[Synonymous Codon Usage-a Guide for Co-Translational Protein Folding in the Cell].

In the cell, protein folding begins during protein synthesis/translation and thus is a co-translational process. Co-translational protein folding is tightly linked to translation elongation, which is not a uniform process. While there are many reasons for translation non-uniformity, it is generally believed that non-uniform synonymous codon usage is one of the key factors modulating translation elongation rates. Frequent/optimal codons as a rule are translated more rapidly than infrequently used ones and vice versa. Over 30 years ago, it was hypothesized that changes in synonymous codon usage affecting translation elongation rates could impinge on co-translation protein folding and that many synonymous codons are strategically placed within mRNA to ensure a particular translation kinetics facilitating productive step-by-step co-translational folding of proteins. It was suggested that this particular translation kinetics (and, specifically, translation pause sites) may define the window of opportunity for the protein parts to fold locally, particularly at the critical points where folding is far from equilibrium. It was thus hypothesized that synonymous codons may provide a secondary code for protein folding in the cell. Although, mostly accepted now, this hypothesis appeared to be difficult to prove and many convincing results were obtained only relatively recently. Here, I review the progress in the field and explain, why this simple idea appeared to be so challenging to prove.

The importance of mRNA structure in determining the pathogenicity of synonymous and non-synonymous mutations in haemophilia.

INTRODUCTION: Mutational analysis is commonly used to support the diagnosis and management of haemophilia. This has allowed for the generation of large mutation databases which provide unparalleled insight into genotype-phenotype relationships. Haemophilia is associated with inversions, deletions, insertions, nonsense and missense mutations. Both synonymous and non-synonymous mutations influence the base pairing of messenger RNA (mRNA), which can alter mRNA structure, cellular half-life and ribosome processivity/elongation. However, the role of mRNA structure in determining the pathogenicity of point mutations in haemophilia has not been evaluated. AIM: To evaluate mRNA thermodynamic stability and associated RNA prediction software as a means to distinguish between neutral and disease-associated mutations in haemophilia. METHODS: Five mRNA structure prediction software programs were used to assess the thermodynamic stability of mRNA fragments carrying neutral vs. disease-associated and synonymous vs. non-synonymous point mutations in F8, F9 and a third X-linked gene, DMD (dystrophin). RESULTS: In F8 and DMD, disease-associated mutations tend to occur in more structurally stable mRNA regions, represented by lower MFE (minimum free energy) levels. In comparing multiple software packages for mRNA structure prediction, a 101-151 nucleotide fragment length appears to be a feasible range for structuring future studies. CONCLUSION: mRNA thermodynamic stability is one predictive characteristic, which when combined with other RNA and protein features, may offer significant insight when screening sequencing data for novel disease-associated mutations. Our results also suggest potential utility in evaluating the mRNA thermodynamic stability profile of a gene when determining the viability of interchanging codons for biological and therapeutic applications.

ITPR1 gene p.Val1553Met mutation in Russian family with mild Spinocerebellar ataxia.

BACKGROUND: Spinocerebellar ataxias (SСAs) are a highly heterogeneous group of inherited neurological disorders. The symptoms of ataxia vary in individual patients and even within the same SCA subtype. A study of a four-generation family with autosomal dominant (AD) non-progressive SCA with mild symptoms was conducted. The genotyping of this family revealed no frequent pathogenic mutations. So the objective of this study was to identify the genetic causes of the disease in this family with the technology of whole-exome sequencing (WES). METHODS AND RESULTS: WES, candidate variant analysis with further Sanger sequencing, mRNA secondary structure prediction, and RSCU analysis were performed; a heterozygous missense mutation in ITPR1 was identified. CONCLUSION: Our study confirms the fact that ITPR1 gene plays a certain role in the pathogenesis of SCAs, and, therefore, we suggest that c.4657G>A p.Val1553Met) is a disease-causing mutation in the family studied.

Factor IX oligomerization underlies reduced activity upon disruption of physiological conditions.

Coagulation factor IX (FIX) is a serine protease that plays a pivotal role in the blood coagulation cascade. FIX deficiency leads to a blood clotting disorder known as haemophilia B. FIX, synthesized as a prepro-peptide of 461 amino acids, is processed and secreted into plasma. The protein undergoes numerous modifications, including, but not limited to glycosylation, γ-carboxylation and disulphide bond formation. Upon processing and limited proteolysis, the protein is converted into an active protease. Under physiological conditions, the FIX zymogen is a monomer. The purpose of this work was to analyse the conditions that may affect FIX monomeric state and promote and/or reduce oligomerization. Using native gel electrophoresis and size exclusion chromatography, we found that under decreased pH and ionic strength conditions, the FIX zymogen can oligomerize, resulting in the formation of higher molecular weight species, with a concomitant reduction in specific activity. Similarly, FIX oligomers formed readily with low bovine serum albumin (BSA) concentrations; however, increased BSA concentrations impeded FIX oligomerization. We hypothesize that normal blood physiological conditions are critical for maintaining active FIX monomers. Under conditions of stress associated with acidosis, electrolyte imbalance and low albumin levels, FIX oligomerization is expected to take place thus leading to compromised activity. Furthermore, albumin, which is commonly used as a drug stabilizer, may enhance the efficacy of FIX biological drugs by reducing oligomerization.

Analysis of F9 point mutations and their correlation to severity of haemophilia B disease.

Haemophilia B is an X-linked recessive disorder caused by deficiency of functional coagulation factor IX, which results almost exclusively from mutations in the F9 gene. We sought to determine features, which could distinguish between mutations that cause severe disease symptoms from those that cause non-severe disease symptoms. Towards this objective, we have performed a statistical analysis of reported point mutations in F9. These include: potential local changes in mRNA free energy, codon usage, charge and type of mutated amino acid, location of the mutation with regard to protein secondary structure and functional domain and amino acids' evolutionary conservation scores. Wilcoxon signed-rank tests showed highly significant differences between severe and non-severe disease causing mutations in their effect on free energy of small mRNA fragments and evolutionarily conserved amino acids. Our results suggest that information at the mRNA level as well as conservation of the amino acid correlate well with disease severity. This study demonstrates that computational tools may be used to characterize the severity of haemophilia B associated with point mutations and suggests their utility in predicting the outcome of sequence changes in recombinant proteins.

Spirulina enhances the viability of Lactobacillus rhamnosus E/N after freeze-drying in a protective medium of sucrose and lactulose.

AIMS: Response surface methodology (RSM) was used to optimize a protective medium for enhancing the viability of Lactobacillus rhamnosus E/N cells during lyophilization. METHODS AND RESULTS: Spirulina, sucrose and lactulose were selected, on the basis of a Plackett-Burman factorial design, as important protectants having the following protective effects on cell viability: 102.025, 36.885 and -34.42, respectively. A full-factorial central composite design was applied to determine optimal levels of three used agents. CONCLUSION: The optimal protective medium composition was determined to be: Spirulina 1.304% (w/v), lactulose 5.48% (w/v), and sucrose 13.04% (w/v) (Polish Patent P-393189). The predictive value of cell viability in this medium was 89.619%, and experimental viability obtained during freeze-drying was 87.5%. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, Spirulina was used for the first time as the protective agent in freeze-drying medium, significantly increasing lactobacilli viability and giving synbiotic character of the final product.

[Features of the binding of the fluorescent probe K-35 to albumin].

The binding of the fluorescent probe K-35 (CAPIDAB, N-(carboxyphenyl)imide of 4-(dimethylamino)naphthalic acid), which is used as an indicator of albumin structural changes in pathology, to human serum albumin has been studied. Based on the data on the fluorescence decay of the probe, four types of sites of binding of K-35 to albumin have been recoonized, which differ by fluorescence decay time (tau) and binding constants (K). Probe molecules bound to the first type of sites have a decay time close to 8-10 ns; this value corresponds to a high fluorescence quantum yield of about 0.7. These sites have a maximal binding constant, K1 = 5 x 10(4) M(-1). Tau2 of the second type of sites is close to 3.6 ns and K2 = 1 x 10(4) M(-1), which is much lower than K1; however, the number of the sites is several times greater. The number of sites of the third type and the binding constant are close to those of the second type, but the decay time tau3 is equal to 1 ns, which is significantly lower than tau2. The binding of K-35 to sites of the second and the third types is characterized by a positive cooperativity. Their properties are similar but not completely identical. The total number of sites of these three types is about 2 per one HSA molecule. There are one to two sites of the fourth type where bound K-35 molecules have a very low decay time tau4 << 1; i.e. they are virtually nonfluorescent, and K4 = 1 x 10(4) M(-1). The major contribution to the steady-state fluorescence is made by probe molecules bound to sites of the first and second types. As a rule, the concentration of albumin binding sites in blood is significantly higher than the concentration of metabolites and xenobiotics transferred by albumin. Therefore, this metabolite or the probe in these experiments, is distributed between different sites in accordance with their K(i)n(i) values (n(i) is the number of sites of the ith type per albumin molecule). It was shown that the low occupation of the sites leads to an approximately equal number of K-35 molecules bound to different sites of types 1, 2, and 3. The competition of K-35 with phenylbutazone, a marker of the albumin drug-binding site I, allows one to suggest that the K-35 site of the first type is localized near the drug site I, while the sites of the second and third types are close to it.

9-Aminoacridine-based anticancer drugs target the PI3K/AKT/mTOR, NF-kappaB and p53 pathways.

Acquisition of a transformed phenotype involves deregulation of several signal transduction pathways contributing to unconstrained cell growth. Understanding the interplay of different cancer-related signaling pathways is important for development of efficacious multitargeted anticancer drugs. The small molecule 9-aminoacridine (9AA) and its derivative, the antimalaria drug quinacrine, have selective toxicity for tumor cells and can simultaneously suppress nuclear factor-kappaB (NF-kappaB) and activate p53 signaling. To investigate the mechanism underlying these drug activities, we used a combination of two-dimensional protein separation by gel electrophoresis and mass spectrometry to identify proteins whose expression is altered in tumor cells by 9AA treatment. We found that 9AA treatment results in selective downregulation of a specific catalytic subunit of the phosphoinositide 3-kinase (PI3K) family, p110 gamma. Further exploration of this observation demonstrated that the mechanism of action of 9AA involves inhibition of the prosurvival AKT/mammalian target of rapamycin (mTOR) pathway that lies downstream of PI3K. p110 gamma translation appears to be regulated by mTOR and feeds back to further modulate mTOR and AKT, thereby impacting the p53 and NF-kappaB pathways as well. These results reveal functional interplay among the PI3K/AKT/mTOR, p53 and NF-kappaB pathways that are frequently deregulated in cancer and suggest that their simultaneous targeting by a single small molecule such as 9AA could result in efficacious and selective killing of transformed cells.

Embryotoxicity of magainin-2-amide and its enhancement by cyclodextrin, albumin, hydrogen peroxide and acidification.

BACKGROUND: The channel-forming antimicrobial peptide, magainin-2-amide, interacts preferentially with negatively charged, non cholesterol-containing membranes, including those of sperm, oocytes and cells of pre-implantation embryos. Cyclodextrin and albumin remove membrane cholesterol and together with hydrogen peroxide (H2O2) are potential enhancers of embryotoxicity. METHODS: Two-cell murine embryos were cultured in vitro with magainin-2-amide at a high effective concentration (250 microg/ml) and at subthreshold concentrations (166 and 200 microg/ml). Embryos treated with sub-threshold concentrations of magainin were additionally treated with cyclodextrin, bovine serum albumin or H2O2 or were cultured under acidified conditions. Cell viability was verified with propidium iodide and fluorescein diacetate. RESULTS: The embryotoxic effect of magainin and H2O2 was dose- and time-dependent. Cyclodextrin, H2O2, acidification of the medium, and to a lesser extent albumin, enhanced the embryotoxicity of magainin at sub-threshold concentrations. CONCLUSION: Magainin on its own is highly embryotoxic. Its embryotoxicity is enhanced by cyclodextrin, albumin, H2O2 and acidification. Thus, magainin which has antibacterial, antifungal and antiprotozoal activity may also have a potential role as a contraceptive agent. The harmful effects of various concentrations of the exogenous H2O2 on 2-cell stage mouse embryos are reported here, to the best of our knowledge, for the first time.

Stability, folding, dimerization, and assembly properties of the yeast prion Ure2p.

The [URE3] factor of Saccharomyces cerevisiae propagates by a prion-like mechanism and corresponds to the loss of the function of the cellular protein Ure2. The molecular basis of the propagation of this phenotype is unknown. We recently expressed Ure2p in Escherichia coli and demonstrated that the N-terminal region of the protein is flexible and unstructured, while its C-terminal region is compactly folded. Ure2p oligomerizes in solution to form mainly dimers that assemble into fibrils [Thual et al. (1999) J. Biol. Chem. 274, 13666-13674]. To determine the role played by each domain of Ure2p in the overall properties of the protein, specifically, its stability, conformation, and capacity to assemble into fibrils, we have further analyzed the properties of Ure2p N- and C-terminal regions. We show here that Ure2p dimerizes through its C-terminal region. We also show that the N-terminal region is essential for directing the assembly of the protein into a particular pathway that yields amyloid fibrils. A full-length Ure2p variant that possesses an additional tryptophan residue in its N-terminal moiety was generated to follow conformational changes affecting this domain. Comparison of the overall conformation, folding, and unfolding properties, and the behavior upon proteolytic treatments of full-length Ure2p, Ure2pW37 variant, and Ure2p C-terminal fragment reveals that Ure2p N-terminal domain confers no additional stability to the protein. This study reveals the existence of a stable unfolding intermediate of Ure2p under conditions where the protein assembles into amyloid fibrils. Our results contradict the intramolecular interaction between the N- and C-terminal moieties of Ure2p and the single unfolding transitions reported in a number of previous studies.

Characterisation of epithelial cell line from rat cornea.

PURPOSE: Characterisation of RtCE-1 cells, newly established rat corneal epithelial cell line. METHODS: Morphology of RtCE-1 cells was characterised by light and electron microscopy. Expression of cytokeratins was studied by polyacrylamide electrophoresis and Western blotting. Characterisation of RtCE-1 cells also included karyotype analysis and in vitro study of growth kinetics. RESULTS: The line shows morphological similarities to normal corneal epithelium. The cells express cornea-specific cytokeratins. Karyotype analysis revealed that the cells are polyploid with modal number of chromosomes at passage 30 equalling 72 +/- 14. Growth of the line is partially dependent on EGF. CONCLUSION: RtCE-1 cells may constitute a model for the evaluation of proliferation, function and differentiation of corneal epithelium in vitro.

Gastric acid secretion in aquaporin-4 knockout mice.

The aquaporin-4 (AQP4) water channel has been proposed to play a role in gastric acid secretion. Immunocytochemistry using anti-AQP4 antibodies showed strong AQP4 protein expression at the basolateral membrane of gastric parietal cells in wild-type (+/+) mice. AQP4 involvement in gastric acid secretion was studied using transgenic null (-/-) mice deficient in AQP4 protein. -/- Mice had grossly normal growth and appearance and showed no differences in gastric morphology by light microscopy. Gastric acid secretion was measured in anesthetized mice in which the stomach was luminally perfused (0. 3 ml/min) with 0.9% NaCl containing [(14)C]polyethylene glycol ([(14)C]PEG) as a volume marker. Collected effluent was assayed for titratable acid content and [(14)C]PEG radioactivity. After 45-min baseline perfusion, acid secretion was stimulated by pentagastrin (200 microg. kg(-1). h(-1) iv) for 1 h or histamine (0.23 mg/kg iv) + intraluminal carbachol (20 mg/l). Baseline gastric acid secretion (means +/- SE, n = 25) was 0.06 +/- 0.03 and 0.03 +/- 0.02 microeq/15 min in +/+ and -/- mice, respectively. Pentagastrin-stimulated acid secretion was 0.59 +/- 0.14 and 0.70 +/- 0.15 microeq/15 min in +/+ and -/- mice, respectively. Histamine plus carbachol-stimulated acid secretion was 7.0 +/- 1.9 and 8.0 +/- 1.8 microeq/15 min in +/+ and -/- mice, respectively. In addition, AQP4 deletion did not affect gastric fluid secretion, gastric pH, or fasting serum gastrin concentrations. These results provide direct evidence against a role of AQP4 in gastric acid secretion.

Structural characterization of Saccharomyces cerevisiae prion-like protein Ure2.

Sacchromyces cerevisiae prion-like protein Ure2 was expressed in Escherichia coli and was purified to homogeneity. We show here that Ure2p is a soluble protein that can assemble into fibers that are similar to the fibers observed in the case of PrP in its scrapie prion filaments form or that form on Sup35 self-assembly. Ure2p self-assembly is a cooperative process where one can distinguish a lag phase followed by an elongation phase preceding a plateau. A combination of size exclusion chromatography, sedimentation velocity, and electron microscopy demonstrates that the soluble form of Ure2p consists at least of three forms of the protein as follows: a monomeric, dimeric, and tetrameric form whose abundance is concentration-dependent. By the use of limited proteolysis, intrinsic fluorescence, and circular dichroism measurements, we bring strong evidence for the existence of at least two structural domains in Ure2p molecules. Indeed, Ure2p NH2-terminal region is found poorly structured, whereas its COOH-terminal domain appears to be compactly folded. Finally, we show that only slight conformational changes accompany Ure2p assembly into insoluble high molecular weight oligomers. These changes essentially affect the COOH-terminal part of the molecule. The properties of Ure2p are compared in the discussion to that of other prion-like proteins such as Sup35 and mammalian prion protein PrP.

Characterization of the interaction domains of Ure2p, a prion-like protein of yeast.

In the yeast Saccharomyces cerevisiae, the non-Mendelian inherited genetic element [URE3] behaves as a prion. A hypothesis has been put forward which states that [URE3] arises spontaneously from its cellular isoform Ure2p (the product of the URE2 gene), and propagates through interactions of the N-terminal domain of the protein, thus leading to its aggregation and loss of function. In the present study, various N- and C-terminal deletion mutants of Ure2p were constructed and their cross-interactions were tested in vitro and in vivo using affinity binding and a two-hybrid analysis. We show that the self-interaction of the protein is mediated by at least two domains, corresponding to the first third of the protein (the so-called prion-forming domain) and the C-terminal catalytic domain.

The [URE3] yeast prion: from genetics to biochemistry.

[URE3] is a non-Mendelian genetic element of the yeast Saccharomyces cerevisiae, an altered prion form of Ure2 protein. We show that recombinant Ure2p is a soluble protein that can assemble in vitro into dimers, tetramers, and octamers or form insoluble fibrils observed for PrP in its filamentous form or for Sup35p upon self-assembling, suggesting a similar mechanism for all prions. Computational, genetic, biochemical, and structural data allow us to specify a new boundary between the so-called prion-forming and nitrogen regulator (catalytic) domains of the protein and to map this boundary to Met-94. We bring strong evidence that the COOH-terminal (94-354) part of the protein forms a tightly folded domain, while the NH2-terminal (1-94) part is unstructured. These domains (or various parts of these domains) were shown (by means of the two-hybrid system approach and affinity binding experiments) to interact with each other (both in vivo and in vitro). We bring also evidence that the COOH-terminal (94-354) catalytically active part of the protein can be synthesized (both in vitro and in vivo) via an internal ribosome-binding mechanism, independently of the production of the full-length protein. We finally show that Ure2p aggregation in vivo (monitored by fluorescence of Ure2p--GFP fusion) does not necessarily give rise to [URE3] phenotype. The significance of these findings for the appearance and propagation of the yeast prion [URE3] is discussed.

Synonymous codon substitutions affect ribosome traffic and protein folding during in vitro translation.

To investigate the possible influence of the local rates of translation on protein folding, 16 consecutive rare (in Escherichia coli) codons in the chloramphenicol acetyltransferase (CAT) gene have been replaced by frequent ones. Site-directed silent mutagenesis reduced the pauses in translation of CAT in E. coli S30 extract cell-free system and led to the acceleration of the overall rate of CAT protein synthesis. At the same time, the silently mutated protein (with unaltered protein sequence) synthesized in the E. coli S30 extract system was shown to possess 20% lower specific activity. The data suggest that kinetics of protein translation can affect the in vivo protein-folding pathway, leading to increased levels of protein misfolding.

Enhanced expression of the yeast Ure2 protein in Escherichia coli: the effect of synonymous codon substitutions at a selected place in the gene.

The expression of the yeast Ure2 protein and its two N- and C-terminal HA-(YPYPVDYA) epitope and His-tag fusions has been enhanced in E. coli by selected silent mutagenesis of the URE2 gene. The two Arg-AGA codons at positions 253 and 254 of the URE2 gene coding sequence were exchanged by CGT codons accordingly. This has allowed an increased yield (up to 100-fold) of the full-length protein synthesized. Western blotting with HA-epitope-specific antibodies using N- and C-terminal Ure2p-HA(epitope)-His-tag fusion constructs confirmed the integrity of the recombinant proteins. The N-(C-) terminal tagged proteins were shown to possess biological activity of the natural Ure2 protein.

Differential resistance to proteinase K digestion of the yeast prion-like (Ure2p) protein synthesized in vitro in wheat germ extract and rabbit reticulocyte lysate cell-free translation systems.

The Ure2p yeast prion-like protein was translated in vitro in the presence of labeled [35S]methionine in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE) cell-free systems. When subjected to proteinase K digestion, the Ure2p protein synthesized in WGE was proteolysed much more slowly compared to that synthesized in RRL; this displays fragments of about 31-34 kDa, persisting over 8 min. Thus, the digestion rate and pattern of the protein synthesized in WGE, unlike that synthesized in RRL, revealed characteristic features of the [URE3] prion-like isoform of the Ure2p protein [Masison, D.C. and Wickner, R.B. (1995) Science 270, 93-95]. Chloramphenicol acetyltransferase, synthesized under the same conditions, differed fundamentally in its proteolytic sensitivity toward proteinase K (PK); in the RRL system it was more slowly digested than in WGE, proving specific PK inhibitors to be absent in both systems. Posttranslational addition of the WGE to the RRL-synthesized Ure2p does not protect Ure2p from efficient PK degradation either. The differences in Ure2p degradation may be ascribed to a specific structure or specific states of association of Ure2p synthesized in WGE; obviously, they yield a protein that mimics the behavior of the Ure2p in [URE3] yeast strains. The present data suggest that particular conditions of the Ure2p protein translation and/or certain cellular components (accessory proteins and extrinsic factors), as well as the nature of the translation process itself, could affect the intracellular folding pathway of Ure2p leading to the de novo formation of the prion [URE3] isoform.

Cotranslational folding of globin.

Globin synthesis in a wheat germ cell-free translation system was performed in the presence of [3H]hemin and [35S]methionine to determine the minimal length of the nascent ribosome-bound globin chain capable of heme binding. Nascent polypeptides of predetermined size were synthesized on ribosomes by translation of truncated mRNA molecules. Analysis with the use of sucrose gradient centrifugation and puromycin reaction revealed that the ribosome-bound N-terminal alpha-globin fragments of 140, 100, and 86 amino acid residues are capable of an efficient heme binding, whereas those of 75, 65, and 34 amino acid residues display a significantly weaker, or just nonspecific, affinity to heme. This indicates that the ribosome-bound nascent chain of 86 amino acid residues has already acquired a spatial structure that allows its interaction with the heme group or that heme attachment promotes the formation of the proper tertiary structure in the ribosome-bound nascent peptide. In any case the cotranslational folding of globin is suggested.

Apoptosis induced in L1210 leukaemia cells by an inhibitor of the chymotrypsin-like activity of the proteasome.

Of a number of factors involved in apoptosis, protease activity may play a crucial role. We show that N-benzyloxycarbonyl-Ile-Glu( O-t-butyl)-Ala-leucinal (PSI), a selective inhibitor of the chymotrypsin-like activity of the proteasome, induces massive apoptosis in murine leukaemia L1210 cells. At 50 nM concentration, PSI induces a block of cytokinesis, while higher concentrations (500 nM) cause S phase block and massive apoptosis. Z-Leu-leucinal, a specific calpain inhibitor, did not induce apoptosis. In contrast to previous reports, TNF-alpha did not enhance apoptosis when combined with PSI. Our results suggest that proteasome inhibitors may be considered as potential anti-neoplastic agents.