Claude Reiss

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folding, 16 consecutive rare (in Escherichia coli) codons in the chloramphenicol acetyltransferase (CAT) gene have been replaced by frequent ones.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.

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.

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.

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.

Scientific toxicity assessment of pesticides, drugs and other chemicals

People living in developed countries are exposed to over 100 000 chemically pure, man-made substances (and an immense number of their combinations). 98% of these chemicals have never been tested for their effects on our health or environment. Traditional toxicity testing in animal models has proven unreliable. With the help of concepts, methods and tools developed in modern biology, scientific toxicity assessment is at present possible. Various fields of toxicology are reviewed and the scientific methods allowing their reliable assessments are summarized. The implementation of scientific toxicity testing is expected to save yearly millions of lives in developed countries.

Dynamic Monitoring of Neurological Diseases by CSF Clinical Markers

Neurological symptoms and neurodegeneration are silent features of many neuropathological conditions. These include acute metabolic and physiological insult, chronic motor and dementia disorders as well as inflammatory diseases represting important causes of morbidity and mortality in humans. Studies dining the last decade have provided convincing evidence that numerous neuroactive molecules play a vital role in chemical signaling and neurodegenerative processes of the nervous system.1 These substances before mainly represented putative transmitters and neuropeptides. Investigations on oxidative stress involving reactive oxygen species (ROS) and reactive nitrogen species (NOS) are now largely believed to be in part responsible for the induction of neurogenic lesions not only by producing neurotoxins but also leading to DNA adduct formation.2 The interdependence of neuropeptide/neurotransmitter pathways and their co-existence in the same neuron is a tremendous advance to understand how neurosecretion can control the processes of transmitter synthesis, release and metabolism. Receptor active opioid peptides in human brain were demonstrated more than 20 years ago.3 Regardless of our advance knowledge on the neurochemitsry of neuropeptide/neurotransmitter and free radicals, it still remains extremely difficult to have a precise diagnosis of the early onset of neurodegenerative processes and only the post-mortem histopathological and laboratory tests can provide the true neuropathological picture.

Molecular Biology of Conformational Diseases

An important family of diseases is characterized by the presence of a disease-specific protein that accumulates as “amyloid” fibers and thereby becomes toxic to the cell. Although the protein has the authentic primary sequence (except in familial cases), it adopts a non-native conformation which resists proteolysis, allowing the protein to accumulate and form fibers. Non-familial amyloid conditions are clearly epigenetic.