Effects of temperature upon the cell-free translation system from Coxiella burnetii

The rate and extent of coliphage Q beta RNA translation by cell-free extracts prepared from Coxiella burnetii were studied. When translations were conducted at temperatures elevated above 37 degrees C, both polypeptide elongation and frequency of initiation were by comparison increased. The ratios of products synthesized from the polycistronic phage mRNA also changed upon increases in translation temperature, especially at 45 degrees C. Although the organism is a moderate acidophile, initiation of protein synthesis in extracts did not occur below pH 6.2, and was superior when the pH was 6.8-7.2. The results are discussed in context with the known physiological characteristics of this obligate intracellular bacterium.     

PDZ domains facilitate binding of high temperature requirement protease A (HtrA) and tail-specific protease (Tsp) to heterologous substrates through recognition of the small stable RNA A (ssrA)-encoded peptide

The Escherichia coli protease HtrA has two PDZ domains, and sequence alignments predict that the E. coli protease Tsp has a single PDZ domain. PDZ domains are composed of short sequences (80-100 amino acids) that have been implicated in a range of protein:protein interactions. The PDZ-like domain of Tsp may be involved in binding to the extreme COOH-terminal sequence of its substrate, whereas the HtrA PDZ domains are involved in subunit assembly and are predicted to be responsible for substrate binding and subsequent translocation into the active site. E. coli has a system of protein quality control surveillance mediated by the ssrA-encoded peptide tagging system. This system tags misfolded proteins or protein fragments with an 11-amino acid peptide that is recognized by a battery of cytoplasmic and periplasmic proteases as a degradation signal. Here we show that both HtrA and Tsp are able to recognize the ssrA-encoded peptide tag with apparent K(D) values of approximately 5 and 390 nm, respectively, and that their PDZ-like domains mediate this recognition. Fusion of the ssrA-encoded peptide tag to the COOH terminus of a heterologous protein (glutathione S-transferase) renders it sensitive to digestion by Tsp but not HtrA. These observations support the prediction that the HtrA PDZ domains facilitate substrate binding and the differential proteolytic responses of HtrA and Tsp to SsrA-tagged glutathione S-transferase are interpreted in terms of the structure of HtrA.     

Peptide selectivity between the PDZ domains of human pregnancy‐related serine proteases (HtrA1, HtrA2, HtrA3, and HtrA4) can be reshaped by different halogen probes

The human HtrA family of serine proteases (HtrA1, HtrA2, HtrA3, and HtrA4) are the key enzymes associated with pregnancy and closely related to the development and progression of many pathological events. Previously, it was found that halogen substitution at the indole moiety of peptide Trp‐1 residue can form a geometrically satisfactory halogen bond with the Drosophila discs large, zona occludens‐1 (PDZ) domain of HtrA proteases. Here, we attempt to systematically investigate the effect of substitution with 4 halogen types and 2 indole positions on the binding affinity and specificity of peptide ligands to the 4 HtrA PDZ domains. The complex structures, interaction energies, halogen‐bonding strength, and binding affinity of domain‐peptide systems were modeled, analyzed, and measured via computational modeling and fluorescence‐based assay. It is revealed that there is a compromise between the local rearrangement of halogen bond involving different halogen atoms and the global optimization of domain‐peptide interaction; the substitution position is fundamentally important for peptide‐binding affinity, while the halogen type can effectively shift peptide selectivity between the 4 domains. The HtrA1‐PDZ and HtrA4‐PDZ as well as HtrA2‐PDZ and HtrA3‐PDZ respond similarly to different halogen substitutions of peptide; –Br substitution at R2‐position and –I substitution at R4‐position are most effective in improving peptide selectivity for HtrA1‐PDZ/HtrA4‐PDZ and HtrA2‐PDZ/HtrA3‐PDZ, respectively; –F substitution would not address substantial effect on peptide selectivity for all the 4 domains. Consequently, the binding affinities of a native peptide ligand DSRIWWV^–COOH as well as its 4 R2‐halogenated counterparts were determined as 1.9, 1.4, 0.5, 0.27, and 0.92 μM, which are basically consistent with computational analysis. This study would help to rationally design selective peptide inhibitors of HtrA family members by using different halogen substitutions.     

Picornavirus IRESes and the poly(A) tail jointly promote cap-independent translation in a mammalian cell-free system

In eukaryotic cells, efficient translation of most cellular mRNAs requires the synergistic interplay between the m7GpppN cap structure and the poly(A) tail during initiation. We have developed and characterized a cell-free system from human HeLa cells that recapitulates this important feature, displaying more than one order of magnitude of translational synergism between the cap structure and the poly(A) tail. The stimulation of cap-dependent translation by the poly(A) tail is length-dependent, but not mediated by changes in mRNA stability. Using this system, we investigated the effect of the poly(A) tail on the translation of picornaviral RNAs, which are naturally polyadenylated but initiate translation via internal ribosome entry sites (IRESs). We show that translation driven by the IRESs of poliovirus (PV), encephalomyocarditis virus (EMCV), and hepatitis A virus is also significantly augmented by a poly(A) tail, ranging from an approximately 3-fold stimulation for the EMCV-IRES to a more than 10-fold effect for the PV IRES. These results raise interesting questions concerning the underlying molecular mechanism(s). The cell-free system described here should prove useful in studying these questions as well as providing a general biochemical tool to examine the translation initiation pathway in a more physiological setting.     

Modulation of the selective translation of mRNA in cell-free system exposed to tRNA

The homo- and heterologous tRNA were studied for their influence on the synthesis rate of globin and ovalbumin under simultaneous translation of their mRNAs in the tRNA-dependent cell-free system from wheat germ. In the presence of the fixed concentrations of one of the mRNAs the different amounts of the other mRNA (from 6 up to saturation of the system) were added to the samples. The ratio of the synthesis rate of the translation products after their electrophoretic characterization is determined on the basis of the autoradiogram scanning. The competition among mRNAs in question for some components of translation apparatus has been found: globin mRNA has a higher competitiveness than mRNA for ovalbumin. Modulation of the simultaneous translation of these mRNAs has been shown to depend on the nature of tRNA. The presence of homologous tRNA in the system increases a competitiveness of the corresponding mRNA, but heterologous tRNA reduces it. This regularity is retained in a wide range of the concentration ratios for the both mRNAs.     

Human high temperature requirement serine protease A1 (HTRA1) degrades tau protein aggregates

Protective proteases are key elements of protein quality control pathways that are up-regulated, for example, under various protein folding stresses. These proteases are employed to prevent the accumulation and aggregation of misfolded proteins that can impose severe damage to cells. The high temperature requirement A (HtrA) family of serine proteases has evolved to perform important aspects of ATP-independent protein quality control. So far, however, no HtrA protease is known that degrades protein aggregates. We show here that human HTRA1 degrades aggregated and fibrillar tau, a protein that is critically involved in various neurological disorders. Neuronal cells and patient brains accumulate less tau, neurofibrillary tangles, and neuritic plaques, respectively, when HTRA1 is expressed at elevated levels. Furthermore, HTRA1 mRNA and HTRA1 activity are up-regulated in response to elevated tau concentrations. These data suggest that HTRA1 is performing regulated proteolysis during protein quality control, the implications of which are discussed.     

Effect of streptomycin on the stoichiometry of GTP hydrolysis in a poly(U)-dependent cell-free translation system

The technique of Sepharose-bound template translation has been used to estimate the stoichiometry of GTP hydrolysis during peptide elongation in the presence of streptomycin. The presence of streptomycin has been shown to have no great effect on the elongation rate and the stoichiometry of GTP hydrolysis during codon-specific peptide elongation in the poly(U)-directed translation system: the molar ratio of hydrolysed GTP to incorporated phenylalanine was about 2. At the same time streptomycin exerted a significant effect during misreading when a ribosome-bound peptide in the poly(U)-programmed system was elongated by leucine or isoleucine residues: the miselongation was stimulated and hence the ratio of hydrolysed GTP per peptide bond was strongly reduced, as compared with the excessive GTP hydrolysis which is characteristic of the misreading system in the absence of streptomycin [(1984) FEBS Lett. 178, 283-287]. The conclusion has been made that streptomycin blocks the stage of correction ('proof-reading') following GTP hydrolysis during EF-Tu-dependent aminoacyl-tRNA binding.     

A cell-free protein synthesis system as an investigational tool for the translation stop processes

Using Escherichia coli cell-free protein synthesis system and aminoacylated amber suppressor tRNA, we successfully inserted an unnatural amino acid S-(2-nitrobenzyl)cysteine into human erythropoietin. Three different types of translation stop suppression were observed and each of the three types was easily discerned with SDS-PAGE. Optimal conditions were established for correct stop and programmed suppressions. Since this system differentiates proteins produced by misreading of codons from those produced by programmed suppression, we conclude that this cell-free translation system that we describe in this paper will be of a great use for future investigations on translation stop processes.     

Stabilization of mRNA in the cell-free translation system from Escherichia coli.

In the RNA directed cell-free protein synthesizing system from E. coli, there is a problem of contaminating 3'-exonucleases which attack the mRNAs. Thus, we tried the following two methods to stabilize mRNAs against nucleases: (A) To use mRNAs having hairpin structures at their termini and (B) To hybridize mRNAs with small DNA fragments to the 3'-termini of mRNAs. It was found that degradation of a mRNA was inhibited by the method B rather than the method A in the translation system.     

Poly(A)-binding protein-interacting protein 1 binds to eukaryotic translation initiation factor 3 to stimulate translation

Poly(A)-binding protein (PABP) stimulates translation initiation by binding simultaneously to the mRNA poly(A) tail and eukaryotic translation initiation factor 4G (eIF4G). PABP activity is regulated by PABP-interacting (Paip) proteins. Paip1 binds PABP and stimulates translation by an unknown mechanism. Here, we describe the interaction between Paip1 and eIF3, which is direct, RNA independent, and mediated via the eIF3g (p44) subunit. Stimulation of translation by Paip1 in vivo was decreased upon deletion of the N-terminal sequence containing the eIF3-binding domain and upon silencing of PABP or several eIF3 subunits. We also show the formation of ternary complexes composed of Paip1-PABP-eIF4G and Paip1-eIF3-eIF4G. Taken together, these data demonstrate that the eIF3-Paip1 interaction promotes translation. We propose that eIF3-Paip1 stabilizes the interaction between PABP and eIF4G, which brings about the circularization of the mRNA.     

Cap-Poly(A) synergy in mammalian cell-free extracts. Investigation of the requirements for poly(A)-mediated stimulation of translation initiation

The 5' cap and 3' poly(A) tail of eukaryotic mRNAs cooperate to stimulate synergistically translation initiation in vivo, a phenomenon observed to date in vitro only in translation systems containing endogenous competitor mRNAs. Here we describe nuclease-treated rabbit reticulocyte lysates and HeLa cell cytoplasmic extracts that reproduce cap-poly(A) synergy in the absence of such competitor RNAs. Extracts were rendered poly(A)-dependent by ultracentrifugation to partially deplete them of ribosomes and associated initiation factors. Under optimal conditions, values for synergy in reticulocyte lysates approached 10-fold. By using this system, we investigated the molecular mechanism of poly(A) stimulation of translation. Maximal cap-poly(A) cooperativity required the integrity of the eukaryotic initiation factor 4G-poly(A)-binding protein (eIF4G-PABP) interaction, suggesting that synergy results from mRNA circularization. In addition, polyadenylation stimulated uncapped cellular mRNA translation and that driven by the encephalomyocarditis virus internal ribosome entry segment (IRES). These effects of poly(A) were also sensitive to disruption of the eIF4G-PABP interaction, suggesting that 5'-3' end cross-talk is functionally conserved between classical mRNAs and an IRES-containing mRNA. Finally, we demonstrate that a rotaviral non-structural protein that evicts PABP from eIF4G is capable of provoking the shut-off of host cell translation seen during rotavirus infection.     

A cell-free translation and proteoliposome reconstitution system for functional analysis of plant solute transporters

We describe here a novel proteoliposome reconstitution system for functional analysis of plant membrane transporters that is based on a modified wheat germ cell-free translation system. We established optimized conditions for the reconstitution system with Arabidopsis thaliana phosphoenolpyruvate/phosphate translocator 1 (AtPPT1) as a model transporter. A high activity of AtPPT1 was achieved by synthesis of the protein in the presence of both a detergent such as Brij35 and liposomes. We also determined the substrate specificities of three putative rice PPT homologs with this system. The cell-free proteoliposome reconstitution system provides a valuable tool for functional analysis of transporter proteins.     

Detrimental role for human high temperature requirement serine protease A1 (HTRA1) in the pathogenesis of intervertebral disc (IVD) degeneration

Human HTRA1 is a highly conserved secreted serine protease that degrades numerous extracellular matrix proteins. We have previously identified HTRA1 as being up-regulated in osteoarthritic patients and as having the potential to regulate matrix metalloproteinase (MMP) expression in synovial fibroblasts through the generation of fibronectin fragments. In the present report, we have extended these studies and investigated the role of HTRA1 in the pathogenesis of intervertebral disc (IVD) degeneration. HTRA1 mRNA expression was significantly elevated in degenerated disc tissue and was associated with increased protein levels. However, these increases did not correlate with the appearance of rs11200638 single nucleotide polymorphism in the promoter region of the HTRA1 gene, as has previously been suggested. Recombinant HTRA1 induced MMP production in IVD cell cultures through a mechanism critically dependent on MEK but independent of IL-1β signaling. The use of a catalytically inactive mutant confirmed these effects to be primarily due to HTRA1 serine protease activity. HTRA1-induced fibronectin proteolysis resulted in the generation of various sized fragments, which when added to IVD cells in culture, caused a significant increase in MMP expression. Furthermore, one of these fragments was identified as being the amino-terminal fibrin- and heparin-binding domain and was also found to be increased within HTRA1-treated IVD cell cultures as well as in disc tissue from patients with IVD degeneration. Our results therefore support a scenario in which HTRA1 promotes IVD degeneration through the proteolytic cleavage of fibronectin and subsequent activation of resident disc cells.     

5'-poly(A) sequence as an effective leader for translation in eukaryotic cell-free systems

Poly(A) sequence of 25 adenylic residues placed immediately before the start codons of the green fluorescent protein (GFP) and firefly luciferase (Luc) mRNAs is shown to provide a high rate of translation of the heterologous messages in eukaryotic cell-free translation systems. Also the poly(A) leader is found to provide the abolition of the inhibition of translation at excess mRNA concentrations. The possibility of the practical use of the constructs with the poly(A) leader for preparative protein production is demonstrated in the wheat germ continuous-exchange cell-free (CECF) translation system.     

A fed-batch strategy to produce high poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) terpolymer yield with enhanced mechanical properties in bioreactor

Bioprocess and Biosystems Engineering - This study reports an efficient fed-batch strategy to improve poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate) [P(3HB-co-3HV-co-4HB)]...