Identification of a novel site specific endonuclease produced by Mycoplasma fermentans: discovery while characterizing DNA binding proteins in T lymphocyte cell lines.

We have discovered a new restriction endonuclease, MfeI, in nuclear extracts from T cells contaminated with Mycoplasma fermentans. This endonuclease was identified while studying proteins binding to the interleukin-2 receptor alpha chain gene promoter. MfeI cuts at the recognition sequence C'AATTG generating EcoRI compatible cohesive ends. Potential applications are discussed.     

Identification of pre-osteonectin produced by cell-free translation of fetal porcine calvarial mRNA

The cell-free biosynthesis of the bone protein osteonectin was studied using mRNA from fetal porcine calvariae. Total RNA was extracted from the calvariae with guanidinium thiocyanate and was partially purified by precipitation with acid/ethanol. Translations were performed using the reticulocyte lysate system and were optimized with respect to mRNA concentration and K+ (70 mM) and Mg2+ (0.6 mM) concentration. Cell-free synthesized osteonectin, radiolabeled with [35S]methionine, was specifically immunoprecipitated with rabbit antiserum to porcine osteonectin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. When analyzed under reduced conditions, the translated protein migrated with an Mr 45,000 compared to an Mr 39,000 for cell-synthesized osteonectin. When translated in the presence of microsomal membranes, the immunoprecipitated osteonectin co-migrated with the cell-synthesized osteonectin, indicating that a signal sequence of about 45-50 amino acids (Mr 6,000) had been removed. Under nonreduced conditions the pre-osteonectin co-migrated with osteonectin (Mr 39,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that a highly folded structure is retained by disulfide bridges under denaturing conditions. The relationship between the immunoprecipitated pre-osteonectin from the cell-free translations and both the cell-synthesized and tissue-extracted osteonectin was confirmed by one-dimensional peptide mapping of Staphylococcus aureus V-8 protease digestions. The results indicate that porcine osteonectin is synthesized on polysomes in a pre-osteonectin form which is translocated vectorially into microsomal vesicles and cotranslationally processed by the removal of a signal peptide.     

Development of a minimal cell-free translation system for the synthesis of presecretory and integral membrane proteins

By combining translation and membrane integration/translocation systems, we have constructed a novel cell-free system for the production of presecretory and integral membrane proteins in vitro. A totally defined, cell-free system reconstituted from a minimal number of translation factors was supplemented with urea-washed inverted membrane vesicles (U-INVs) prepared from Escherichia coli, as well as with purified proteins mediating membrane targeting of presecretory and integral membrane proteins. Initially, efficient membrane translocation of a presecretory protein (pOmpA) was obtained simply by the addition of only SecA and SecB. Proteinase K digestion clearly showed the successful translocation of pOmpA inside the vesicles. Next, integration of an inner membrane protein (MtlA) into U-INVs was achieved in the presence of only SRP (Ffh) and SR (FtsY). Finally, a membrane protein possessing a large periplasmic region (FtsQ) and therefore requiring both factors (SRP/SR and SecA/SecB) for membrane integration/translocation was also shown to be integrated correctly in this cell-free system. Thus, our novel cell-free system provides not only an efficient strategy for the production of membrane-related proteins but also an improved platform for the biological study of protein translocation and integration mechanisms.     

A novel inhibitory mechanism of mitochondrion-dependent apoptosis by a herpesviral protein

Upon viral infection, cells undergo apoptosis as a defense against viral replication. Viruses, in turn, have evolved elaborate mechanisms to subvert apoptotic processes. Here, we report that a novel viral mitochondrial anti-apoptotic protein (vMAP) of murine gamma-herpesvirus 68 (gammaHV-68) interacts with Bcl-2 and voltage-dependent anion channel 1 (VDAC1) in a genetically separable manner. The N-terminal region of vMAP interacted with Bcl-2, and this interaction markedly increased not only Bcl-2 recruitment to mitochondria but also its avidity for BH3-only pro-apoptotic proteins, thereby suppressing Bax mitochondrial translocation and activation. In addition, the central and C-terminal hydrophobic regions of vMAP interacted with VDAC1. Consequently, these interactions resulted in the effective inhibition of cytochrome c release, leading to the comprehensive inhibition of mitochondrion-mediated apoptosis. Finally, vMAP gene was required for efficient gammaHV-68 lytic replication in normal cells, but not in mitochondrial apoptosis-deficient cells. These results demonstrate that gammaHV-68 vMAP independently targets two important regulators of mitochondrial apoptosis-mediated intracellular innate immunity, allowing efficient viral lytic replication.     

Control of oskar mRNA translation by Bruno in a novel cell-free system from Drosophila ovaries

The coupled regulation of oskar mRNA localization and translation in time and space is critical for correct anteroposterior patterning of the Drosophila embryo. Localization-dependent translation of oskar mRNA, a mechanism whereby oskar RNA localized at the posterior of the oocyte is selectively translated and the unlocalized RNA remains in a translationally repressed state, ensures that Oskar activity is present exclusively at the posterior pole. Genetic experiments indicate that translational repression involves the binding of Bruno protein to multiple sites, the Bruno Response Elements (BRE), in the 3' untranslated region (UTR) of oskar mRNA. We have established a cell-free translation system derived from Drosophila ovaries, which faithfully reproduces critical features of mRNA translation in vivo, namely cap structure and poly(A) tail dependence. We show that this ovary extract, containing endogenous Bruno, is able to recapitulate oskar mRNA regulation in a BRE-dependent way. Thus, the assembly of a ribonucleoprotein (RNP) complex leading to the translationally repressed state occurs in vitro. Moreover, we show that a Drosophila embryo extract lacking Bruno efficiently translates oskar mRNA. Addition of recombinant Bruno to this extract establishes the repressed state in a BRE-dependent manner, providing a direct biochemical demonstration of the critical role of Bruno in oskar mRNA translation. The approach that we describe opens new avenues to investigate translational regulation in Drosophila oogenesis at a biochemical level.     

tRNA-mediated labelling of proteins with biotin. A nonradioactive method for the detection of cell-free translation products

We have developed a new method for the rapid and sensitive detection of cell-free translation products. Biotinylated lysine is incorporated into newly synthesized proteins by means of lysyl-tRNA that is modified in the epsilon-position. After electrophoresis in a dodecyl sulfate gel and blotting onto nitrocellulose, the translation products can be identified by probing with streptavidin and biotinylated alkaline phosphatase, followed by incubation with a chromogenic enzyme substrate. The non-radioactive labelling by biotin approaches in its sensitivity that obtained by radioactive amino acids. The products are absolutely stable and can be rapidly identified. The new method has been tested with different mRNAs in the cell-free translation systems of wheat germ and reticulocytes. Neither the interaction of secretory proteins with the signal recognition particle nor the in vitro translocation across the endoplasmic reticulum membrane or core glycosylation of nascent polypeptides are prevented by the incorporation of biotinylated lysine residues. The results indicate that both the ribosome and the endoplasmic reticulum membrane permit the passage of polypeptides carrying bulky groups attached to the amino acids (by atomic models it was estimated that the size of the side chain of lysine changes from approximately equal to 0.8 nm to approximately equal to 2 nm after modification.     

Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 ± 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 ± 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 ± 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 ± 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the importance of this domain.     

Fluorescence labeling of the C-terminus of proteins with a puromycin analogue in cell-free translation systems

L-Arginine uptake and Ca(2+) changes in unstirred platelets activated by thrombin, collagen and Ca(2+) ionophore A23187 were evaluated. Thrombin did not affect L-arginine uptake at short incubation times (2-15 min), but at prolonged times slowed down the amino acid transport. Collagen was ineffective. A23187 decreased the L-arginine uptake in a dose-dependent manner, producing the maximal inhibition at 5 microM. In FURA 2-loaded platelets collagen did not modify Ca(2+) basal level, thrombin induced a late Ca(2+) rise and A23187 dose-dependently increased cytosolic Ca(2+), eliciting the highest increase at 5 microM. It is likely that L-arginine uptake is inversely modulated by Ca(2+) concentrations and is inhibited during platelet stimulation with agonists which induce cytosolic Ca(2+) elevation.     

In vitro translation in a cell-free system from Trypanosoma brucei yields glycosylated and glycosylphosphatidylinositol-anchored proteins.

African trypanosomes escape many cellular and unspecific immune reactions by the expression of a protective barrier formed from a repertoire of several hundred genes encoding immunologically distinct variant surface glycoproteins (VSGs). All mature VSGs are glycosylphosphatidylionositol-anchored and N-glycosylated. To study trypanosome-specific post-translational modifications of VSG, a cell-free system capable of in vitro translation, translocation into the rough endoplasmic reticulum, N-glycosylation and glycosylphosphatidylinositol-anchor addition was established using lysates of the bloodstream form of Trypanosoma brucei. Monitoring protein synthesis by [35S]methionine incorporation, labeled protein bands were readily detected by fluorography following SDS/PAGE. Appearance of these bands increased during a time-course of 45 min and was sensitive to cycloheximide but not chloramphenicol treatment. Efficiency of this system, in terms of incorporation of radiolabeled amino acids into newly formed proteins, is similar to reticulocyte lysates. The system does not, however, allow initiation of protein synthesis. Depending on the clone used, immunoprecipitation revealed one or two newly formed VSG bands. Upon digestion with N-glycosidase F these bands resulted in a single band of a lower apparent molecular mass, indicating that newly synthesized VSG underwent translocation and glycosylation in the cell-free system. Biotinylation of VSG and a combination of precipitation with immobilized avidin and detection of VSG using antibodies specific for clones and cross-reacting determinants revealed that newly formed VSG contained the glycosylphosphatidylinositol anchor.     

Heating RNA before cell-free translation is essential for the efficient and reproducible synthesis of several peroxisomal proteins.

Total RNA, extracted with guanidinium thiocyanate from liver of clofibrate-treated rats, was translated in vitro. Heating the RNA at 60 degrees C for 5 min before translation increased the synthesis of three peroxisomal polypeptides 10-100-fold. Preproalbumin synthesis increased 10-fold. Total incorporation of [35S]methionine into proteins merely doubled. Heating is essential for reproducible and adequate translation of mRNAs coding for peroxisomal and some other proteins.     

A new method for mapping discontinuous antibody epitopes to reveal structural features of proteins.

Antibodies that bind to protein surfaces of interest can be used to report the three-dimensional structure of the protein as follows: Proteins are composed of linear polypeptide chains that fold together in complex spatial patterns to create the native protein structure. These folded structures form binding sites for antibodies. Antibody binding sites are typically "assembled" on the protein surface from segments that are far apart in the primary amino acid sequence of the target proteins. Short amino acid probe sequences that bind to the active region of each antibody can be used as witnesses to the antibody epitope surface and these probes can be efficiently selected from random sequence peptide libraries. This paper presents a new method to align these antibody epitopes to discontinuous regions of the one-dimensional amino acid sequence of a target protein. Such alignments of the epitopes indicate how segments of the protein sequence must be folded together in space and thus provide long-range constraints for solving the 3-D protein structure. This new antibody-based approach is applicable to the large fraction of proteins that are refractory to current approaches for structure determination and has the additional advantage of requiring very small amounts of the target protein. The binding site of an antibody is a surface, not just a continuous linear sequence, so the epitope mapping alignment problem is outside the scope of classical string alignment algorithms, such as Smith-Waterman. We formalize the alignment problem that is at the heart of this new approach, prove that the epitope mapping alignment problem is NP-complete, and give some initial results using a branch-and-bound algorithm to map two real-life cases. Initial results for two validation cases are presented for a graph-based protein surface neighbor mapping procedure that promises to provide additional spatial proximity information for the amino acid residues on the protein surface.     

Human transcortin synthesis by a cell-free translation of hepatic mRNA

To evaluate the site of synthesis and to characterize the translated transcortin, poly (A)-containing RNA (mRNA) from human liver was translated in a cell-free system derived from rabbit reticulocyte lysate. The in vitro synthesized product was identified as transcortin by immuno-precipitation with its specific antiserum. This translated transcortin could be displaced from the antibody by unlabeled purified transcortin obtained from plasma. Furthermore, when the translation mixture was applied to a cortisol-Sepharose column, the translated transcortin was bound to the matrix in a specific manner, indicating that this product binds to cortisol. The molecular weight of the translated transcortin was estimated to be 45,700 by its mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis, while that of plasma transcortin was 53,800. The difference in molecular weight between the translated transcortin and plasma transcortin was probably due to the presence of pre-sequence (signal peptide) in addition to the absence of carbohydrate moiety in the former. In conclusion, human liver mRNA directed the synthesis of transcortin, and the translated transcortin binds to cortisol in spite of the absence of carbohydrate moiety.     

Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system

Background  

Recently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system.     

Enterocin T, a novel class IIa bacteriocin produced by Enterococcus sp. 812

Enterococcus sp. 812, isolated from fresh broccoli, was previously found to produce a bacteriocin active against a number of Gram-positive bacteria, including Listeria monocytogenes. Bacteriocin activity decreased slightly after autoclaving (121 °C for 15 min), but was inactivated by protease K. Mass spectrometry analysis revealed the bacteriocin mass to be approximately 4,521.34 Da. N-terminal amino acid sequencing yielded a partial sequence, NH₂-ATYYGNGVYXDKKKXWVEWGQA, by Edman degradation, which contained the consensus class IIa bacteriocin motif YGNGV in the N-terminal region. The obtained partial sequence showed high homology with some enterococcal bacteriocins; however, no identical peptide or protein was found. This peptide was therefore considered to be a novel bacteriocin produced by Enterococcus sp. 812 and was termed enterocin T.     

Further mapping of late adenovirus genes by cell-free translation of RNA selected by hybridization to specific DNA fragments

RNA isolated from the cytoplasm of human cells at late times after infection by adenovirus type 2 (Ad2) has been fractionated by hybridization to fragments of Ad2 DNA which were produced by digestion with the restriction endonucleases Hpa I, Eco RI, Bam HI and Hind III. Cell-free translation of these partially purified mRNAs indicates that the genes for the late Ad2 proteins lie within the following intervals on the conventional Ad2 map: 15K (4.4–17.0 map units), IX and IVa₂ (7.5–17.0), IIIa (29.1–40.9), III and V (29.1–57.0), pVIII (40.9–57.0), pVI and II (40.9–70.7), 100K (59.0–83.4), pVIII (70.7–83.4) and IV (85.0–100). In addition to the primary hybridization of the late Ad2 mRNAs to the regions indicated above, most late Ad2 mRNAs (except those for 15K, IX and IVa₂) exhibited some hybridization to a secondary site between 17.0 and 29.1 map units.