Quantitative Protein Localization Signatures Reveal an Association between Spatial and Functional Divergences of Proteins

<div><p>Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast <i>Saccharomyces cerevisiae</i> as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein functions and how these functions were acquired in cells from different organisms or species. A public web interface of PLAST is available at <a href="http://plast.bii.a-star.edu.sg" target="_blank">http://plast.bii.a-star.edu.sg</a>.</p></div

Functional analysis of ankrd55, a multiple sclerosis risk gene with unknown function.

213 p.El objetivo de este trabajo consiste en la caracterización de ANKRD55, un gen de función desconocida asociado a la esclerosis múltiple (EM). Para ello, por un lado, se realizó un análisis basado en DNA y expresión génica que consistió en la identificación de la principal fuente celular de ANKRD55 en PBMC, donde los resultados mostraron que los tres transcritos de ANKRD55 se expresaron de forma única y elevada en los linfocitos T CD4+. Además, el genotipo de riesgo para la variante intrónica asociada a EM mostró niveles significativamente más altos de dos transcritos de ANKRD55 en células T CD4+. A continuación, utilizando el sistema CRISPR/deadCas9 se analizaron 5 variantes de DNA presentes en ANKRD55 por su posible actividad enhancer, donde se observó que la región que contiene la variante rs71624119 regula la expresión de uno de los tres transcritos de ANKRD55. Por otro lado, mediante el análisis basado en proteína, se estudió la localización subcelular y la red de interacciones proteína-proteína de ANKRD55. Se observó que la localización intracelular de la forma endógena de ANKRD55 era principalmente nuclear en células inmunes y no inmunes, mientras que las formas recombinantes se encontraban tanto en el núcleo como en orgánulos membranosos y citosol de las líneas celulares HEK293 y HeLa. A través de la inmunoprecipitación (IP) de una de las formas recombinantes de ANKRD55 y análisis mediante espectrometría de masas, se identificaron 158 proteínas en el extracto proteico total y 22 en extracto nuclear que interaccionan con ANKRD55, de las cuales ocho se validaron por microscopía confocal e IP y posterior immunoblot

Functional analysis of ankrd55, a multiple sclerosis risk gene with unknown function.

213 p.El objetivo de este trabajo consiste en la caracterización de ANKRD55, un gen de función desconocida asociado a la esclerosis múltiple (EM). Para ello, por un lado, se realizó un análisis basado en DNA y expresión génica que consistió en la identificación de la principal fuente celular de ANKRD55 en PBMC, donde los resultados mostraron que los tres transcritos de ANKRD55 se expresaron de forma única y elevada en los linfocitos T CD4+. Además, el genotipo de riesgo para la variante intrónica asociada a EM mostró niveles significativamente más altos de dos transcritos de ANKRD55 en células T CD4+. A continuación, utilizando el sistema CRISPR/deadCas9 se analizaron 5 variantes de DNA presentes en ANKRD55 por su posible actividad enhancer, donde se observó que la región que contiene la variante rs71624119 regula la expresión de uno de los tres transcritos de ANKRD55. Por otro lado, mediante el análisis basado en proteína, se estudió la localización subcelular y la red de interacciones proteína-proteína de ANKRD55. Se observó que la localización intracelular de la forma endógena de ANKRD55 era principalmente nuclear en células inmunes y no inmunes, mientras que las formas recombinantes se encontraban tanto en el núcleo como en orgánulos membranosos y citosol de las líneas celulares HEK293 y HeLa. A través de la inmunoprecipitación (IP) de una de las formas recombinantes de ANKRD55 y análisis mediante espectrometría de masas, se identificaron 158 proteínas en el extracto proteico total y 22 en extracto nuclear que interaccionan con ANKRD55, de las cuales ocho se validaron por microscopía confocal e IP y posterior immunoblot

Quantified phenotype analysis in a cell model for Autosomal Dominant Retinitis Pigmentosa

Modern cell biology relies greatly on microscopy to assess distribution and dynamics of fluorescently labelled cellular proteins. Quantified image analysis allows not only measurement of clear differences between individual phenotypes and time points, but also discovery of subtle changes which are not obvious to an observer by visual inspection. In this dissertation, we quantitatively characterise wild type and mutations P23H/A/L in rhodopsin in a cellular model for autosomal dominant retinitis pigmentosa in stable HEK 293S cell lines and in GMK cells. Autosomal dominant retinitis pigmentosa is a genetic disorder which can lead to photoreceptor cell death and result in reduced vision and complete blindness. Endoplasmic Reticulum chaperone calnexin was also quantified from both immune-labelled fixed cells, and from transient co-transfection of live cells. The clinically relevant severity of rhodopsin mutations was in keeping with the phenotypes of the cellular model. The severe mutation P23H showed the lowest volume of rhodopsin-GFP in both cell lines in comparison to wild-type. We also reported a significantly higher calnexin volume in HEK293 and GMK expressing P23H rhodopsin (with p<0.05). Less severe mutants had a phenotype more similar to wildtype. Colocalisation was assessed using a simple approach of overlapping volume. As co-expression of rhodopsin and calnexin during time-lapse acquisition induced cytotoxicity and accelerated cellular death, we assessed phototoxicity caused by blue light illumination. We quantified motility and division rates in PC3 and GMK mammalian cell cultures, respectively. A surprisingly low phototoxicity threshold of 13.9 J/cm2 was determined for imaging unlabelled GMK cells without inducing mitotic delay. To assess the production of reactive oxygen species, which are key to phototoxicity in fluorescence microscopy, the end-product hydrogen peroxide was monitored using a ratiometric biosensor. Finally, all findings are synthesised as practical guidelines for end users