Ricardo Ehrlich

Silent mutations affect in vivo protein folding in Escherichia coli

As an approach to investigate the molecular mechanism of in vivo protein folding and the role of translation kinetics on specific folding pathways, we made codon substitutions in the EgFABP1 (Echinococcus granulosus fatty acid binding protein1) gene that replaced five minor codons with their synonymous major ones. The altered region corresponds to a turn between two short alpha helices. One of the silent mutations of EgFABP1 markedly decreased the solubility of the protein when expressed in Escherichia coli. Expression of this protein also caused strong activation of a reporter gene designed to detect misfolded proteins, suggesting that the turn region seems to have special translation kinetic requirements that ensure proper folding of the protein. Our results highlight the importance of codon usage in the in vivo protein folding.

A new potent antigen from Echinococcus granulosus associated with muscles and tegument

An immunoscreening of a cDNA library derived from the adult stage of the parasitic platyhelminth Echinococcus granulosus has been carried out with sera from infected dogs. The EgA31 clone encodes a fibrous protein which shares some sequence elements with paramyosins. The corresponding gene is present as a single copy in the genome. As revealed by an antibody obtained against a fusion protein produced in bacteria, the polypeptide has a molecular weight of 66 kDa. This polypeptide is present at all developmental stages studied and is a potent antigen during an infection by the adult stage in the dog and during cyst growth in human patients. By immunohistology, it was shown that it is present in the tegument and subtegumental parenchyma of the adult with a main location in the region of the suckers where it rapidly accumulates at the time of the head evagination. It is also present in the germinal layer of the cyst and on the protoscolex.

Genomic structure and expression of a gene coding for a new fatty acid binding protein from Echinococcus granulosus.

This work describes a new gene coding for a fatty acid binding protein (FABP) in the parasite Echinococcus granulosus, named EgFABP2. The complete gene structure, including the promoter sequence, is reported. The genomic coding domain organisation of the previously reported E. granulosus FABP gene (EgFABP1) has been also determined. The corresponding polypeptide chains share 76% of identical residues and an overall 96% of similarity. The two EgFABPs present the highest amino acid homologies with the mammalian FABP subfamily containing heart-FABPs (H-FABPs). The coding sequences of both genes are interrupted by a single intron located in the position of the third intron reported for vertebrate FABP genes. Both genes are expressed in the protoscolex stage of the parasite. The promoter region of EgFABP2 presents several consensus putative cis-acting elements found in other members of the family, suggesting interesting possible mechanisms involved in the host-parasite adaptation.

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.

Fluorimetric study of the complex between yeast phenylalanyl-tRNA synthetase and tRNA-Phe. 2. Evidence for an asymmetric behaviour of the enzyme.

The variations of several spectroscopic properties of yeast tRNA-Phe and phenylalanyl-tRNA synthetase upon complex formation, were used to study the stoichiometry of the complex in different experimental conditions. In all cases, for the tRNA-Phe-enzyme complex, in the absence of other ligands, the saturations of the different conformational changes monitored for both macromolecules, are achieved at a 2:1 tRNA/enzyme stoichiometry. Phenylalanine does not modify this saturation. In contrast, the presence of 1 mM ATP induces an asymmetric behaviour of the synthetase: two tRNAs are still bound per enzyme molecule but the conformational change of the latter is completed upon binding of a single tRNA molecule.

A tropomyosin gene is differentially expressed in the larval stage of Echinococcus granulosus.

We report the isolation of a differentially expressed gene, EgTrp, which is expressed in the protoscoleces (PS) of Echinococcus granulosus. The experimental design was based on a differential immuno-screening of a PS kgt11 cDNA library. We used antisera raised against PS and against germinal layers, selecting those clones exclusively reactive against the former antiserum. The insert of one of the selected clones was subcloned in pBluescript II KS+ and sequenced. A single major open reading frame coding for 177 amino acids was found, with 66–77% identity with a-tropomyosin (a-TM) of various species, but lacking a 5’ terminal domain. The nucleotide sequence data was lodged in GenBank with the accession number AAB65799. The highest identity score was with a Schistosoma japonicum tropomyosin. The computer-predicted secondary structure suggests that EgTrp is organized mainly in a-helix. The deduced amino acid sequence shows the expected heptapeptide typical pattern for a a-helical coiled-coil structure (Fig. 1; Stone et al. 1975). The highly conserved amino acids of tropomyosins are present in the E. granulosus protein. The kEgTrp fragment was amplified by PCR, subcloned and expressed in the pQIA system; it appeared that rEgTrp is highly immunogenic. Immunohistochemical analysis of PS sections revealed that the E. granulosus tropomyosin is expressed in the suckers and at the subtegumental level (Fig. 2). No signal was detected in the germinative layer. These results suggest that EgTrp could be a late marker during E. granulosus PS development. Western blot analysis of total extracts of PS revealed several bands, indicating the presence of at least six members of the tropomyosin family in E. granulosus, with molecular masses ranging over 33–66 kDa. Several isoforms of the a-TM genes were described in vertebrates and invertebrates. The corresponding proteins

Interaction between tRNA and Aminoacyl-tRNA Synthetase in the Valine and Phenylalanine Systems from Yeast

Much work has been devoted to studies of the interaction between tRNAs and aminoacyl-tRNA synthetases. The aim was to understand the mechanisms that govern the specificity of the tRNA aminoacylation reaction (for recent general reviews, see Kisselev and Favorova 1974; Soll and Schimmel 1974; Goddard 1977; Ofengand 1977). In the early days, indirect approaches to this problem were used; recently, direct approaches have been introduced. The indirect approaches included perturbing the primary structure of tRNA by chemical or genetic means, excising small sequences in tRNA and correlating these perturbations with the activity of the modified molecules, or comparing the sequences of different tRNAs recognized by the same aminoacyl-tRNA synthetase. The direct approaches include measuring the inhibition of the tRNA aminoacyl-tRNA synthetase interaction caused by various factors (e.g., oligonucleotides or ionic strength) or studying the shielding of some areas of the tRNA by the aminoacyl-tRNA synthetase against RNases, oligonucleotides, or chemicals; they also include measurements by physical means of the thermodynamic parameters involved in the interaction between the aminoacyl-tRNA synthetase and intact or fragmented tRNA and the determination of the contact zones between the two macromolecules by cross-linking methods (for additional details, see reviews by Loftfield 1972; Schimmel 1977; Smith 1977). The interpretation of the results of most of these approaches has been greatly facilitated by knowledge of the structures of tRNA (Rich and RajBhandary 1976; Dirheimer et al., this volume) and aminoacyl-tRNA synthetase (Irwin et al. 1976; Zelwer et al. 1976; Winter and Hartley 1977). At this stage, however, this...

Silent Polymorphisms: Can the tRNA Population Explain Changes in Protein Properties?

Silent mutations are being intensively studied. We previously showed that the estrogen receptor alpha Ala87’s synonymous polymorphism affects its functional properties. Whereas a link has been clearly established between the effect of silent mutations, tRNA abundance and protein folding in prokaryotes, this connection remains controversial in eukaryotic systems. Although a synonymous polymorphism can affect mRNA structure or the interaction with specific ligands, it seems that the relative frequencies of isoacceptor tRNAs could play a key role in the protein-folding process, possibly through modulation of translation kinetics. Conformational changes could be subtle but enough to cause alterations in solubility, proteolysis profiles, functional parameters or intracellular targeting. Interestingly, recent advances describe dramatic changes in the tRNA population associated with proliferation, differentiation or response to chemical, physical or biological stress. In addition, several reports reveal changes in tRNAs’ posttranscriptional modifications in different physiological or pathological conditions. In consequence, since changes in the cell state imply quantitative and/or qualitative changes in the tRNA pool, they could increase the likelihood of protein conformational variants, related to a particular codon usage during translation, with consequences of diverse significance. These observations emphasize the importance of genetic code flexibility in the co-translational protein-folding process.

Echinococcus granulosus: preparation of protein extracts from protoscolex nuclei for mobility-shift assays.

Abstract Nuclei from Echinococcus granulosus protoscolices were isolated from infected sheep. Protein extracts were prepared for analysis of DNA-protein interactions involving specific transcriptional regulatory factors. Gel mobility-shift assays were done using a heterologous probe containing binding sites for widespread transcription factors. A fragment of the promoter of GATA-1 transcription factor from the chicken was selected. When nuclear extracts from E. granulosus protoscolices were assayed a specific band shift was observed. The methodologies developed in this study could provide an important contribution for the characterization of the DNA-protein interactions involved in transcriptional regulation within the context of recent developments in the molecular biology of this parasite.

Conformational diseases and the protein folding problem: Evidence for species-independent link between protein folding and genetic code degeneracy in ribosomal proteins

The family of conformational diseases is expanding rapidly, including not only neurodegenerative conditions like Alzheimers and Parkinsons diseases, but also certain autoimmune diseases and cancers. The corresponding pathologies have in common their link to misfolded proteins, which accumulate in the cytosol, the rough ER or built up extracellularily, thereby inducing apoptosis, abolishing cell to cell communication, eliciting the immune response or cancelling proper enzymatic (i.e. tumour suppressor) activities. Understanding the mechanism leading to protein misconformation is essential for early diagnosis, efficient therapy and reliable prognosis of these devastating conditions. In previous articles, we showed that, in addition to the primary sequence, an important determinant of the protein structure is the local translation rate, controlled mainly by the choice of synonymous codons along the coding sequence. We found that in coding sequences of E. coli, amino acids having a propensity to appear in particular secondary structures are located in surroundings strongly constraint in fast, or slowly translated codons. To extend this observation to other species, we analyse here all ribosomal protein genes in four species (H. sapiens, S. cerevisiae, B. subtilis and E. coli), for constraints of particular amino acids within sequences of 5 to 21 codons, translated fastest or slowest on average. Although the species investigated make rather different usage of synonymous codons, each species utilizes its synonymous codon repertoire so as to achieve amino-acid constraints in these sequences similar to those observed in E. coli. This suggests that the amino-acid constraints within gene sequences translated at extreme rates are biological invariants to which the species have to conform. Although the constraints are more relaxed in sequences translated at less extreme rates, the general conclusion is that the local translation rate makes, at places at least, important contributions to the protein conformation. Conversely, modifications the local translation rate can at places interfere with native folding and lead to misfolding. Such modifications could be the consequence of spontaneous defects in the translation machinery of the cell, or of its unscheduled, excessive solicitation. This may be one of the routes to conformational diseases.