Mutational and computational characterization of transmembrane domains in the fungal G protein-coupled pheromone receptors STE2 and Mam2

G protein-coupled receptors (GPCRs) comprise the largest family of cell-surface receptors involved in sensing a multitude of ligands and are consequently attractive pharmacological targets. Their study is complicated by cross-talk between signalling pathways and altered receptor pharmacology due to, for instance, receptor oligomerization. Difficulties in obtaining structural information of the receptors hinder the understanding of oligomerization and therefore it is desirable to develop alternative approaches in which to study this phenomenon. The fungal pheromone GPCRs, STE2 and Mam2, from Saccharomyces cerevisiae and Schizosaccharomyces pombe respectively are both known to oligomerize and a GxxxG motif in the first transmembrane (TM) domain of STE2 has previously been shown to mediate receptor oligomerization. Previous work on polytopic proteins suggest that individual TM helices may be treated as individually stable domains, and it may therefore be possible to study oligomerization via single TM peptides as opposed to full-length receptor. This thesis describes the use of STE2 and Mam2 to explore TM helix oligomerization and the effects of mutations on receptor trafficking, localization and cellular signalling. The development of a luminescent reporter assay for Sz. pombe, which proved more sensitive than previously used assays and is capable of generating high-throughput data, is also discussed. It was found that STE2 could couple to the Sz. pombe pheromone-response pathway and mutations in the GxxxG dimerization motif affected both signalling and trafficking. Expression of the first TM GxxxG containing domain of STE2 was insufficient for oligomerization, in line with previous reports suggesting that the presence of the second domain is required for receptor oligomerization. In Mam2, a motif was identified that appeared homologous to the STE2 dimerization motif and mutations of this motif also affected trafficking and signalling. This domain could oligomerize in isolation, and mutations of the motif abolished oligomerization. In contrast the study of more polar TM domains appeared more complicated. These findings suggest that relatively hydrophobic TM domains can be studied as individually stable units, whereas more polar domains may require the presence of other TM domains

Model organism databases: essential resources that need the support of both funders and users.

Modern biomedical research depends critically on access to databases that house and disseminate genetic, genomic, molecular, and cell biological knowledge. Even as the explosion of available genome sequences and associated genome-scale data continues apace, the sustainability of professionally maintained biological databases is under threat due to policy changes by major funding agencies. Here, we focus on model organism databases to demonstrate the myriad ways in which biological databases not only act as repositories but actively facilitate advances in research. We present data that show that reducing financial support to model organism databases could prove to be not just scientifically, but also economically, unsound

Canto: an online tool for community literature curation.

MOTIVATION: Detailed curation of published molecular data is essential for any model organism database. Community curation enables researchers to contribute data from their papers directly to databases, supplementing the activity of professional curators and improving coverage of a growing body of literature. We have developed Canto, a web-based tool that provides an intuitive curation interface for both curators and researchers, to support community curation in the fission yeast database, PomBase. Canto supports curation using OBO ontologies, and can be easily configured for use with any species. AVAILABILITY: Canto code and documentation are available under an Open Source license from http://curation.pombase.org/. Canto is a component of the Generic Model Organism Database (GMOD) project (http://www.gmod.org/)

Rotational dynamics of optically trapped polymeric nanofibers

The optical trapping of polymeric nanofibers and the characterization of the rotational dynamics are reported. A strategy to apply a torque to a polymer nanofiber, by tilting the trapped fibers using a symmetrical linear polarized Gaussian beam is demonstrated. Rotation frequencies up to 10 Hz are measured, depending on the trapping power, the fiber length and the tilt angle. A comparison of the experimental rotation frequencies in the different trapping configurations with calculations based on optical trapping and rotation of linear nanostructures through a T-Matrix formalism, accurately reproduce the measured data, providing a comprehensive description of the trapping and rotation dynamics.Comment: (21 pages, 5 figures

PomBase 2015: updates to the fission yeast database.

PomBase (http://www.pombase.org) is the model organism database for the fission yeast Schizosaccharomyces pombe. PomBase provides a central hub for the fission yeast community, supporting both exploratory and hypothesis-driven research. It provides users easy access to data ranging from the sequence level, to molecular and phenotypic annotations, through to the display of genome-wide high-throughput studies. Recent improvements to the site extend annotation specificity, improve usability and allow for monthly data updates. Both in-house curators and community researchers provide manually curated data to PomBase. The genome browser provides access to published high-throughput data sets and the genomes of three additional Schizosaccharomyces species (Schizosaccharomyces cryophilus, Schizosaccharomyces japonicus and Schizosaccharomyces octosporus)

Hidden in plain sight: what remains to be discovered in the eukaryotic proteome?

The first decade of genome sequencing stimulated an explosion in the characterization of unknown proteins. More recently, the pace of functional discovery has slowed, leaving around 20% of the proteins even in well-studied model organisms without informative descriptions of their biological roles. Remarkably, many uncharacterized proteins are conserved from yeasts to human, suggesting that they contribute to fundamental biological processes (BP). To fully understand biological systems in health and disease, we need to account for every part of the system. Unstudied proteins thus represent a collective blind spot that limits the progress of both basic and applied biosciences. We use a simple yet powerful metric based on Gene Ontology BP terms to define characterized and uncharacterized proteins for human, budding yeast and fission yeast. We then identify a set of conserved but unstudied proteins in S. pombe, and classify them based on a combination of orthogonal attributes determined by large-scale experimental and comparative methods. Finally, we explore possible reasons why these proteins remain neglected, and propose courses of action to raise their profile and thereby reap the benefits of completing the catalogue of proteins' biological roles

A method for increasing expressivity of Gene Ontology annotations using a compositional approach.

BACKGROUND: The Gene Ontology project integrates data about the function of gene products across a diverse range of organisms, allowing the transfer of knowledge from model organisms to humans, and enabling computational analyses for interpretation of high-throughput experimental and clinical data. The core data structure is the annotation, an association between a gene product and a term from one of the three ontologies comprising the GO. Historically, it has not been possible to provide additional information about the context of a GO term, such as the target gene or the location of a molecular function. This has limited the specificity of knowledge that can be expressed by GO annotations. RESULTS: The GO Consortium has introduced annotation extensions that enable manually curated GO annotations to capture additional contextual details. Extensions represent effector-target relationships such as localization dependencies, substrates of protein modifiers and regulation targets of signaling pathways and transcription factors as well as spatial and temporal aspects of processes such as cell or tissue type or developmental stage. We describe the content and structure of annotation extensions, provide examples, and summarize the current usage of annotation extensions. CONCLUSIONS: The additional contextual information captured by annotation extensions improves the utility of functional annotation by representing dependencies between annotations to terms in the different ontologies of GO, external ontologies, or an organism's gene products. These enhanced annotations can also support sophisticated queries and reasoning, and will provide curated, directional links between many gene products to support pathway and network reconstruction