Modeling the Structural Consequences of \u3cem\u3eBEST1\u3c/em\u3e Missense Mutations

Mutations in the bestrophin-1 gene (BEST1) are an important cause of inherited retinal disorders. Hitherto, over 100 unique allelic variants have been linked to the human BEST1 (hBEST1), and associated with disease phenotypes, broadly termed as bestrophinopathies. A spontaneous animal model recapitulating BEST1-related phenotypes, canine multifocal retinopathy (cmr), is caused by mutations in the canine gene ortholog (cBEST1). We have recently characterized molecular consequences of cmr, demonstrating defective protein trafficking as a result of G161D (cmr2) mutation. To further investigate the pathological effects of BEST1 missense mutations, canine and human peptide fragments derived from the protein sequence have been studied in silico as models for early events in the protein folding. The results showed that G161D as well as I201T substitutions cause severe conformational changes in the structure of bestrophin-1, suggesting protein misfolding as an underlying disease mechanism. The comparative modeling studies expand our insights into BEST1 pathogenesis

The N-terminal intrinsically disordered domain of mgm101p is localized to the mitochondrial nucleoid.

The mitochondrial genome maintenance gene, MGM101, is essential for yeasts that depend on mitochondrial DNA replication. Previously, in Saccharomyces cerevisiae, it has been found that the carboxy-terminal two-thirds of Mgm101p has a functional core. Furthermore, there is a high level of amino acid sequence conservation in this region from widely diverse species. By contrast, the amino-terminal region, that is also essential for function, does not have recognizable conservation. Using a bioinformatic approach we find that the functional core from yeast and a corresponding region of Mgm101p from the coral Acropora millepora have an ordered structure, while the N-terminal domains of sequences from yeast and coral are predicted to be disordered. To examine whether ordered and disordered domains of Mgm101p have specific or general functions we made chimeric proteins from yeast and coral by swapping the two regions. We find, by an in vivo assay in S.cerevisiae, that the ordered domain of A.millepora can functionally replace the yeast core region but the disordered domain of the coral protein cannot substitute for its yeast counterpart. Mgm101p is found in the mitochondrial nucleoid along with enzymes and proteins involved in mtDNA replication. By attaching green fluorescent protein to the N-terminal disordered domain of yeast Mgm101p we find that GFP is still directed to the mitochondrial nucleoid where full-length Mgm101p-GFP is targeted

Comparative analysis of an experimental subcellular protein localization assay and in silico prediction methods

The subcellular localization of a protein can provide important information about its function within the cell. As eukaryotic cells and particularly mammalian cells are characterized by a high degree of compartmentalization, most protein activities can be assigned to particular cellular compartments. The categorization of proteins by their subcellular localization is therefore one of the essential goals of the functional annotation of the human genome. We previously performed a subcellular localization screen of 52 proteins encoded on human chromosome 21. In the current study, we compared the experimental localization data to the in silico results generated by nine leading software packages with different prediction resolutions. The comparison revealed striking differences between the programs in the accuracy of their subcellular protein localization predictions. Our results strongly suggest that the recently developed predictors utilizing multiple prediction methods tend to provide significantly better performance over purely sequence-based or homology-based predictions

A broad distribution of the alternative oxidase in microsporidian parasites

Microsporidia are a group of obligate intracellular parasitic eukaryotes that were considered to be amitochondriate until the recent discovery of highly reduced mitochondrial organelles called mitosomes. Analysis of the complete genome of Encephalitozoon cuniculi revealed a highly reduced set of proteins in the organelle, mostly related to the assembly of ironsulphur clusters. Oxidative phosphorylation and the Krebs cycle proteins were absent, in keeping with the notion that the microsporidia and their mitosomes are anaerobic, as is the case for other mitosome bearing eukaryotes, such as Giardia. Here we provide evidence opening the possibility that mitosomes in a number of microsporidian lineages are not completely anaerobic. Specifically, we have identified and characterized a gene encoding the alternative oxidase (AOX), a typically mitochondrial terminal oxidase in eukaryotes, in the genomes of several distantly related microsporidian species, even though this gene is absent from the complete genome of E. cuniculi. In order to confirm that these genes encode functional proteins, AOX genes from both A. locustae and T. hominis were over-expressed in E. coli and AOX activity measured spectrophotometrically using ubiquinol-1 (UQ-1) as substrate. Both A. locustae and T. hominis AOX proteins reduced UQ-1 in a cyanide and antimycin-resistant manner that was sensitive to ascofuranone, a potent inhibitor of the trypanosomal AOX. The physiological role of AOX microsporidia may be to reoxidise reducing equivalents produced by glycolysis, in a manner comparable to that observed in trypanosome

MemBrain: Improving the Accuracy of Predicting Transmembrane Helices

Prediction of transmembrane helices (TMH) in α helical membrane proteins provides valuable information about the protein topology when the high resolution structures are not available. Many predictors have been developed based on either amino acid hydrophobicity scale or pure statistical approaches. While these predictors perform reasonably well in identifying the number of TMHs in a protein, they are generally inaccurate in predicting the ends of TMHs, or TMHs of unusual length. To improve the accuracy of TMH detection, we developed a machine-learning based predictor, MemBrain, which integrates a number of modern bioinformatics approaches including sequence representation by multiple sequence alignment matrix, the optimized evidence-theoretic K-nearest neighbor prediction algorithm, fusion of multiple prediction window sizes, and classification by dynamic threshold. MemBrain demonstrates an overall improvement of about 20% in prediction accuracy, particularly, in predicting the ends of TMHs and TMHs that are shorter than 15 residues. It also has the capability to detect N-terminal signal peptides. The MemBrain predictor is a useful sequence-based analysis tool for functional and structural characterization of helical membrane proteins; it is freely available at http://chou.med.harvard.edu/bioinf/MemBrain/

A Combination of Compositional Index and Genetic Algorithm for Predicting Transmembrane Helical Segments

Transmembrane helix (TMH) topology prediction is becoming a focal problem in bioinformatics because the structure of TM proteins is difficult to determine using experimental methods. Therefore, methods that can computationally predict the topology of helical membrane proteins are highly desirable. In this paper we introduce TMHindex, a method for detecting TMH segments using only the amino acid sequence information. Each amino acid in a protein sequence is represented by a Compositional Index, which is deduced from a combination of the difference in amino acid occurrences in TMH and non-TMH segments in training protein sequences and the amino acid composition information. Furthermore, a genetic algorithm was employed to find the optimal threshold value for the separation of TMH segments from non-TMH segments. The method successfully predicted 376 out of the 378 TMH segments in a dataset consisting of 70 test protein sequences. The sensitivity and specificity for classifying each amino acid in every protein sequence in the dataset was 0.901 and 0.865, respectively. To assess the generality of TMHindex, we also tested the approach on another standard 73-protein 3D helix dataset. TMHindex correctly predicted 91.8% of proteins based on TM segments. The level of the accuracy achieved using TMHindex in comparison to other recent approaches for predicting the topology of TM proteins is a strong argument in favor of our proposed method. Availability: The datasets, software together with supplementary materials are available at: http://faculty.uaeu.ac.ae/nzaki/TMHindex.htm

Localization of Human RNase Z Isoforms: Dual Nuclear/Mitochondrial Targeting of the ELAC2 Gene Product by Alternative Translation Initiation

RNase Z is an endonuclease responsible for the removal of 3′ extensions from tRNA precursors, an essential step in tRNA biogenesis. Human cells contain a long form (RNase ZL) encoded by ELAC2, and a short form (RNase ZS; ELAC1). We studied their subcellular localization by expression of proteins fused to green fluorescent protein. RNase ZS was found in the cytosol, whereas RNase ZL localized to the nucleus and mitochondria. We show that alternative translation initiation is responsible for the dual targeting of RNase ZL. Due to the unfavorable context of the first AUG of ELAC2, translation apparently also starts from the second AUG, whereby the mitochondrial targeting sequence is lost and the protein is instead routed to the nucleus. Our data suggest that RNase ZL is the enzyme involved in both, nuclear and mitochondrial tRNA 3′ end maturation

Recovery and resilience of tropical forests after disturbance

The time taken for forested tropical ecosystems to re-establish post-disturbance is of widespread interest. Yet to date there has been no comparative study across tropical biomes to determine rates of forest re-growth, and how they vary through space and time. Here we present results from a meta-analysis of palaeoecological records that use fossil pollen as a proxy for vegetation change over the past 20,000 years. A total of 283 forest disturbance and recovery events, reported in 71 studies, are identified across four tropical regions. Results indicate that forests in Central America and Africa generally recover faster from past disturbances than those in South America and Asia, as do forests exposed to natural large infrequent disturbances compared with post-climatic and human impacts. Results also demonstrate that increasing frequency of disturbance events at a site through time elevates recovery rates, indicating a degree of resilience in forests exposed to recurrent past disturbance

Integrative Identification of Arabidopsis Mitochondrial Proteome and Its Function Exploitation through Protein Interaction Network

Mitochondria are major players on the production of energy, and host several key reactions involved in basic metabolism and biosynthesis of essential molecules. Currently, the majority of nucleus-encoded mitochondrial proteins are unknown even for model plant Arabidopsis. We reported a computational framework for predicting Arabidopsis mitochondrial proteins based on a probabilistic model, called Naive Bayesian Network, which integrates disparate genomic data generated from eight bioinformatics tools, multiple orthologous mappings, protein domain properties and co-expression patterns using 1,027 microarray profiles. Through this approach, we predicted 2,311 candidate mitochondrial proteins with 84.67% accuracy and 2.53% FPR performances. Together with those experimental confirmed proteins, 2,585 mitochondria proteins (named CoreMitoP) were identified, we explored those proteins with unknown functions based on protein-protein interaction network (PIN) and annotated novel functions for 26.65% CoreMitoP proteins. Moreover, we found newly predicted mitochondrial proteins embedded in particular subnetworks of the PIN, mainly functioning in response to diverse environmental stresses, like salt, draught, cold, and wound etc. Candidate mitochondrial proteins involved in those physiological acitivites provide useful targets for further investigation. Assigned functions also provide comprehensive information for Arabidopsis mitochondrial proteome

A Mutation in Intracellular Loop 4 Affects the Drug-Efflux Activity of the Yeast Multidrug Resistance ABC Transporter Pdr5p

Multidrug resistance protein Pdr5p is a yeast ATP-binding cassette (ABC) transporter in the plasma membrane. It confers multidrug resistance by active efflux of intracellular drugs. However, the highly polymorphic Pdr5p from clinical strain YJM789 loses its ability to expel azole and cyclohexmide. To investigate the role of amino acid changes in this functional change, PDR5 chimeras were constructed by segmental replacement of homologous BY4741 PDR5 fragments. Functions of PDR5 chimeras were evaluated by fluconazole and cycloheximide resistance assays. Their expression, ATPase activity, and efflux efficiency for other substrates were also analyzed. Using multiple lines of evidence, we show that an alanine-to-methionine mutation at position 1352 located in the predicted short intracellular loop 4 significantly contributes to the observed transport deficiency. The degree of impairment is likely correlated to the size of the mutant residue