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Evolutionary survey of druggable protein targets with respect to their subcellular localizations.

ABSTRACT: The druggable subset of the human genome, termed the "druggable genome," provides the pharmaceutical industry with a unique opportunity for the advancement of new therapeutic interventions for a multitude of diseases and disorders. To date, there is no systematic assessment of the evolutionary history and nature of the defined druggable proteins derived from the contemporary druggable genome (i.e., proteins that bind or are predicted to bind with high affinity to a biologic). An understanding of drug-protein target interactions in specific cellular compartments is crucial for the optimal therapeutic delivery of pharmaceutical agents, as well as for preclinical drug trials in model animals. This study applied the concept of pharmacophylogenomics, the study of genes, evolution, and drug targets, to conduct an evolutionary survey of drug targets with respect to their subcellular localizations. Using multiple models and modes of druggable genome comparison, the results concordantly indicated that orthologous drug targets with a nuclear localization in the human, macaque, mouse, and rat showed a higher trend for evolutionary conservation compared with drug targets in the cell membrane and the extracellular compartment. As such, this study provides important information regarding druggable protein targets and the druggable genome at the pharmacophylogenomics level.

SUBMITTER: Wang X

PROVIDER: S-EPMC3730344 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Evolutionary survey of druggable protein targets with respect to their subcellular localizations.

Wang Xiaotong X Wang Rui R Zhang Yanfeng Y Zhang Hao H

Genome biology and evolution 20130101 7

The druggable subset of the human genome, termed the "druggable genome," provides the pharmaceutical industry with a unique opportunity for the advancement of new therapeutic interventions for a multitude of diseases and disorders. To date, there is no systematic assessment of the evolutionary history and nature of the defined druggable proteins derived from the contemporary druggable genome (i.e., proteins that bind or are predicted to bind with high affinity to a biologic). An understanding of ...[more]

PMID: 23749117

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Project description:The flavivirus capsid protein is considered to be essential for the formation of nucleocapsid complexes with viral genomic RNA at the viral replication organelle that appears on the endoplasmic reticulum (ER), as well as for incorporation into virus particles. However, this protein is also detected at the lipid droplet (LD) and nucleolus, and physiological roles of these off-site localizations are still unclear. In this study, we made a series of alanine substitution mutants of Japanese encephalitis virus (JEV) capsid protein that cover all polar and hydrophobic amino acid residues to identify the molecular surfaces required for virus particle formation and for localization at the LD and nucleolus. Five mutants exhibited a defect in the formation of infectious particles, and two of these mutants failed to be incorporated into the subviral particles (SVP). Three mutants lost the ability to localize to the nucleolus, and only a single mutant, with mutations at α2, was unable to localize to the LD. Unlike the cytoplasmic capsid protein, the nucleolar capsid protein was resistant to detergent treatment, and the α2 mutant was hypersensitive to detergent treatment. To scrutinize the relationship between these localizations and viral particle formation, we made eight additional alanine substitution mutants and found that all the mutants that did not localize at the LD or nucleolus failed to form normal viral particles. These results support the functional correlation between LD or nucleolus localization of the flaviviral capsid protein and the formation of infectious viral particles.IMPORTANCE This study is the first to report the comprehensive mutagenesis of a flavivirus capsid protein. We assessed the requirement of each molecular surface for infectious viral particle formation as well as for LD and nucleolar localization and found functional relationships between the subcellular localization of the virus capsid protein and infectious virus particle formation. We developed a system to independently assess the packaging of viral RNA and that of the capsid protein and found a molecular surface of the capsid protein that is crucial for packaging of viral RNA but not for packaging of the capsid protein itself. We also characterized the biochemical properties of capsid protein mutants and found that the capsid protein localizes at the nucleolus in a different manner than for its localization to the LD. Our comprehensive alanine-scanning mutagenesis study will aid in the development of antiflavivirus small molecules that can target the flavivirus capsid protein.

Project description:Computational methods have been widely used for the prediction of protein subcellular localization. However, these predictions are rarely validated experimentally and as a result remain questionable. Therefore, experimental validation of the predicted localizations is needed to assess the accuracy of predictions so that such methods can be confidently used to annotate the proteins of unknown localization. Previously, we published a method called ngLOC that predicts the localization of proteins targeted to ten different subcellular organelles. In this short report, we describe the accuracy of these predictions using experimental validations.We have experimentally validated the predicted subcellular localizations of 114 human proteins corresponding to nine different organelles in normal breast and breast cancer cell lines using live cell imaging/confocal microscopy. Target genes were cloned into expression vectors as GFP fusions and cotransfected with RFP-tagged organelle-specific gene marker into normal breast epithelial and breast cancer cell lines. Subcellular localization of each target protein is confirmed by colocalization with a co-expressed organelle-specific protein marker. Our results showed that about 82.5% of the predicted subcellular localizations coincided with the experimentally validated localizations. The highest agreement was found in the endoplasmic reticulum proteins, while the cytoplasmic location showed the least concordance. With the exclusion of cytoplasmic location, the average prediction accuracy increased to 90.4%. In addition, there was no difference observed in the protein subcellular localization between normal and cancer breast cell lines.The experimentally validated accuracy of ngLOC method with (82.5%) or without cytoplasmic location (90.4%) nears the prediction accuracy of 89%. These results demonstrate that the ngLOC method can be very useful for large-scale annotation of the unknown subcellular localization of proteins.

Dataset Information

Evolutionary survey of druggable protein targets with respect to their subcellular localizations.

Publications

Evolutionary survey of druggable protein targets with respect to their subcellular localizations.

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