MobiDB 2.0: an improved database of intrinsically disordered and mobile proteins.

MobiDB (http://mobidb.bio.unipd.it/) is a database of intrinsically disordered and mobile proteins. Intrinsically disordered regions are key for the function of numerous proteins. Here we provide a new version of MobiDB, a centralized source aimed at providing the most complete picture on different flavors of disorder in protein structures covering all UniProt sequences (currently over 80 million). The database features three levels of annotation: manually curated, indirect and predicted. Manually curated data is extracted from the DisProt database. Indirect data is inferred from PDB structures that are considered an indication of intrinsic disorder. The 10 predictors currently included (three ESpritz flavors, two IUPred flavors, two DisEMBL flavors, GlobPlot, VSL2b and JRONN) enable MobiDB to provide disorder annotations for every protein in absence of more reliable data. The new version also features a consensus annotation and classification for long disordered regions. In order to complement the disorder annotations, MobiDB features additional annotations from external sources. Annotations from the UniProt database include post-translational modifications and linear motifs. Pfam annotations are displayed in graphical form and are link-enabled, allowing the user to visit the corresponding Pfam page for further information. Experimental protein-protein interactions from STRING are also classified for disorder content.

RepeatsDB: a database of tandem repeat protein structures.

RepeatsDB (http://repeatsdb.bio.unipd.it/) is a database of annotated tandem repeat protein structures. Tandem repeats pose a difficult problem for the analysis of protein structures, as the underlying sequence can be highly degenerate. Several repeat types haven been studied over the years, but their annotation was done in a case-by-case basis, thus making large-scale analysis difficult. We developed RepeatsDB to fill this gap. Using state-of-the-art repeat detection methods and manual curation, we systematically annotated the Protein Data Bank, predicting 10,745 repeat structures. In all, 2797 structures were classified according to a recently proposed classification schema, which was expanded to accommodate new findings. In addition, detailed annotations were performed in a subset of 321 proteins. These annotations feature information on start and end positions for the repeat regions and units. RepeatsDB is an ongoing effort to systematically classify and annotate structural protein repeats in a consistent way. It provides users with the possibility to access and download high-quality datasets either interactively or programmatically through web services.

Exploration of alternative splicing events in ten different grapevine cultivars.

BACKGROUND: The complex dynamics of gene regulation in plants are still far from being fully understood. Among many factors involved, alternative splicing (AS) in particular is one of the least well documented. For many years, AS has been considered of less relevant in plants, especially when compared to animals, however, since the introduction of next generation sequencing techniques the number of plant genes believed to be alternatively spliced has increased exponentially. RESULTS: Here, we performed a comprehensive high-throughput transcript sequencing of ten different grapevine cultivars, which resulted in the first high coverage atlas of the grape berry transcriptome. We also developed findAS, a software tool for the analysis of alternatively spliced junctions. We demonstrate that at least 44% of multi-exonic genes undergo AS and a large number of low abundance splice variants is present within the 131.622 splice junctions we have annotated from Pinot noir. CONCLUSIONS: Our analysis shows that ~70% of AS events have relatively low expression levels, furthermore alternative splice sites seem to be enriched near the constitutive ones in some extent showing the noise of the splicing mechanisms. However, AS seems to be extensively conserved among the 10 cultivars.

Structural protein reorganization and fold emergence investigated through amino acid sequence permutations.

Correlation between random amino acid sequences and protein folds suggests that proteins autonomously evolved the most stable folds, with stability and function evolving subsequently, suggesting the existence of common protein ancestors from which all modern proteins evolved. To test this hypothesis, we shuffled the sequences of 10 natural proteins and obtained 40 different and apparently unrelated folds. Our results suggest that shuffled sequences are sufficiently stable and may act as a basis to evolve functional proteins. The common secondary structure of modern proteins is well represented by a small set of permuted sequences, which also show the emergence of intrinsic disorder and aggregation-prone stretches of the polypeptide chain.

Isoform-specific interactions of the von Hippel-Lindau tumor suppressor protein.

Deregulation of the von Hippel-Lindau tumor suppressor protein (pVHL) is considered one of the main causes for malignant renal clear-cell carcinoma (ccRCC) insurgence. In human, pVHL exists in two isoforms, pVHL19 and pVHL30 respectively, displaying comparable tumor suppressor abilities. Mutations of the p53 tumor suppressor gene have been also correlated with ccRCC insurgence and ineffectiveness of treatment. A recent proteomic analysis linked full length pVHL30 with p53 pathway regulation through complex formation with the p14ARF oncosuppressor. The alternatively spliced pVHL19, missing the first 53 residues, lacks this interaction and suggests an asymmetric function of the two pVHL isoforms. Here, we present an integrative bioinformatics and experimental characterization of the pVHL oncosuppressor isoforms. Predictions of the pVHL30 N-terminus three-dimensional structure suggest that it may exist as an ensemble of structured and disordered forms. The results were used to guide Yeast two hybrid experiments to highlight isoform-specific binding properties. We observed that the physical pVHL/p14ARF interaction is specifically mediated by the 53 residue long pVHL30 N-terminal region, suggesting that this N-terminus acts as a further pVHL interaction interface. Of note, we also observed that the shorter pVHL19 isoform shows an unexpected high tendency to form homodimers, suggesting an additional isoform-specific binding specialization.

Identification of low temperature stress regulated transcript sequences and gene families in Italian cypress.

Cold acclimation is a complex transcriptionally controlled process regulated by many different genes and genic-interactions in plants. The northward spreading of woody species is mainly limited by winter harshness. To increase our knowledge about the biological processes underlying cold acclimation, plants evolved in warmer climates can serve as models. In this work, a Suppression Subtractive Hybridization approach using PCR-select was used to isolate Italian cypress (Cupressus sempervirens L.) transcript sequences putatively expressed under low temperature stress. After assessing the reliability of the subtractive step, a total of 388 clones were selected and sequenced. Following sequence assembly and removal of the redundant cDNAs, 156 unique transcripts were identified and annotated in order to assign them a putative functional class. Most of the identified transcripts were functionally classified pertaining to stress in cellular and chloroplast membranes, which are previously known to be severely damaged by cold treatment. Among the identified functional gene families, the extensively represented ones were dehydrins, early light-inducible proteins, senescence-associated genes and oleosins. The last three gene families were further selected for phylogenetic analysis, with the corresponding protein sequences across the complete genomes of the model plants Populus trichocarpa, Vitis vinifera, Physcomitrella patens, and Arabidopsis thaliana. The relationship with the ortholog sequences coming from these species and their further implications are discussed.

Type 3 metallothioneins respond to water deficit in leaf and in the cambial zone of white poplar (Populus alba).

The involvement of metallothioneins (MTs) in response to plant water stress and recovery was assessed by analyzing gene expression in leaves and in the cambial zone of white poplar. One-year-old plants were submitted to two different watering regimes: irrigation was withheld for 9d and then resumed until day 17, or soil moisture was maintained to field capacity by irrigation during the experiment. Changes in leaves and stem water relations, gas exchange and CO(2) assimilation were recorded. The expression profiles of MT genes were analyzed in developing leaves and the cambial zone at maximum stress levels and after recovery and compared with the watered controls. Whole-plant water relations were significantly affected by water deprivation, though a complete recovery of plant water status was reached after resumption of watering. Withholding irrigation resulted in a significant decrease of leaf turgor potential and relative water content without a significant increase of the osmotic potential at full turgor. Similarly, stem water content decreased, leading to a marked increase of stem shrinkage, confirming that mild water stress affected primarily tissue water status. Following water depletion, the transcript analysis of MT genes revealed increased expression of type 3a and 3b MT genes in cambial tissues, and particularly in leaves. After water resumption, transcription decreased, suggesting that the changes in gene expression were related to water deficit. The results indicate that in leaves and, for the first time, in the cambial zone, type 3 MTs respond in a specific manner to changes in water status. These results are consistent with the regulatory cis-elements present in the 5' flanking region of type 3 MT genes.