An enhanced workflow for variant interpretation in UniProtKB/Swiss-Prot improves consistency and reuse in ClinVar.

Personalized genomic medicine depends on integrated analyses that combine genetic and phenotypic data from individual patients with reference knowledge of the functional and clinical significance of sequence variants. Sources of this reference knowledge include the ClinVar repository of human genetic variants, a community resource that accepts submissions from external groups, and UniProtKB/Swiss-Prot, an expert-curated resource of protein sequences and functional annotation. UniProtKB/Swiss-Prot provides knowledge on the functional impact and clinical significance of over 30 000 human protein-coding sequence variants, curated from peer-reviewed literature reports. Here we present a pilot study that lays the groundwork for the integration of curated knowledge of protein sequence variation from UniProtKB/Swiss-Prot with ClinVar. We show that existing interpretations of variant pathogenicity in UniProtKB/Swiss-Prot and ClinVar are highly concordant, with 88% of variants that are common to the two resources having interpretations of clinical significance that agree. Re-curation of a subset of UniProtKB/Swiss-Prot variants according to American College of Medical Genetics and Genomics (ACMG) guidelines using ClinGen tools further increases this level of agreement, mainly due to the reclassification of supposedly pathogenic variants as benign, based on newly available population frequency data. We have now incorporated ACMG guidelines and ClinGen tools into the UniProt Knowledgebase (UniProtKB) curation workflow and routinely submit variant data from UniProtKB/Swiss-Prot to ClinVar. These efforts will increase the usability and utilization of UniProtKB variant data and will facilitate the continuing (re-)evaluation of clinical variant interpretations as data sets and knowledge evolve.

Gene Ontology annotations and resources.

The Gene Ontology (GO) Consortium (GOC, http://www.geneontology.org) is a community-based bioinformatics resource that classifies gene product function through the use of structured, controlled vocabularies. Over the past year, the GOC has implemented several processes to increase the quantity, quality and specificity of GO annotations. First, the number of manual, literature-based annotations has grown at an increasing rate. Second, as a result of a new 'phylogenetic annotation' process, manually reviewed, homology-based annotations are becoming available for a broad range of species. Third, the quality of GO annotations has been improved through a streamlined process for, and automated quality checks of, GO annotations deposited by different annotation groups. Fourth, the consistency and correctness of the ontology itself has increased by using automated reasoning tools. Finally, the GO has been expanded not only to cover new areas of biology through focused interaction with experts, but also to capture greater specificity in all areas of the ontology using tools for adding new combinatorial terms. The GOC works closely with other ontology developers to support integrated use of terminologies. The GOC supports its user community through the use of e-mail lists, social media and web-based resources.

Transport and function of syntaxin 3 in human epithelial intestinal cells.

To follow the transport of human syntaxin (Syn) 3 to the apical surface of intestinal cells, we produced and expressed in Caco-2 cells a chimera made of the entire Syn3 coding sequence and the extracellular domain of the human transferrin receptor (TfR). This chimera (Syn3TfR) was localized to the apical membrane and was transported along the direct apical pathway, suggesting that this is also the case for endogenous Syn3. To test the potential role of Syn3 in apical transport, we overexpressed it in Caco-2 cells and measured the efficiency of apical and basolateral delivery of several endogenous markers. We observed a strong inhibition of apical delivery of sucrase-isomaltase (SI), an apical transmembrane protein, and of alpha-glucosidase, an apically secreted protein. No effect was observed on the basolateral delivery of Ag525, a basolateral antigen, strongly suggesting that Syn3 is necessary for efficient delivery of proteins to the apical surface of intestinal cells.

Different functional recognition of basolateral signals in Caco-2 and MDCK cells.

Using the basolateral mutant PS of the normally apical neurotrophin receptor p75 (p75NTR) we have identified two cytoplasmic determinants responsible for this reversed localization in the human intestinal cell line, Caco2. These signals are based on two consecutive leucines (322-323) and a tyrosine (Y308). Truncation of the cytoplasmic tail removing the two leucines or their replacement by alanines led to a nonpolarized expression of the resulting mutants in Caco2 cells. To our surprise, the same mutations had no effect on the basolateral localization of the mutant PS in MDCK cells. In MDCK cells, the basolateral localization was entirely dependent on a cytoplasmic tyrosine Y308, while in Caco-2 cells this tyrosine signal was functional as a basolateral signal only when the cytoplasmic domain of PS was truncated shortly after it. These data indicate for the first time that there is a differential recognition of basolateral signals between MDCK and Caco-2 cells.

[Identification of signals and mechanisms of sorting of plasma membrane proteins in intestinal epithelial cells]. / Identification de signaux et de mécanismes de tri des protéines de la membrane plasmique dans les cellules épithéliales intestinales.

In epithelial cells the plasma membrane is divided into domains that are biochemically and functionally different. In intestinal cells for example the apical domain is facing the intestinal lumen and is involved in the uptake of nutriments while the basolateral domain is mediating cell-cell adhesion and signalisation. We are interested in deciphering the mechanisms underlying the creation and maintenance of such specialized domains. As an epithelial model we have used the intestinal cell line Caco-2 and we have studied the transport and sorting of the human neurotrophin receptor (p75 NTR) in these cells. Newly synthesized p75 NTR is first transported to the basolateral membrane and then is accumulated on the apical membrane after transcytosis. This final apical localization is controlled by the presence of a membrane anchor and a cluster of O-glycosylation sites located in the part of the ectodomain close to the membrane. Among the mechanisms likely to be involved in the sorting of apical components we have looked for a role of lipid-protein microdomain formation in the Golgi apparatus. These membrane microdomains are highly enriched in glycosylphosphatidyl inositol (GPI) anchored proteins, glycosphingolipids and apical proteins such as sucrase isomaltase (SI). Such a composition is also found for endocytic structures called caveolae which are made of caveolin 1. We have expressed caveolin 1 in Caco-2 cells which do not express it and also caveolin 2, a related protein of unknown function. Expression of caveolin 1 led to formation of caveolae indicating that this protein is necessary for caveolae formation while caveolin 2 is restricted to the Golgi apparatus and has no effect on caveolae formation. However Caveolin 2 increased the amount of SI incorporated in microdomains suggesting a role in recruitment into the apical pathway. The choice for a site of fusion for transport vesicles is the last step of control during exocytosis. To identify proteins involved in that step we have cloned and characterized two members of the t-SNARE family, namely syntaxin 3 and SNAP23. Syntaxin 3 is present on the apical membrane and forms a complex with SNAP23 which is also localized on the basolateral membrane where it forms a complex with syntaxin 4. Overexpression of syntaxin 3 in Caco-2 led to a decrease of SI exocytosis towards the apical membrane confirming that syntaxin 3 is involved in targeting the fusion of apical transport vesicles to the apical pole of the cells.

Putative O-glycosylation sites and a membrane anchor are necessary for apical delivery of the human neurotrophin receptor in Caco-2 cells.

We have expressed the human neurotrophin receptor p75 (p75(NTR)) in the intestinal epithelial cell line Caco-2 as a model to study intracellular transport and subcellular sorting signals in intestinal cells. p75(NTR) was localized at the apical membrane of Caco-2 cells and reached this membrane mainly via an indirect pathway. Apical localization, intracellular routing, and basolateral to apical transcytosis were not affected by truncation of the cytoplasmic domain or replacement of the transmembrane domain by a glycosyl phosphatidylinositol anchor. Removal of membrane anchoring resulted in basolateral secretion of the ectodomain of p75(NTR) in Caco-2 cells but in apical secretion in Madin-Darby canine kidney (MDCK) cells. Substitution of potential O-glycosylation sites present in the stalk of p75(NTR) led to intracellular cleavage and secretion of the ectodomain into the basolateral medium both in Caco-2 and MDCK cells. These results suggest that the stalk of p75(NTR) carries an apical sorting information that is recognized efficiently by Caco-2 cells only when attached to the membrane. This apical sorting information is linked to the presence of predicted O-glycosylation sites in that region. These putative O-glycosylation sites also play a role in the regulation of p75(NTR) transport to the cell surface and in the prevention of rapid degradation by cleavage of the stalk domain.

Human syntaxin 3 is localized apically in human intestinal cells.

To understand the molecular mechanisms underlying sorting of apical and basolateral membrane components in human intestinal epithelial cells, we have cloned the human homolog of rat syntaxin 3 and looked for its subcellular localization. Endogenous human syntaxin 3 was found to be localized at the apical membrane of colon epithelial and Caco-2 cells. This apical localization was confirmed by confocal microscopy after transfection of the cDNA coding for either full length or N-terminally truncated human syntaxin 3 in Caco-2 cells. Furthermore the signal(s) and machinery targeting human syntaxin 3 to the apical membrane of epithelial cells are conserved between species since human syntaxin 3 was also localized at the apical membrane of canine MDCK cells and of epithelial cells in transgenic Drosophila melanogaster.