Translational errors: from yeast to new therapeutic targets

Errors occur randomly and at low frequency during the translation of mRNA. However, such errors may also be programmed by the sequence and structure of the mRNA. These programmed events are called 'recoding' and are found mostly in viruses, in which they are usually essential for viral replication. Translational errors at a stop codon may also be induced by drugs, raising the possibility of developing new treatment protocols for genetic diseases on the basis of nonsense mutations. Many studies have been carried out, but the molecular mechanisms governing these events remain largely unknown. Studies on the yeast Saccharomyces cerevisiae have contributed to characterization of the HIV-1 frameshifting site and have demonstrated that frameshifting is conserved from yeast to humans. Yeast has also proved a particularly useful model organism for deciphering the mechanisms of translation termination in eukaryotes and identifying the factors required to obtain a high level of natural suppression. These findings open up new possibilities for large-scale screening in yeast to identify new drugs for blocking HIV replication by inhibiting frameshifting or restoring production of the full-length protein from a gene inactivated by a premature termination codon. We explore these two aspects of the contribution of yeast studies to human medicine in this review.     

New adhesin functions of surface-exposed pneumococcal proteins

Background

Streptococcus pneumoniae is a widely distributed commensal Gram-positive bacteria of the upper respiratory tract. Pneumococcal colonization can progress to invasive disease, and thus become lethal, reason why antibiotics and vaccines are designed to limit the dramatic effects of the bacteria in such cases. As a consequence, pneumococcus has developed efficient antibiotic resistance, and the use of vaccines covering a limited number of serotypes such as Pneumovax^® and Prevnar^® results in the expansion of non-covered serotypes. Pneumococcal surface proteins represent challenging candidates for the development of new therapeutic targets against the bacteria. Despite the number of described virulence factors, we believe that the majority of them remain to be characterized. This is the reason why pneumococcus invasion processes are still largely unknown.

Results

Availability of genome sequences facilitated the identification of pneumococcal surface proteins bearing characteristic motifs such as choline-binding proteins (Cbp) and peptidoglycan binding (LPXTG) proteins. We designed a medium throughput approach to systematically test for interactions between these pneumococcal surface proteins and host proteins (extracellular matrix proteins, circulating proteins or immunity related proteins). We cloned, expressed and purified 28 pneumococcal surface proteins. Interactions were tested in a solid phase assay, which led to the identification of 23 protein-protein interactions among which 20 are new.

Conclusions

We conclude that whether peptidoglycan binding proteins do not appear to be major adhesins, most of the choline-binding proteins interact with host proteins (elastin and C reactive proteins are the major Cbp partners). These newly identified interactions open the way to a better understanding of host-pneumococcal interactions.

     

Solution structure and backbone dynamics of the cysteine 103 to serine mutant of the N-terminal domain of DsbD from Neisseria meningitidis

The DsbD protein is essential for electron transfer from the cytoplasm to the periplasm of Gram-negative bacteria. Its N-terminal domain dispatches electrons coming from cytoplasmic thioredoxin (Trx), via its central transmembrane and C-terminal domains, to its periplasmic partners: DsbC, DsbE/CcmG, and DsbG. Previous structural studies described the latter proteins as Trx-like folds possessing a characteristic C-X-X-C motif able to generate a disulfide bond upon oxidation. The Escherichia coli nDsbD displays an immunoglobulin-like fold in which two cysteine residues (Cys103 and Cys109) allow a disulfide bond exchange with its biological partners.We have determined the structure in solution and the backbone dynamics of the C103S mutant of the N-terminal domain of DsbD from Neisseria meningitidis. Our results highlight significant structural changes concerning the beta-sheets and the local topology of the active site compared with the oxidized form of the E. coli nDsbD. The structure reveals a "cap loop" covering the active site, similar to the oxidized E. coli nDsbD X-ray structure. However, regions featuring enhanced mobility were observed both near to and distant from the active site, revealing a capacity of structural adjustments in the active site and in putative interaction areas with nDsbD biological partners. Results are discussed in terms of functional consequences.     

Increased expression of tryptophan and tyrosine tRNAs elevates stop codon readthrough of reporter systems in human cell lines

Regulation of translation via stop codon readthrough (SC-RT) expands not only tissue-specific but also viral proteomes in humans and, therefore, represents an important subject of study. Understanding this mechanism and all involved players is critical also from a point of view of prospective medical therapies of hereditary diseases caused by a premature termination codon. tRNAs were considered for a long time to be just passive players delivering amino acid residues according to the genetic code to ribosomes without any active regulatory roles. In contrast, our recent yeast work identified several endogenous tRNAs implicated in the regulation of SC-RT. Swiftly emerging studies of human tRNA-ome also advocate that tRNAs have unprecedented regulatory potential. Here, we developed a universal U6 promotor-based system expressing various human endogenous tRNA iso-decoders to study consequences of their increased dosage on SC-RT employing various reporter systems in vivo. This system combined with siRNA-mediated downregulations of selected aminoacyl-tRNA synthetases demonstrated that changing levels of human tryptophan and tyrosine tRNAs do modulate efficiency of SC-RT. Overall, our results suggest that tissue-to-tissue specific levels of selected near-cognate tRNAs may have a vital potential to fine-tune the final landscape of the human proteome, as well as that of its viral pathogens.     

Influence of Affective Stimuli on Leg Power Output and Associated Neuromuscular Parameters during Repeated High Intensity Cycling Exercises

The aim of this study was to examine the impact of emotional eliciting pictures on neuromuscular performance during repetitive supramaximal cycling exercises (RSE). In a randomized order, twelve male participants were asked to perform five 6-s cycle sprints (interspaced by 24 s of recovery) on a cycle ergometer in front of neutral, pleasant or unpleasant pictures. During each RSE, mean power output (MPO) and electromyographic activity [root mean square (RMS) and median frequency (MF)] of the vastus lateralis and vastus medialis muscles were analyzed. Neuromuscular efficiency (NME) was calculated as the ratio of MPO to RMS. Higher RMS (232.17 ± 1.17 vs. 201.90 ± 0.47 μV) and MF (68.56 ± 1.78 vs. 64.18 ± 2.17 Hz) were obtained in pleasant compared to unpleasant conditions (p < 0.05). This emotional effect persisted from the first to the last sprint. Higher MPO was obtained in pleasant than in unpleasant conditions (690.65 ± 38.23 vs. 656.73 ± 35.95 W, p < 0.05). However, this emotional effect on MPO was observed only for the two first sprints. NME decreased from the third sprint (p < 0.05), which indicated the occurrence of peripheral fatigue after the two first sprints. These results suggested that, compared with unpleasant pictures, pleasant ones increased the neuromuscular performance during RSE. Moreover, the disappearance of the beneficial effect of pleasant emotion on mechanical output from the third sprint appears to be due to peripheral fatigue.     

Influenza A viruses limit NLRP3‐NEK7‐complex formation and pyroptosis in human macrophages

Pyroptosis is a fulminant form of macrophage cell death, contributing to release of pro‐inflammatory cytokines. In humans, it depends on caspase 1/4‐activation of gasdermin D and is characterized by the release of cytoplasmic content. Pathogens apply strategies to avoid or antagonize this host response. We demonstrate here that a small accessory protein (PB1‐F2) of contemporary H5N1 and H3N2 influenza A viruses (IAV) curtails fulminant cell death of infected human macrophages. Infection of macrophages with a PB1‐F2‐deficient mutant of a contemporary IAV resulted in higher levels of caspase‐1 activation, cleavage of gasdermin D, and release of LDH and IL‐1β. Mechanistically, PB1‐F2 limits transition of NLRP3 from its auto‐repressed and closed confirmation into its active state. Consequently, interaction of a recently identified licensing kinase NEK7 with NLRP3 is diminished, which is required to initiate inflammasome assembly.     

The problem of characters susceptible to parallel evolution in phylogenetic analysis: a reply to Marquès and Gnaspini (2001) with emphasis on cave life phenotypic evolution

Marquès and Gnaspini [Cladistics 17 (2001) 371–381] analyzed the problem of the phylogenetic treatment of characters submitted to parallel evolution. Their proposal aimed at preserving the potential phylogenetic significance of supposedly homoplastic characters and considering them for phylogeny reconstruction. As an example, they used the troglobiomorphic features of animals restricted to caves; according to their preliminary hypothesis of homoplasy, troglobitic animals would exhibit a particular phenotype, referred to as troglobiomorphic, which they would acquire under similar selective pressures from the subterranean environment. We examined Marquès and Gnaspini's approach not from the point of view of its technical flaws, but from the point of view of the authors’ basic assertions on the treatment of troglobiomorphic characters and more generally the inclusion/exclusion/transformation of characters prior to phylogenetic analysis. In the present paper, we argue that this approach is invalidated by the repeated use of ad hoc hypotheses, which are supported neither by an existing phylogenetic pattern nor by available data. We consequently contest the adequateness of this approach with a cladistic analysis of historical questions.     

The rat kidney acylase I, characterization and molecular cloning. Differences with other acylases I.

The soluble acylase I from rat kidney was purified to homogeneity using a five-step procedure. As the resulting protein was found to have a relative molecular mass of 125 kDa based on size-exclusion chromatography and 44 kDa based on SDS/PAGE, the native protein was taken to consist of three subunits. The amino-acid sequence of a peptide resulting from limited proteolysis of the polypeptide chain with proteinase K, which was determined by microsequencing (RHEFHALRAGFALDEGLA), was found to be very similar to the corresponding sequence of porcine kidney acylase I. However, as N-furyl-acryloyl-L-methionine, a synthetic substrate for porcine acylases, was not hydrolyzed by the rat enzyme, it was suggested that the polypeptide chain might differ in other respects from those of the other acylases I. A full length cDNA coding for the rat kidney acylase I was therefore isolated and found to contain a 1224-bp open reading frame encoding a protein consisting of 408 amino-acid residues, which corresponded to a calculated molecular mass of 45 847 Da per subunit. The deduced amino-acid sequence showed 93.6% and 87.2% identity with that of the human liver and porcine kidney, respectively.     

Activation of a dimeric metabotropic glutamate receptor by intersubunit rearrangement

Although many G protein-coupled receptors (GPCRs) can form dimers, a possible role of this phenomenon in their activation remains elusive. A recent and exciting proposal is that a dynamic intersubunit interplay may contribute to GPCR activation. Here, we examined this possibility using dimeric metabotropic glutamate receptors (mGluRs). We first developed a system to perfectly control their subunit composition and show that mGluR dimers do not form larger oligomers. We then examined an mGluR dimer containing one subunit in which the extracellular agonist-binding domain was uncoupled from the G protein-activating transmembrane domain. Despite this uncoupling in one protomer, agonist stimulation resulted in symmetric activation of either transmembrane domain in the dimer with the same efficiency. This, plus other data, can only be explained by an intersubunit rearrangement as the activation mechanism. Although well established for other types of receptors such as tyrosine kinase and guanylate cyclase receptors, this is the first clear demonstration that such a mechanism may also apply to GPCRs.