were established by centrifugation methods and compared with the distributions of marker enzymes of the membrane compartments involved in endocytosis: alkaline phosphodiesterase (plasma membrane) and cathepsin C (lysosomes). RESULTS: (35)S-DNA uptake by the liver is similar when a small or a large volume of injection is used but its degradation is markedly slower after a 2.0 ml injection. When a small volume of injection is given, distribution of radioactivity after differential centrifugation indicates that the plasmid DNA is endocytosed and reaches lysosomes where it is hydrolysed. After a large volume injection, part of (35)S-DNA has the same fate, another part remains acid-precipitable for at least 1 h and is associated with structures sedimenting at low centrifugation speed in the nuclear fraction N. Analysis of that fraction by gradient centrifugation suggests that these structures are plasma membrane fragments that could originate from the apical domain of hepatocytes. The proportion of (35)S-DNA associated with hepatocytes is about doubled after a large volume injection. Fractionation of isolated hepatocytes by centrifugation confirms results obtained on the whole liver. Treatment of the N fraction or isolated hepatocytes with pancreatic DNAse illustrates that (35)S-DNA that remains bound to plasma membrane after a large volume injection is located on the outer face. CONCLUSIONS: The fact that after an hydrodynamic injection (35)S-DNA remains bound to the outside face of the plasma membrane for at least 1 h indicates that it is not, or very slowly, internalised during that period. The relatively small difference in the amount of DNA picked up by hepatocytes depending on the type of injection could not explain the absence of expression after a conventional injection and the strong expression after a hydrodynamic injection. If DNA enters the cells by endocytosis, even after an hydrodynamic injection, its persistence at the outside face of the plasma membrane could favour transfection by allowing hepatocytes to dispose for a relatively long time of a reservoir of intact DNA.

Arginine operator binding by heterologous and chimeric ArgR repressors from Escherichia coli and Bacillus stearothermophilus.

Bacillus stearothermophilus ArgR binds efficiently to the Escherichia coli carAB operator, whereas the E. coli repressor binds very poorly to the argCo operator of B. stearothermophilus. In order to elucidate this contradictory behavior between ArgRs, we constructed chimeric proteins by swapping N-terminal DNA-binding and C-terminal oligomerization domains or by exchanging the linker peptide. Chimeras carrying the E. coli DNA-binding domain and the B. stearothermophilus oligomerization domain showed sequence-nonspecific rather than sequence-specific interactions with arg operators. Chimeras carrying the B. stearothermophilus DNA-binding domain and E. coli oligomerization domain exhibited a high DNA-binding affinity for the B. stearothermophilus argCo and E. coli carAB operators and repressed the reporter-gene transcription from the B. stearothermophilus PargCo control region in vitro; arginine had no effect on, and indeed even decreased, their DNA-binding affinity. With the protein array method, we showed that the wild-type B. stearothermophilus ArgR and derivatives of it containing only the exchanged linker from E. coli ArgR or carrying the B. stearothermophilus DNA-binding domain along with the linker and the alpha4 regions were able to bind argCo containing the single Arg box. This binding was weaker than binding to the two-box operator but was no longer arginine dependent. Several lines of observations indicate that the alpha4 helix in the oligomerization domain and the linker peptide can contribute to the recognition of single or double Arg boxes and therefore to the operator DNA-binding specificity in similar but not identical ArgR repressors from two distant bacteria.

Genes and enzymes of the acetyl cycle of arginine biosynthesis in Corynebacterium glutamicum: enzyme evolution in the early steps of the arginine pathway.

A cluster of arginine biosynthetic genes of Corynebacterium glutamicum ATCC 13032, comprising argJ, argB and argD as well as part of argC and argF, has been cloned by heterologous complementation of an Escherichia coli argE mutant. The gene order has been established as argCJBDF by sequencing the entire 4.4 kb cloned DNA fragment. The C. glutamicum argB gene can be transcribed in E. coli cells from an internal promoter located in the coding part of the preceding argJ gene, whereas transcription of the argJ gene appears vector-dependent. Expression of the corynebacterial argB gene is repressed by arginine in the native host but not in recombinant E. coli cells. Feedback inhibition of the corresponding N-acetylglutamate kinase activity was observed both in cell extracts of C. glutamicum and in recombinant E. coli argB auxotrophic strains. Extracts of E. coli cells carrying cloned corynebacterial DNA display an ornithine acetyltransferase activity (encoded by argJ) which alleviates the acetylornithinase (encoded by argE) deficiency of the enterobacterial host. In contrast to Bacillus stearothermophilus ornithine acetyltransferase which also exhibits acetylglutamate synthase activity, C. glutamicum ornithine acetyltransferase appears monofunctional. ArgA and ArgB proteins from different sources share highly significant similarities. The evolutionary implications of these data are discussed.