Proteome-wide Structural Analysis of PTM Hotspots Reveals Regulatory Elements Predicted to Impact Biological Function and Disease.

Post-translational modifications (PTMs) regulate protein behavior through modulation of protein-protein interactions, enzymatic activity, and protein stability essential in the translation of genotype to phenotype in eukaryotes. Currently, less than 4% of all eukaryotic PTMs are reported to have biological function - a statistic that continues to decrease with an increasing rate of PTM detection. Previously, we developed SAPH-ire (Structural Analysis of PTM Hotspots) - a method for the prioritization of PTM function potential that has been used effectively to reveal novel PTM regulatory elements in discrete protein families (Dewhurst et al., 2015). Here, we apply SAPH-ire to the set of eukaryotic protein families containing experimental PTM and 3D structure data - capturing 1,325 protein families with 50,839 unique PTM sites organized into 31,747 modified alignment positions (MAPs), of which 2010 (∼6%) possess known biological function. Here, we show that using an artificial neural network model (SAPH-ire NN) trained to identify MAP hotspots with biological function results in prediction outcomes that far surpass the use of single hotspot features, including nearest neighbor PTM clustering methods. We find the greatest enhancement in prediction for positions with PTM counts of five or less, which represent 98% of all MAPs in the eukaryotic proteome and 90% of all MAPs found to have biological function. Analysis of the top 1092 MAP hotspots revealed 267 of truly unknown function (containing 5443 distinct PTMs). Of these, 165 hotspots could be mapped to human KEGG pathways for normal and/or disease physiology. Many high-ranking hotspots were also found to be disease-associated pathogenic sites of amino acid substitution despite the lack of observable PTM in the human protein family member. Taken together, these experiments demonstrate that the functional relevance of a PTM can be predicted very effectively by neural network models, revealing a large but testable body of potential regulatory elements that impact hundreds of different biological processes important in eukaryotic biology and human health.

Structural Analysis of PTM Hotspots (SAPH-ire)--A Quantitative Informatics Method Enabling the Discovery of Novel Regulatory Elements in Protein Families.

Predicting the biological function potential of post-translational modifications (PTMs) is becoming increasingly important in light of the exponential increase in available PTM data from high-throughput proteomics. We developed structural analysis of PTM hotspots (SAPH-ire)--a quantitative PTM ranking method that integrates experimental PTM observations, sequence conservation, protein structure, and interaction data to allow rank order comparisons within or between protein families. Here, we applied SAPH-ire to the study of PTMs in diverse G protein families, a conserved and ubiquitous class of proteins essential for maintenance of intracellular structure (tubulins) and signal transduction (large and small Ras-like G proteins). A total of 1728 experimentally verified PTMs from eight unique G protein families were clustered into 451 unique hotspots, 51 of which have a known and cited biological function or response. Using customized software, the hotspots were analyzed in the context of 598 unique protein structures. By comparing distributions of hotspots with known versus unknown function, we show that SAPH-ire analysis is predictive for PTM biological function. Notably, SAPH-ire revealed high-ranking hotspots for which a functional impact has not yet been determined, including phosphorylation hotspots in the N-terminal tails of G protein gamma subunits--conserved protein structures never before reported as regulators of G protein coupled receptor signaling. To validate this prediction we used the yeast model system for G protein coupled receptor signaling, revealing that gamma subunit-N-terminal tail phosphorylation is activated in response to G protein coupled receptor stimulation and regulates protein stability in vivo. These results demonstrate the utility of integrating protein structural and sequence features into PTM prioritization schemes that can improve the analysis and functional power of modification-specific proteomics data.

Systematic analysis of non-structural protein features for the prediction of PTM function potential by artificial neural networks.

Post-translational modifications (PTMs) provide an extensible framework for regulation of protein behavior beyond the diversity represented within the genome alone. While the rate of identification of PTMs has rapidly increased in recent years, our knowledge of PTM functionality encompasses less than 5% of this data. We previously developed SAPH-ire (Structural Analysis of PTM Hotspots) for the prioritization of eukaryotic PTMs based on function potential of discrete modified alignment positions (MAPs) in a set of 8 protein families. A proteome-wide expansion of the dataset to all families of PTM-bearing, eukaryotic proteins with a representational crystal structure and the application of artificial neural network (ANN) models demonstrated the broader applicability of this approach. Although structural features of proteins have been repeatedly demonstrated to be predictive of PTM functionality, the availability of adequately resolved 3D structures in the Protein Data Bank (PDB) limits the scope of these methods. In order to bridge this gap and capture the larger set of PTM-bearing proteins without an available, homologous structure, we explored all available MAP features as ANN inputs to identify predictive models that do not rely on 3D protein structural data. This systematic, algorithmic approach explores 8 available input features in exhaustive combinations (247 models; size 2-8). To control for potential bias in random sampling for holdback in training sets, we iterated each model across 100 randomized, sample training and testing sets-yielding 24,700 individual ANNs. The size of the analyzed dataset and iterative generation of ANNs represents the largest and most thorough investigation of predictive models for PTM functionality to date. Comparison of input layer combinations allows us to quantify ANN performance with a high degree of confidence and subsequently select a top-ranked, robust fit model which highlights 3,687 MAPs, including 10,933 PTMs with a high probability of biological impact but without a currently known functional role.