The complex impact of cancer-related missense mutations on the stability and on the biophysical and biochemical properties of MAPK1 and MAPK3 somatic variants.

Mitogen-activated protein kinases 1 and 3 (MAPK1 and MAPK3), also called extracellular regulated kinases (ERK2 and ERK1), are serine/threonine kinase activated downstream by the Ras/Raf/MEK/ERK signal transduction cascade that regulates a variety of cellular processes. A dysregulation of MAPK cascade is frequently associated to missense mutations on its protein components and may be related to many pathologies, including cancer. In this study we selected from COSMIC database a set of MAPK1 and MAPK3 somatic variants found in cancer tissues carrying missense mutations distributed all over the MAPK1 and MAPK3 sequences. The proteins were expressed as pure recombinant proteins, and their biochemical and biophysical properties have been studied in comparison with the wild type. The missense mutations lead to changes in the tertiary arrangements of all the variants. The thermodynamic stability of the wild type and variants has been investigated in the non-phosphorylated and in the phosphorylated form. Significant differences in the thermal stabilities of most of the variants have been observed, as well as changes in the catalytic efficiencies. The energetics of the catalytic reaction is affected for all the variants for both the MAPK proteins. The stability changes and the variation in the enzyme catalysis observed for most of MAPK1/3 variants suggest that a local change in a residue, distant from the catalytic site, may have long-distance effects that reflect globally on enzyme stability and functions.

K-Pro: Kinetics Data on Proteins and Mutants.

The study of protein folding plays a crucial role in improving our understanding of protein function and of the relationship between genetics and phenotypes. In particular, understanding the thermodynamics and kinetics of the folding process is important for uncovering the mechanisms behind human disorders caused by protein misfolding. To address this issue, it is essential to collect and curate experimental kinetic and thermodynamic data on protein folding. K-Pro is a new database designed for collecting and storing experimental kinetic data on monomeric proteins, with a two-state folding mechanism. With 1,529 records from 62 proteins corresponding to 65 structures, K-Pro contains various kinetic parameters such as the logarithm of the folding and unfolding rates, Tanford's ß and the ϕ values. When available, the database also includes thermodynamic parameters associated with the kinetic data. K-Pro features a user-friendly interface that allows browsing and downloading kinetic data of interest. The graphical interface provides a visual representation of the protein and mutants, and it is cross-linked to key databases such as PDB, UniProt, and PubMed. K-Pro is open and freely accessible through https://folding.biofold.org/k-pro and supports the latest versions of popular browsers.

Identification of Driver Epistatic Gene Pairs Combining Germline and Somatic Mutations in Cancer.

Cancer arises from the complex interplay of various factors. Traditionally, the identification of driver genes focuses primarily on the analysis of somatic mutations. We describe a new method for the detection of driver gene pairs based on an epistasis analysis that considers both germline and somatic variations. Specifically, the identification of significantly mutated gene pairs entails the calculation of a contingency table, wherein one of the co-mutated genes can exhibit a germline variant. By adopting this approach, it is possible to select gene pairs in which the individual genes do not exhibit significant associations with cancer. Finally, a survival analysis is used to select clinically relevant gene pairs. To test the efficacy of the new algorithm, we analyzed the colon adenocarcinoma (COAD) and lung adenocarcinoma (LUAD) samples available at The Cancer Genome Atlas (TCGA). In the analysis of the COAD and LUAD samples, we identify epistatic gene pairs significantly mutated in tumor tissue with respect to normal tissue. We believe that further analysis of the gene pairs detected by our method will unveil new biological insights, enhancing a better description of the cancer mechanism.

PhD-SNPg: updating a webserver and lightweight tool for scoring nucleotide variants.

One of the primary challenges in human genetics is determining the functional impact of single nucleotide variants (SNVs) and insertion and deletions (InDels), whether coding or noncoding. In the past, methods have been created to detect disease-related single amino acid changes, but only some can assess the influence of noncoding variations. CADD is the most commonly used and advanced algorithm for predicting the diverse effects of genome variations. It employs a combination of sequence conservation and functional features derived from the ENCODE project data. To use CADD, a large set of pre-calculated information must be downloaded during the installation process. To streamline the variant annotation process, we developed PhD-SNPg, a machine-learning tool that is easy to install and lightweight, relying solely on sequence-based features. Here we present an updated version, trained on a larger dataset, that can also predict the impact of the InDel variations. Despite its simplicity, PhD-SNPg performs similarly to CADD, making it ideal for rapid genome interpretation and as a benchmark for tool development.

Resources and tools for rare disease variant interpretation.

Collectively, rare genetic disorders affect a substantial portion of the world's population. In most cases, those affected face difficulties in receiving a clinical diagnosis and genetic characterization. The understanding of the molecular mechanisms of these diseases and the development of therapeutic treatments for patients are also challenging. However, the application of recent advancements in genome sequencing/analysis technologies and computer-aided tools for predicting phenotype-genotype associations can bring significant benefits to this field. In this review, we highlight the most relevant online resources and computational tools for genome interpretation that can enhance the diagnosis, clinical management, and development of treatments for rare disorders. Our focus is on resources for interpreting single nucleotide variants. Additionally, we present use cases for interpreting genetic variants in clinical settings and review the limitations of these results and prediction tools. Finally, we have compiled a curated set of core resources and tools for analyzing rare disease genomes. Such resources and tools can be utilized to develop standardized protocols that will enhance the accuracy and effectiveness of rare disease diagnosis.

Turning Failures into Applications: The Problem of Protein ΔΔG Prediction.

After nearly two decades of research in the field of computational methods based on machine learning and knowledge-based potentials for ΔG and ΔΔG prediction upon variations, we now realize that all the approaches are poorly performing when tested on specific cases and that there is large space for improvement. Why this is so? Is it wrong the underlying assumption that experimental protein thermodynamics in solution reflects the thermodynamics of a single protein? Both machine learning and knowledge-based computational methods are rigorous and we know the solid theory behind. We are now in a critical situation, which suggests that predictions of protein instability upon variation should be considered with care. In the following, we will show how to cope with the problem of understanding which protein positions may be of interest for biotechnological and biomedical purposes. By applying a consensus procedure, we indicate possible strategies for the result interpretation.

DDGun: an untrained predictor of protein stability changes upon amino acid variants.

Estimating the functional effect of single amino acid variants in proteins is fundamental for predicting the change in the thermodynamic stability, measured as the difference in the Gibbs free energy of unfolding, between the wild-type and the variant protein (ΔΔG). Here, we present the web-server of the DDGun method, which was previously developed for the ΔΔG prediction upon amino acid variants. DDGun is an untrained method based on basic features derived from evolutionary information. It is antisymmetric, as it predicts opposite ΔΔG values for direct (A → B) and reverse (B → A) single and multiple site variants. DDGun is available in two versions, one based on only sequence information and the other one based on sequence and structure information. Despite being untrained, DDGun reaches prediction performances comparable to those of trained methods. Here we make DDGun available as a web server. For the web server version, we updated the protein sequence database used for the computation of the evolutionary features, and we compiled two new data sets of protein variants to do a blind test of its performances. On these blind data sets of single and multiple site variants, DDGun confirms its prediction performance, reaching an average correlation coefficient between experimental and predicted ΔΔG of 0.45 and 0.49 for the sequence-based and structure-based versions, respectively. Besides being used for the prediction of ΔΔG, we suggest that DDGun should be adopted as a benchmark method to assess the predictive capabilities of newly developed methods. Releasing DDGun as a web-server, stand-alone program and docker image will facilitate the necessary process of method comparison to improve ΔΔG prediction.

Evaluating the relevance of sequence conservation in the prediction of pathogenic missense variants.

Evolutionary information is the primary tool for detecting functional conservation in nucleic acid and protein. This information has been extensively used to predict structure, interactions and functions in macromolecules. Pathogenicity prediction models rely on multiple sequence alignment information at different levels. However, most accurate genome-wide variant deleteriousness ranking algorithms consider different features to assess the impact of variants. Here, we analyze three different ways of extracting evolutionary information from sequence alignments in the context of pathogenicity predictions at DNA and protein levels. We showed that protein sequence-based information is slightly more informative in the annotation of Clinvar missense variants than those obtained at the DNA level. Furthermore, to achieve the performance of state-of-the-art methods, such as CADD and REVEL, the conservation of reference and variant, encoded as frequencies of reference/alternate alleles or wild-type/mutant residues, should be included. Our results on a large set of missense variants show that a basic method based on three input features derived from the protein sequence profile performs similarly to the CADD algorithm which uses hundreds of genomic features. As expected, our method results in ~ 3% lower area under the receiver-operating characteristic curve (AUC). When compared with an ensemble-based algorithm (REVEL). Nevertheless, the combination of predictions of multiple methods can help to identify more reliable predictions. These observations indicate that for missense variants, evolutionary information, when properly encoded, plays the primary role in ranking pathogenicity.

Predicting protein stability changes upon single-point mutation: a thorough comparison of the available tools on a new dataset.

Predicting the difference in thermodynamic stability between protein variants is crucial for protein design and understanding the genotype-phenotype relationships. So far, several computational tools have been created to address this task. Nevertheless, most of them have been trained or optimized on the same and 'all' available data, making a fair comparison unfeasible. Here, we introduce a novel dataset, collected and manually cleaned from the latest version of the ThermoMutDB database, consisting of 669 variants not included in the most widely used training datasets. The prediction performance and the ability to satisfy the antisymmetry property by considering both direct and reverse variants were evaluated across 21 different tools. The Pearson correlations of the tested tools were in the ranges of 0.21-0.5 and 0-0.45 for the direct and reverse variants, respectively. When both direct and reverse variants are considered, the antisymmetric methods perform better achieving a Pearson correlation in the range of 0.51-0.62. The tested methods seem relatively insensitive to the physiological conditions, performing well also on the variants measured with more extreme pH and temperature values. A common issue with all the tested methods is the compression of the $\Delta \Delta G$ predictions toward zero. Furthermore, the thermodynamic stability of the most significantly stabilizing variants was found to be more challenging to predict. This study is the most extensive comparisons of prediction methods using an entirely novel set of variants never tested before.

Challenges in predicting stabilizing variations: An exploration.

An open challenge of computational and experimental biology is understanding the impact of non-synonymous DNA variations on protein function and, subsequently, human health. The effects of these variants on protein stability can be measured as the difference in the free energy of unfolding (ΔΔG) between the mutated structure of the protein and its wild-type form. Throughout the years, bioinformaticians have developed a wide variety of tools and approaches to predict the ΔΔG. Although the performance of these tools is highly variable, overall they are less accurate in predicting ΔΔG stabilizing variations rather than the destabilizing ones. Here, we analyze the possible reasons for this difference by focusing on the relationship between experimentally-measured ΔΔG and seven protein properties on three widely-used datasets (S2648, VariBench, Ssym) and a recently introduced one (S669). These properties include protein structural information, different physical properties and statistical potentials. We found that two highly used input features, i.e., hydrophobicity and the Blosum62 substitution matrix, show a performance close to random choice when trying to separate stabilizing variants from either neutral or destabilizing ones. We then speculate that, since destabilizing variations are the most abundant class in the available datasets, the overall performance of the methods is higher when including features that improve the prediction for the destabilizing variants at the expense of the stabilizing ones. These findings highlight the need of designing predictive methods able to exploit also input features highly correlated with the stabilizing variants. New tools should also be tested on a not-artificially balanced dataset, reporting the performance on all the three classes (i.e., stabilizing, neutral and destabilizing variants) and not only the overall results.

Network-based strategies for protein characterization.

Protein structure characterization is fundamental to understand protein properties, such as folding process and protein resistance to thermal stress, up to unveiling organism pathologies (e.g., prion disease). In this chapter, we provide an overview on how the spectral properties of the networks reconstructed from the Protein Contact Map (PCM) can be used to generate informative observables. As a specific case study, we apply two different network approaches to an example protein dataset, for the aim of discriminating protein folding state, and for the reconstruction of protein 3D structure.

A Deep-Learning Sequence-Based Method to Predict Protein Stability Changes Upon Genetic Variations.

Several studies have linked disruptions of protein stability and its normal functions to disease. Therefore, during the last few decades, many tools have been developed to predict the free energy changes upon protein residue variations. Most of these methods require both sequence and structure information to obtain reliable predictions. However, the lower number of protein structures available with respect to their sequences, due to experimental issues, drastically limits the application of these tools. In addition, current methodologies ignore the antisymmetric property characterizing the thermodynamics of the protein stability: a variation from wild-type to a mutated form of the protein structure (XW→XM) and its reverse process (XM→XW) must have opposite values of the free energy difference (ΔΔGWM=-ΔΔGMW). Here we propose ACDC-NN-Seq, a deep neural network system that exploits the sequence information and is able to incorporate into its architecture the antisymmetry property. To our knowledge, this is the first convolutional neural network to predict protein stability changes relying solely on the protein sequence. We show that ACDC-NN-Seq compares favorably with the existing sequence-based methods.

Analysis and Interpretation of the Impact of Missense Variants in Cancer.

Large scale genome sequencing allowed the identification of a massive number of genetic variations, whose impact on human health is still unknown. In this review we analyze, by an in silico-based strategy, the impact of missense variants on cancer-related genes, whose effect on protein stability and function was experimentally determined. We collected a set of 164 variants from 11 proteins to analyze the impact of missense mutations at structural and functional levels, and to assess the performance of state-of-the-art methods (FoldX and Meta-SNP) for predicting protein stability change and pathogenicity. The result of our analysis shows that a combination of experimental data on protein stability and in silico pathogenicity predictions allowed the identification of a subset of variants with a high probability of having a deleterious phenotypic effect, as confirmed by the significant enrichment of the subset in variants annotated in the COSMIC database as putative cancer-driving variants. Our analysis suggests that the integration of experimental and computational approaches may contribute to evaluate the risk for complex disorders and develop more effective treatment strategies.

ThermoScan: Semi-automatic Identification of Protein Stability Data From PubMed.

During the last years, the increasing number of DNA sequencing and protein mutagenesis studies has generated a large amount of variation data published in the biomedical literature. The collection of such data has been essential for the development and assessment of tools predicting the impact of protein variants at functional and structural levels. Nevertheless, the collection of manually curated data from literature is a highly time consuming and costly process that requires domain experts. In particular, the development of methods for predicting the effect of amino acid variants on protein stability relies on the thermodynamic data extracted from literature. In the past, such data were deposited in the ProTherm database, which however is no longer maintained since 2013. For facilitating the collection of protein thermodynamic data from literature, we developed the semi-automatic tool ThermoScan. ThermoScan is a text mining approach for the identification of relevant thermodynamic data on protein stability from full-text articles. The method relies on a regular expression searching for groups of words, including the most common conceptual words appearing in experimental studies on protein stability, several thermodynamic variables, and their units of measure. ThermoScan analyzes full-text articles from the PubMed Central Open Access subset and calculates an empiric score that allows the identification of manuscripts reporting thermodynamic data on protein stability. The method was optimized on a set of publications included in the ProTherm database, and tested on a new curated set of articles, manually selected for presence of thermodynamic data. The results show that ThermoScan returns accurate predictions and outperforms recently developed text-mining algorithms based on the analysis of publication abstracts. Availability: The ThermoScan server is freely accessible online at https://folding.biofold.org/thermoscan. The ThermoScan python code and the Google Chrome extension for submitting visualized PMC web pages to the ThermoScan server are available at https://github.com/biofold/ThermoScan.

Protein Stability Perturbation Contributes to the Loss of Function in Haploinsufficient Genes.

Missense variants are among the most studied genome modifications as disease biomarkers. It has been shown that the "perturbation" of the protein stability upon a missense variant (in terms of absolute ΔΔG value, i.e., |ΔΔG|) has a significant, but not predictive, correlation with the pathogenicity of that variant. However, here we show that this correlation becomes significantly amplified in haploinsufficient genes. Moreover, the enrichment of pathogenic variants increases at the increasing protein stability perturbation value. These findings suggest that protein stability perturbation might be considered as a potential cofactor in diseases associated with haploinsufficient genes reporting missense variants.

Calibrating variant-scoring methods for clinical decision making.

SUMMARY: Identifying pathogenic variants and annotating them is a major challenge in human genetics, especially for the non-coding ones. Several tools have been developed and used to predict the functional effect of genetic variants. However, the calibration assessment of the predictions has received little attention. Calibration refers to the idea that if a model predicts a group of variants to be pathogenic with a probability P, it is expected that the same fraction P of true positive is found in the observed set. For instance, a well-calibrated classifier should label the variants such that among the ones to which it gave a probability value close to 0.7, approximately 70% actually belong to the pathogenic class. Poorly calibrated algorithms can be misleading and potentially harmful for clinical decision making. AVALIABILITY AND IMPLEMENTATION: The dataset used for testing the methods is available through the DOI:10.5281/zenodo.4448197. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Limitations and challenges in protein stability prediction upon genome variations: towards future applications in precision medicine.

Protein stability predictions are becoming essential in medicine to develop novel immunotherapeutic agents and for drug discovery. Despite the large number of computational approaches for predicting the protein stability upon mutation, there are still critical unsolved problems: 1) the limited number of thermodynamic measurements for proteins provided by current databases; 2) the large intrinsic variability of ΔΔG values due to different experimental conditions; 3) biases in the development of predictive methods caused by ignoring the anti-symmetry of ΔΔG values between mutant and native protein forms; 4) over-optimistic prediction performance, due to sequence similarity between proteins used in training and test datasets. Here, we review these issues, highlighting new challenges required to improve current tools and to achieve more reliable predictions. In addition, we provide a perspective of how these methods will be beneficial for designing novel precision medicine approaches for several genetic disorders caused by mutations, such as cancer and neurodegenerative diseases.

Assessing computational predictions of the phenotypic effect of cystathionine-beta-synthase variants.

Accurate prediction of the impact of genomic variation on phenotype is a major goal of computational biology and an important contributor to personalized medicine. Computational predictions can lead to a better understanding of the mechanisms underlying genetic diseases, including cancer, but their adoption requires thorough and unbiased assessment. Cystathionine-beta-synthase (CBS) is an enzyme that catalyzes the first step of the transsulfuration pathway, from homocysteine to cystathionine, and in which variations are associated with human hyperhomocysteinemia and homocystinuria. We have created a computational challenge under the CAGI framework to evaluate how well different methods can predict the phenotypic effect(s) of CBS single amino acid substitutions using a blinded experimental data set. CAGI participants were asked to predict yeast growth based on the identity of the mutations. The performance of the methods was evaluated using several metrics. The CBS challenge highlighted the difficulty of predicting the phenotype of an ex vivo system in a model organism when classification models were trained on human disease data. We also discuss the variations in difficulty of prediction for known benign and deleterious variants, as well as identify methodological and experimental constraints with lessons to be learned for future challenges.

DDGun: an untrained method for the prediction of protein stability changes upon single and multiple point variations.

BACKGROUND: Predicting the effect of single point variations on protein stability constitutes a crucial step toward understanding the relationship between protein structure and function. To this end, several methods have been developed to predict changes in the Gibbs free energy of unfolding (∆∆G) between wild type and variant proteins, using sequence and structure information. Most of the available methods however do not exhibit the anti-symmetric prediction property, which guarantees that the predicted ∆∆G value for a variation is the exact opposite of that predicted for the reverse variation, i.e., ∆∆G(A → B) = -∆∆G(B → A), where A and B are amino acids. RESULTS: Here we introduce simple anti-symmetric features, based on evolutionary information, which are combined to define an untrained method, DDGun (DDG untrained). DDGun is a simple approach based on evolutionary information that predicts the ∆∆G for single and multiple variations from sequence and structure information (DDGun3D). Our method achieves remarkable performance without any training on the experimental datasets, reaching Pearson correlation coefficients between predicted and measured ∆∆G values of ~ 0.5 and ~ 0.4 for single and multiple site variations, respectively. Surprisingly, DDGun performances are comparable with those of state of the art methods. DDGun also naturally predicts multiple site variations, thereby defining a benchmark method for both single site and multiple site predictors. DDGun is anti-symmetric by construction predicting the value of the ∆∆G of a reciprocal variation as almost equal (depending on the sequence profile) to -∆∆G of the direct variation. This is a valuable property that is missing in the majority of the methods. CONCLUSIONS: Evolutionary information alone combined in an untrained method can achieve remarkably high performances in the prediction of ∆∆G upon protein mutation. Non-trained approaches like DDGun represent a valid benchmark both for scoring the predictive power of the individual features and for assessing the learning capability of supervised methods.