Protein function annotation by homology-based inference

With many genomes now sequenced, computational annotation methods to characterize genes and proteins from their sequence are increasingly important. The BioSapiens Network has developed tools to address all stages of this process, and here we review progress in the automated prediction of protein function based on protein sequence and structure.     

Exploiting protein structure data to explore the evolution of protein function and biological complexity

New directions in biology are being driven by the complete sequencing of genomes, which has given us the protein repertoires of diverse organisms from all kingdoms of life. In tandem with this accumulation of sequence data, worldwide structural genomics initiatives, advanced by the development of improved technologies in X-ray crystallography and NMR, are expanding our knowledge of structural families and increasing our fold libraries. Methods for detecting remote sequence similarities have also been made more sensitive and this means that we can map domains from these structural families onto genome sequences to understand how these families are distributed throughout the genomes and reveal how they might influence the functional repertoires and biological complexities of the organisms. We have used robust protocols to assign sequences from completed genomes to domain structures in the CATH database, allowing up to 60% of domain sequences in these genomes, depending on the organism, to be assigned to a domain family of known structure. Analysis of the distribution of these families throughout bacterial genomes identified more than 300 universal families, some of which had expanded significantly in proportion to genome size. These highly expanded families are primarily involved in metabolism and regulation and appear to make major contributions to the functional repertoire and complexity of bacterial organisms. When comparisons are made across all kingdoms of life, we find a smaller set of universal domain families (approx. 140), of which families involved in protein biosynthesis are the largest conserved component. Analysis of the behaviour of other families reveals that some (e.g. those involved in metabolism, regulation) have remained highly innovative during evolution, making it harder to trace their evolutionary ancestry. Structural analyses of metabolic families provide some insights into the mechanisms of functional innovation, which include changes in domain partnerships and significant structural embellishments leading to modulation of active sites and protein interactions.     

High-throughput staining for the evaluation of fetal skeletal development in rats and rabbits

Typical developmental toxicity studies require the assessment of fetal skeletal development. Regulatory guidelines require the assessment of bone ossification and indicate preferences for an assessment of both ossified bone as well as cartilaginous elements. Current manual methods to process fetuses for skeletal examination, whether single or double staining, are laborious and time consuming, and ultimately extend the time before study interpretations. There is a definite need for a quick and efficient, yet reliable, procedure to generate stained fetal skeletons for analysis. A non-automated high-throughput method for single and double staining rat and rabbit fetuses for skeletal evaluations is described, which results in excellent quality specimens ready for evaluations in approximately 3 days for rats and 7 days for rabbits.     

Synthesis and CYP26A1 inhibitory activity of novel methyl 3-[4-(arylamino)phenyl]-3-(azole)-2,2-dimethylpropanoates

The role of all-trans-retinoic acid (ATRA) in the development and maintenance of many epithelial and neural tissues has raised great interest in the potential of ATRA and related compounds (retinoids) as pharmacological agents, particularly for the treatment of cancer, skin, neurodegenerative and autoimmune diseases. The use of ATRA or prodrugs as pharmacological agents is limited by a short half-life in vivo resulting from the activity of specific ATRA hydroxylases, CYP26 enzymes, induced by ATRA in liver and target tissues. For this reason retinoic acid metabolism blocking agents (RAMBAs) have been developed for treating cancer and a wide range of other diseases.The synthesis, CYP26A1 inhibitory activity and molecular modeling studies of novel methyl 3-[4-(arylamino)phenyl]-3-(azole)-2,2-dimethylpropanoates are presented. From this series of compounds clear SAR can be derived for 4-substitution of the phenyl ring with electron-donating groups more favourable for inhibitory activity. Both the methylenedioxyphenyl imidazole (17, IC₅₀ = 8 nM) and triazole (18, IC₅₀ = 6.7 nM) derivatives were potent inhibitors with additional binding interactions between the methylenedioxy moiety and the CYP26 active site likely to be the main factor. The 6-bromo-3-pyridine imidazole 15 (IC₅₀ = 5.7 nM) was the most active from this series compared with the standards liarozole (IC₅₀ = 540 nM) and R116010 (IC₅₀ = 10 nM).     

FLORA: A Novel Method to Predict Protein Function from Structure in Diverse Superfamilies

Predicting protein function from structure remains an active area of interest, particularly for the structural genomics initiatives where a substantial number of structures are initially solved with little or no functional characterisation. Although global structure comparison methods can be used to transfer functional annotations, the relationship between fold and function is complex, particularly in functionally diverse superfamilies that have evolved through different secondary structure embellishments to a common structural core. The majority of prediction algorithms employ local templates built on known or predicted functional residues. Here, we present a novel method (FLORA) that automatically generates structural motifs associated with different functional sub-families (FSGs) within functionally diverse domain superfamilies. Templates are created purely on the basis of their specificity for a given FSG, and the method makes no prior prediction of functional sites, nor assumes specific physico-chemical properties of residues. FLORA is able to accurately discriminate between homologous domains with different functions and substantially outperforms (a 2–3 fold increase in coverage at low error rates) popular structure comparison methods and a leading function prediction method. We benchmark FLORA on a large data set of enzyme superfamilies from all three major protein classes (α, β, αβ) and demonstrate the functional relevance of the motifs it identifies. We also provide novel predictions of enzymatic activity for a large number of structures solved by the Protein Structure Initiative. Overall, we show that FLORA is able to effectively detect functionally similar protein domain structures by purely using patterns of structural conservation of all residues.     

A mutant form of PTEN linked to autism

The tumor suppressor, phosphatase, and tensin homologue deleted on chromosome 10 (PTEN), is a phosphoinositide (PI) phosphatase specific for the 3‐position of the inositol ring. PTEN has been implicated in autism for a subset of patients with macrocephaly. Various studies identified patients in this subclass with one normal and one mutated PTEN gene. We characterize the binding, structural properties, activity, and subcellular localization of one of these autism‐related mutants, H93R PTEN. Even though this mutation is located at the phosphatase active site, we find that it affects the functions of neighboring domains. H93R PTEN binding to phosphatidylserine‐bearing model membranes is 5.6‐fold enhanced in comparison to wild‐type PTEN. In contrast, we find that binding to phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P₂) model membranes is 2.5‐fold decreased for the mutant PTEN in comparison to wild‐type PTEN. The structural change previously found for wild‐type PTEN upon interaction with PI(4,5)P₂, is absent for H93R PTEN. Consistent with the increased binding to phosphatidylserine, we find enhanced plasma membrane association of PTEN‐GFP in U87MG cells. However, this enhanced plasma membrane association does not translate into increased PI(3,4,5)P₃ turnover, since in vivo studies show a reduced activity of the H93R PTEN‐GFP mutant. Because the interaction of PI(4,5)P₂ with PTEN's N‐terminal domain is diminished by this mutation, we hypothesize that the interaction of PTEN's N‐terminal domain with the phosphatase domain is impacted by the H93R mutation, preventing PI(4,5)P₂ from inducing the conformational change that activates phosphatase activity.     

Reliability and precision of EMG in leg,torso, and arm muscles during running

Changes in electromyographic (EMG) parameters are used to evaluate timing, amplitude, and fatigue of muscle actions during movement. Little published data describe the reliability and precision of multiple EMG parameters, how these parameters compare to one another, and how these parameters vary between muscles. The purpose of this study was to determine the reliability and precision of four EMG parameters recorded from the legs, torso, and arm muscles during running. Fifteen well-trained male runners performed moderate-intensity treadmill running while EMG data were collected from thirteen muscles. Integrated EMG (iEMG), root mean square EMG (RMS), maximum M-wave, and median power frequency (MPF) were calculated for 25 consecutive strides. Intra-class correlation coefficients (ICC) and standard error of measurement (SEM) for each parameter were calculated for each muscle. Seven muscles displayed good reliability (ICC > 0.80) for all parameters studied. MPF was the most reliable variable, with 12 muscles having ICC > 0.80 and <6% normalized SEM. Reliability and precision differed between muscles of similar function and anatomic region. These data emphasize the need for researchers and clinicians to have reliability and precision measures for all parameters of each muscle, and demonstrates that generalizations must be used cautiously when interpreting EMG data collected during running.