Proteomics analysis for amino acid misincorporation detection: mini review

Protein biosynthesis is a highly accurate biological process essential for life. Amino acid misincorporation errors (mistranslation) normally occur at low levels, but can increase sharply upon amino acid starvation, exposure to drugs, oxidative stress and other physiological perturbations. These processes disrupt protein function and are normally regarded as being deleterious, however, recent work has shown that they can also be regulated to produce advantageous phenotypes in both prokaryotes and eukaryotes. The biology of such unexpected adaptive mistranslation is poorly understood due to technical difficulties in the identification and quantification of amino acid misincorporations. In this mini-review, we describe proteome scale methodologies involving the use of massspectrometry and bioinformatics tools to directly detect and quantify mistranslation events and also indirect functional methods that permit sensitive, flexible and low-cost analysis of site specific amino acid variation.publishe

Transcriptome variability in yeast strains

In order to understand yeast natural genome and transcriptome variability and evaluate whether such variability correlates with phenotypic diversity, we have isolated approximately 1000 yeast strains from Portuguese vineyards, namely from the Bairrada and Minho wine regions, genotyped them and selected the most divergent ones for genome and transcriptome characterization, using comparative genome hybridization on array (aCGH) and mRNA profiling, respectively. For comparative purposes, strains isolated from opportunistic clinical infections and commercial strains used by the wine industry were also included in the study. Approximately 3 % of the ORFeome showed copy number alterations relative to the reference strain S288C, which was associated with sub-telomeric instability and Ty element transposition. Interestingly, one third of the Ty elements identified in the genome of the reference laboratory strain (S288C) were absent in the wine strains, but were present in the clinical isolates. Distinct gene functional classes were affected by copy number changes in wine and clinical strains. However, no correlation was found between genome alterations and geographical origin. Transcriptome profiling of the same strains grown in synthetic must, using a common reference design, showed important variation in mRNA levels during fermentation, in particular in the wine isolates. Some genes showed patterns of activation that were consistent with gene copy number differences, while others pointed to strain specific regulation. Substantial variability in expression levels were detected among genes associated with fermentation in the wine strains. Therefore, transcriptome variability is relevant to understand yeast adaptation to new ecological niches, yeast evolution, and also to design strategies to select strains with particular phenotypes.National Facility for DNAInstitute of Electronics and Telecommunications of the University of Aveiro (IEETA)Microarray - projecto Ref: CTS 12 (Instituto de Investigação da Universidade de Aveiro)Fundação para a Ciência e a Tecnologia (FCT) - Bolsas CONC-REEQ/737/200, POCI/SAU-MMO/55476/2004, FEDER/POCI/BIA-PRO/55472/2004Biocant Institute, Cantanhede, Portugal

Transcriptome variability of yeast strains

Comunicação em forma de painel, apresentado por Laura CarretoThis work was supported by the National Facility for DNA Microarrays, The University of Aveiro and Biocant

Yeast as a model organism for studying the evolution of non-standard genetic codes

During the last 30 years, a number of alterations to the standard genetic code have been uncovered both in prokaryotes and eukaryotic nuclear and mitochondrial genomes. But, the study of the evolutionary pathways and molecular mechanisms of codon identity redefinition has been largely ignored due to the assumption that non-standard genetic codes can only evolve through neutral evolutionary mechanisms and that they have no functional significance. The recent discovery of a genetic code change in the genus Candida that evolved through an ambiguous messenger RNA decoding mechanism is bringing that naive assumption to an abrupt end by showing, in a rather dramatic way, that genetic code changes have profound physiological and evolutionary consequences for the species that redefine codon identity. In this paper, the recent data on the evolution of the Candida genetic code are reviewed and an experimental framework based on forced evolution, molecular genetics and comparative and functional genomics methodologies is put forward for the study of non-standard genetic codes and genetic code ambiguity in general. Additionally, the importance of using Saccharomyces cerevisiae as a model organism for elucidating the evolutionary pathway of the Candida and other genetic code changes is emphasised.publishe

Impact of tRNA modifications and tRNA-modifying enzymes on proteostasis and human disease

Transfer RNAs (tRNAs) are key players of protein synthesis, as they decode the genetic information organized in mRNA codons, translating them into the code of 20 amino acids. To be fully active, tRNAs undergo extensive post-transcriptional modifications, catalyzed by different tRNA-modifying enzymes. Lack of these modifications increases the level of missense errors and affects codon decoding rate, contributing to protein aggregation with deleterious consequences to the cell. Recent works show that tRNA hypomodification and tRNA-modifying-enzyme deregulation occur in several diseases where proteostasis is affected, namely, neurodegenerative and metabolic diseases. In this review, we discuss the recent findings that correlate aberrant tRNA modification with proteostasis imbalances, in particular in neurological and metabolic disorders, and highlight the association between tRNAs, their modifying enzymes, translational decoding, and disease onset.publishe

An integrative approach for codon repeats evolutionary analyses

The relationship between genome characteristics and several human diseases has been a central research goal in genomics. Many studies have shown that specific gene patterns, such as amino acid repetitions, are associated with human diseases. However, several open questions still remain, such as, how these tandem repeats appeared in the evolutionary path or how they have evolved in orthologous genes of related organisms. In this paper, we present a computational solution that facilitates comparative studies of orthologous genes from various organisms. The application uses various web services to gather gene sequence information, local algorithms for tandem repeats identification and similarity measures for gene clustering.publishe

DFU-VGG, a Novel and Improved VGG-19 Network for Diabetic Foot Ulcer Classification

A complication caused by diabetes mellitus is the appearance of lesions in the foot region called Diabetic Foot Ulcers (DFU). Delayed treatment can lead to infection or ulcer ischemia, leading to lower limb amputation in an advanced stage. This article proposes the DFU-VGG, a convolutional neural network (CNN) inspired by convolutional blocks of VGG-19 but with smaller dense layers and batch normalizations operations. To specify the DFU-VGG parameters, we fine-tuned s even different CNN architectures using two image datasets containing 8,250 images with different color, contrast, resolution, and texture features. The proposed evaluation identifies f our c lasses: none, ischemia, infection, and both. Our approach achieved 93.45% of accuracy and an excellent Kappa index of 89.24%

Does proteostasis get lost in translation? Implications for protein aggregation across the lifespan

Protein aggregation is a phenomenon of major relevance in neurodegenerative and neuromuscular disorders, cataracts, diabetes and many other diseases. Research has unveiled that proteins also aggregate in multiple tissues during healthy aging yet, the biological and biomedical relevance of this apparently asymptomatic phenomenon remains to be understood. It is known that proteome homeostasis (proteostasis) is maintained by a balanced protein synthesis rate, high protein synthesis accuracy, efficient protein folding and continual tagging of damaged proteins for degradation, suggesting that protein aggregation during healthy aging may be associated with alterations in both protein synthesis and the proteostasis network (PN) pathways. In particular, dysregulation of protein synthesis and alterations in translation fidelity are hypothesized to lead to the production of misfolded proteins which could explain the occurrence of age-related protein aggregation. Nevertheless, some data on this topic is controversial and the biological mechanisms that lead to widespread protein aggregation remain to be elucidated. We review the recent literature about the age-related decline of proteostasis, highlighting the need to build an integrated view of protein synthesis rate, fidelity and quality control pathways in order to better understand the proteome alterations that occur during aging and in age-related diseases.publishe