Exploring the structural landscape of DNA maintenance proteins.

Evolutionary annotation of genome maintenance (GM) proteins has conventionally been established by remote relationships within protein sequence databases. However, often no significant relationship can be established. Highly sensitive approaches to attain remote homologies based on iterative profile-to-profile methods have been developed. Still, these methods have not been systematically applied in the evolutionary annotation of GM proteins. Here, by applying profile-to-profile models, we systematically survey the repertoire of GM proteins from bacteria to man. We identify multiple GM protein candidates and annotate domains in numerous established GM proteins, among other PARP, OB-fold, Macro, TUDOR, SAP, BRCT, KU, MYB (SANT), and nuclease domains. We experimentally validate OB-fold and MIS18 (Yippee) domains in SPIDR and FAM72 protein families, respectively. Our results indicate that, surprisingly, despite the immense interest and long-term research efforts, the repertoire of genome stability caretakers is still not fully appreciated.

Acidic sphingomyelinase interactions with lysosomal membranes and cation amphiphilic drugs: A molecular dynamics investigation.

Lysosomes are pivotal in cellular functions and disease, influencing cancer progression and therapy resistance with Acid Sphingomyelinase (ASM) governing their membrane integrity. Moreover, cation amphiphilic drugs (CADs) are known as ASM inhibitors and have anti-cancer activity, but the structural mechanisms of their interactions with the lysosomal membrane and ASM are poorly explored. Our study, leveraging all-atom explicit solvent molecular dynamics simulations, delves into the interaction of glycosylated ASM with the lysosomal membrane and the effects of CAD representatives, i.e., ebastine, hydroxyebastine and loratadine, on the membrane and ASM. Our results confirm the ASM association to the membrane through the saposin domain, previously only shown with coarse-grained models. Furthermore, we elucidated the role of specific residues and ASM-induced membrane curvature in lipid recruitment and orientation. CADs also interfere with the association of ASM with the membrane at the level of a loop in the catalytic domain engaging in membrane interactions. Our computational approach, applicable to various CADs or membrane compositions, provides insights into ASM and CAD interaction with the membrane, offering a valuable tool for future studies.

ASM variants in the spotlight: A structure-based atlas for unraveling pathogenic mechanisms in lysosomal acid sphingomyelinase.

Lysosomal acid sphingomyelinase (ASM), a critical enzyme in lipid metabolism encoded by the SMPD1 gene, plays a crucial role in sphingomyelin hydrolysis in lysosomes. ASM deficiency leads to acid sphingomyelinase deficiency, a rare genetic disorder with diverse clinical manifestations, and the protein can be found mutated in other diseases. We employed a structure-based framework to comprehensively understand the functional implications of ASM variants, integrating pathogenicity predictions with molecular insights derived from a molecular dynamics simulation in a lysosomal membrane environment. Our analysis, encompassing over 400 variants, establishes a structural atlas of missense variants of lysosomal ASM, associating mechanistic indicators with pathogenic potential. Our study highlights variants that influence structural stability or exert local and long-range effects at functional sites. To validate our predictions, we compared them to available experimental data on residual catalytic activity in 135 ASM variants. Notably, our findings also suggest applications of the resulting data for identifying cases suited for enzyme replacement therapy. This comprehensive approach enhances the understanding of ASM variants and provides valuable insights for potential therapeutic interventions.

Molecular dynamics-based identification of binding pathways and two distinct high-affinity sites for succinate in succinate receptor 1/GPR91.

SUCNR1 is an auto- and paracrine sensor of the metabolic stress signal succinate. Using unsupervised molecular dynamics (MD) simulations (170.400 ns) and mutagenesis across human, mouse, and rat SUCNR1, we characterize how a five-arginine motif around the extracellular pole of TM-VI determines the initial capture of succinate in the extracellular vestibule (ECV) to either stay or move down to the orthosteric site. Metadynamics demonstrate low-energy succinate binding in both sites, with an energy barrier corresponding to an intermediate stage during which succinate, with an associated water cluster, unlocks the hydrogen-bond-stabilized conformationally constrained extracellular loop (ECL)-2b. Importantly, simultaneous binding of two succinate molecules through either a "sequential" or "bypassing" mode is a frequent endpoint. The mono-carboxylate NF-56-EJ40 antagonist enters SUCNR1 between TM-I and -II and does not unlock ECL-2b. It is proposed that occupancy of both high-affinity sites is required for selective activation of SUCNR1 by high local succinate concentrations.

Prediction of cancer driver genes and mutations: the potential of integrative computational frameworks.

The vast amount of available sequencing data allows the scientific community to explore different genetic alterations that may drive cancer or favor cancer progression. Software developers have proposed a myriad of predictive tools, allowing researchers and clinicians to compare and prioritize driver genes and mutations and their relative pathogenicity. However, there is little consensus on the computational approach or a golden standard for comparison. Hence, benchmarking the different tools depends highly on the input data, indicating that overfitting is still a massive problem. One of the solutions is to limit the scope and usage of specific tools. However, such limitations force researchers to walk on a tightrope between creating and using high-quality tools for a specific purpose and describing the complex alterations driving cancer. While the knowledge of cancer development increases daily, many bioinformatic pipelines rely on single nucleotide variants or alterations in a vacuum without accounting for cellular compartments, mutational burden or disease progression. Even within bioinformatics and computational cancer biology, the research fields work in silos, risking overlooking potential synergies or breakthroughs. Here, we provide an overview of databases and datasets for building or testing predictive cancer driver tools. Furthermore, we introduce predictive tools for driver genes, driver mutations, and the impact of these based on structural analysis. Additionally, we suggest and recommend directions in the field to avoid silo-research, moving towards integrative frameworks.

Computational analysis of five neurodegenerative diseases reveals shared and specific genetic loci.

Neurodegenerative diseases (ND) are heterogeneous disorders of the central nervous system that share a chronic and selective process of neuronal cell death. A computational approach to investigate shared genetic and specific loci was applied to 5 different ND: Amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Multiple sclerosis (MS), and Lewy body dementia (LBD). The datasets were analyzed separately, and then we compared the obtained results. For this purpose, we applied a genetic correlation analysis to genome-wide association datasets and revealed different genetic correlations with several human traits and diseases. In addition, a clumping analysis was carried out to identify SNPs genetically associated with each disease. We found 27 SNPs in AD, 6 SNPs in ALS, 10 SNPs in PD, 17 SNPs in MS, and 3 SNPs in LBD. Most of them are located in non-coding regions, with the exception of 5 SNPs on which a protein structure and stability prediction was performed to verify their impact on disease. Furthermore, an analysis of the differentially expressed miRNAs of the 5 examined pathologies was performed to reveal regulatory mechanisms that could involve genes associated with selected SNPs. In conclusion, the results obtained constitute an important step toward the discovery of diagnostic biomarkers and a better understanding of the diseases.

PDBminer to Find and Annotate Protein Structures for Computational Analysis.

Computational methods relying on protein structure strongly depend on the structure selected for investigation. Typical sources of protein structures include experimental structures available at the Protein Data Bank (PDB) and high-quality in silico model structures, such as those available at the AlphaFold Protein Structure Database. Either option has significant advantages and drawbacks, and exploring the wealth of available structures to identify the most suitable ones for specific applications can be a daunting task. We provide an open-source software package, PDBminer, with the purpose of making structure identification and selection easier, faster, and less error prone. PDBminer searches the AlphaFold Database and the PDB for available structures of interest and provides an up-to-date, quality-ranked table of structures applicable for further use. PDBminer provides an overview of the available protein structures to one or more input proteins, parallelizing the runs if multiple cores are specified. The output table reports the coverage of the protein structures aligned to the UniProt sequence, overcoming numbering differences in PDB structures and providing information regarding model quality, protein complexes, ligands, and nucleic acid chain binding. The PDBminer2coverage and PDBminer2network tools assist in visualizing the results. PDBminer can be applied to overcome the tedious task of choosing a PDB structure without losing the wealth of additional information available in the PDB. Here, we showcase the main functionalities of the package on the p53 tumor suppressor protein. The package is available at http://github.com/ELELAB/PDBminer.

A workflow to study mechanistic indicators for driver gene prediction with Moonlight.

Prediction of driver genes (tumor suppressors and oncogenes) is an essential step in understanding cancer development and discovering potential novel treatments. We recently proposed Moonlight as a bioinformatics framework to predict driver genes and analyze them in a system-biology-oriented manner based on -omics integration. Moonlight uses gene expression as a primary data source and combines it with patterns related to cancer hallmarks and regulatory networks to identify oncogenic mediators. Once the oncogenic mediators are identified, it is important to include extra levels of evidence, called mechanistic indicators, to identify driver genes and to link the observed gene expression changes to the underlying alteration that promotes them. Such a mechanistic indicator could be for example a mutation in the regulatory regions for the candidate gene. Here, we developed new functionalities and released Moonlight2 to provide the user with a mutation-based mechanistic indicator as a second layer of evidence. These functionalities analyze mutations in a cancer cohort to classify them into driver and passenger mutations. Those oncogenic mediators with at least one driver mutation are retained as the final set of driver genes. We applied Moonlight2 to the basal-like breast cancer subtype, lung adenocarcinoma and thyroid carcinoma using data from The Cancer Genome Atlas. For example, in basal-like breast cancer, we found four oncogenes (COPZ2, SF3B4, KRTCAP2 and POLR2J) and nine tumor suppressor genes (KIR2DL4, KIF26B, ARL15, ARHGAP25, EMCN, GMFG, TPK1, NR5A2 and TEK) containing a driver mutation in their promoter region, possibly explaining their deregulation. Moonlight2R is available at https://github.com/ELELAB/Moonlight2R.

PyInteraph2 and PyInKnife2 to Analyze Networks in Protein Structural Ensembles.

Due to the complex nature of noncovalent interactions and their long-range effects, analyzing protein conformations using network theory can be enlightening. Protein Structure Networks (PSNs) provide a convenient formalism to study protein structures in relation to essential properties such as key residues for structural stability, allosteric communication, and the effects of modifications of the protein. PSNs can be defined according to very different principles, and the available tools have limitations in input formats, supported models, and version control. Other outstanding problems are related to the definition of network cutoffs and the assessment of the stability of the network properties. The protein science community could benefit from a common framework to carry out these analyses and make them easier to reproduce, reuse, and evaluate. We here provide two open-source software packages, PyInteraph2 and PyInKnife2, to implement and analyze PSNs in a reproducible and documented manner. PyInteraph2 interfaces with multiple formats for protein ensembles and incorporates different network models with the possibility of integrating them into a macronetwork and performing various downstream analyses, including hubs, connected components, and several other centrality measures, and visualizes the networks or further analyzes them thanks to compatibility with Cytoscape.PyInKnife2 that supports the network models implemented in PyInteraph2. It employs a jackknife resampling approach to estimate the convergence of network properties and streamline the selection of distance cutoffs. We foresee that the modular structure of the code and the supported version control system will promote the transition to a community-driven effort, boost reproducibility, and establish common protocols in the PSN field. As developers, we will guarantee the introduction of new functionalities and maintenance, assistance, and training of new contributors.

Minimum information guidelines for experiments structurally characterizing intrinsically disordered protein regions.

An unambiguous description of an experiment, and the subsequent biological observation, is vital for accurate data interpretation. Minimum information guidelines define the fundamental complement of data that can support an unambiguous conclusion based on experimental observations. We present the Minimum Information About Disorder Experiments (MIADE) guidelines to define the parameters required for the wider scientific community to understand the findings of an experiment studying the structural properties of intrinsically disordered regions (IDRs). MIADE guidelines provide recommendations for data producers to describe the results of their experiments at source, for curators to annotate experimental data to community resources and for database developers maintaining community resources to disseminate the data. The MIADE guidelines will improve the interpretability of experimental results for data consumers, facilitate direct data submission, simplify data curation, improve data exchange among repositories and standardize the dissemination of the key metadata on an IDR experiment by IDR data sources.

Comparison of force fields to study the zinc-finger containing protein NPL4, a target for disulfiram in cancer therapy.

Molecular dynamics (MD) simulations are a powerful approach to studying the structure and dynamics of proteins related to health and disease. Advances in the MD field allow modeling proteins with high accuracy. However, modeling metal ions and their interactions with proteins is still challenging. NPL4 is a zinc-binding protein and works as a cofactor for p97 to regulate protein homeostasis. NPL4 is of biomedical importance and has been proposed as the target of disulfiram, a drug recently repurposed for cancer treatment. Experimental studies proposed that the disulfiram metabolites, bis-(diethyldithiocarbamate)­copper and cupric ions, induce NPL4 misfolding and aggregation. However, the molecular details of their interactions with NPL4 and consequent structural effects are still elusive. Here, biomolecular simulations can help to shed light on the related structural details. To apply MD simulations to NPL4 and its interaction with copper the first important step is identifying a suitable force field to describe the protein in its zinc-bound states. We examined different sets of non-bonded parameters because we want to study the misfolding mechanism and cannot rule out that the zinc may detach from the protein during the process and copper replaces it. We investigated the force-field ability to model the coordination geometry of the metal ions by comparing the results from MD simulations with optimized geometries from quantum mechanics (QM) calculations using model systems of NPL4. Furthermore, we investigated the performance of a force field including bonded parameters to treat copper ions in NPL4 that we obtained based on QM calculations.

TRAP1 S-nitrosylation as a model of population-shift mechanism to study the effects of nitric oxide on redox-sensitive oncoproteins.

S-nitrosylation is a post-translational modification in which nitric oxide (NO) binds to the thiol group of cysteine, generating an S-nitrosothiol (SNO) adduct. S-nitrosylation has different physiological roles, and its alteration has also been linked to a growing list of pathologies, including cancer. SNO can affect the function and stability of different proteins, such as the mitochondrial chaperone TRAP1. Interestingly, the SNO site (C501) of TRAP1 is in the proximity of another cysteine (C527). This feature suggests that the S-nitrosylated C501 could engage in a disulfide bridge with C527 in TRAP1, resembling the well-known ability of S-nitrosylated cysteines to resolve in disulfide bridge with vicinal cysteines. We used enhanced sampling simulations and in-vitro biochemical assays to address the structural mechanisms induced by TRAP1 S-nitrosylation. We showed that the SNO site induces conformational changes in the proximal cysteine and favors conformations suitable for disulfide bridge formation. We explored 4172 known S-nitrosylated proteins using high-throughput structural analyses. Furthermore, we used a coarse-grained model for 44 protein targets to account for protein flexibility. This resulted in the identification of up to 1248 proximal cysteines, which could sense the redox state of the SNO site, opening new perspectives on the biological effects of redox switches. In addition, we devised two bioinformatic workflows ( https://github.com/ELELAB/SNO_investigation_pipelines ) to identify proximal or vicinal cysteines for a SNO site with accompanying structural annotations. Finally, we analyzed mutations in tumor suppressors or oncogenes in connection with the conformational switch induced by S-nitrosylation. We classified the variants as neutral, stabilizing, or destabilizing for the propensity to be S-nitrosylated and undergo the population-shift mechanism. The methods applied here provide a comprehensive toolkit for future high-throughput studies of new protein candidates, variant classification, and a rich data source for the research community in the NO field.

GSNOR deficiency promotes tumor growth via FAK1 S-nitrosylation.

Nitric oxide (NO) production in the tumor microenvironment is a common element in cancer. S-nitrosylation, the post-translational modification of cysteines by NO, is emerging as a key transduction mechanism sustaining tumorigenesis. However, most oncoproteins that are regulated by S-nitrosylation are still unknown. Here we show that S-nitrosoglutathione reductase (GSNOR), the enzyme that deactivates S-nitrosylation, is hypo-expressed in several human malignancies. Using multiple tumor models, we demonstrate that GSNOR deficiency induces S-nitrosylation of focal adhesion kinase 1 (FAK1) at C658. This event enhances FAK1 autophosphorylation and sustains tumorigenicity by providing cancer cells with the ability to survive in suspension (evade anoikis). In line with these results, GSNOR-deficient tumor models are highly susceptible to treatment with FAK1 inhibitors. Altogether, our findings advance our understanding of the oncogenic role of S-nitrosylation, define GSNOR as a tumor suppressor, and point to GSNOR hypo-expression as a therapeutically exploitable vulnerability in cancer.

RosettaDDGPrediction for high-throughput mutational scans: From stability to binding.

Reliable prediction of free energy changes upon amino acid substitutions (ΔΔGs) is crucial to investigate their impact on protein stability and protein-protein interaction. Advances in experimental mutational scans allow high-throughput studies thanks to multiplex techniques. On the other hand, genomics initiatives provide a large amount of data on disease-related variants that can benefit from analyses with structure-based methods. Therefore, the computational field should keep the same pace and provide new tools for fast and accurate high-throughput ΔΔG calculations. In this context, the Rosetta modeling suite implements effective approaches to predict folding/unfolding ΔΔGs in a protein monomer upon amino acid substitutions and calculate the changes in binding free energy in protein complexes. However, their application can be challenging to users without extensive experience with Rosetta. Furthermore, Rosetta protocols for ΔΔG prediction are designed considering one variant at a time, making the setup of high-throughput screenings cumbersome. For these reasons, we devised RosettaDDGPrediction, a customizable Python wrapper designed to run free energy calculations on a set of amino acid substitutions using Rosetta protocols with little intervention from the user. Moreover, RosettaDDGPrediction assists with checking completed runs and aggregates raw data for multiple variants, as well as generates publication-ready graphics. We showed the potential of the tool in four case studies, including variants of uncertain significance in childhood cancer, proteins with known experimental unfolding ΔΔGs values, interactions between target proteins and disordered motifs, and phosphomimetics. RosettaDDGPrediction is available, free of charge and under GNU General Public License v3.0, at https://github.com/ELELAB/RosettaDDGPrediction.

The Cancermuts software package for the prioritization of missense cancer variants: a case study of AMBRA1 in melanoma.

Cancer genomics and cancer mutation databases have made an available wealth of information about missense mutations found in cancer patient samples. Contextualizing by means of annotation and predicting the effect of amino acid change help identify which ones are more likely to have a pathogenic impact. Those can be validated by means of experimental approaches that assess the impact of protein mutations on the cellular functions or their tumorigenic potential. Here, we propose the integrative bioinformatic approach Cancermuts, implemented as a Python package. Cancermuts is able to gather known missense cancer mutations from databases such as cBioPortal and COSMIC, and annotate them with the pathogenicity score REVEL as well as information on their source. It is also able to add annotations about the protein context these mutations are found in, such as post-translational modification sites, structured/unstructured regions, presence of short linear motifs, and more. We applied Cancermuts to the intrinsically disordered protein AMBRA1, a key regulator of many cellular processes frequently deregulated in cancer. By these means, we classified mutations of AMBRA1 in melanoma, where AMBRA1 is highly mutated and displays a tumor-suppressive role. Next, based on REVEL score, position along the sequence, and their local context, we applied cellular and molecular approaches to validate the predicted pathogenicity of a subset of mutations in an in vitro melanoma model. By doing so, we have identified two AMBRA1 mutations which show enhanced tumorigenic potential and are worth further investigation, highlighting the usefulness of the tool. Cancermuts can be used on any protein targets starting from minimal information, and it is available at https://www.github.com/ELELAB/cancermuts as free software.

Directed Evolution of (R)-2-Hydroxyglutarate Dehydrogenase Improves 2-Oxoadipate Reduction by 2 Orders of Magnitude.

Pathway engineering is commonly employed to improve the production of various metabolites but may incur in bottlenecks due to the low catalytic activity of a particular reaction step. The reduction of 2-oxoadipate to (R)-2-hydroxyadipate is a key reaction in metabolic pathways that exploit 2-oxoadipate conversion via α-reduction to produce adipic acid, an industrially important platform chemical. Here, we engineered (R)-2-hydroxyglutarate dehydrogenase from Acidaminococcus fermentans (Hgdh) with the aim of improving 2-oxoadipate reduction. Using a combination of computational analysis, saturation mutagenesis, and random mutagenesis, three mutant variants with a 100-fold higher catalytic efficiency were obtained. As revealed by rational analysis of the mutations found in the variants, this improvement could be ascribed to a general synergistic effect where mutation A206V played a key role since it boosted the enzyme's activity by 4.8-fold. The Hgdh variants with increased activity toward 2-oxoadipate generated within this study pave the way for the bio-based production of adipic acid.

Unraveling membrane properties at the organelle-level with LipidDyn.

Cellular membranes are formed from different lipids in various amounts and proportions depending on the subcellular localization. The lipid composition of membranes is sensitive to changes in the cellular environment, and its alterations are linked to several diseases. Lipids not only form lipid-lipid interactions but also interact with other biomolecules, including proteins. Molecular dynamics (MD) simulations are a powerful tool to study the properties of cellular membranes and membrane-protein interactions on different timescales and resolutions. Over the last few years, software and hardware for biomolecular simulations have been optimized to routinely run long simulations of large and complex biological systems. On the other hand, high-throughput techniques based on lipidomics provide accurate estimates of the composition of cellular membranes at the level of subcellular compartments. Lipidomic data can be analyzed to design biologically relevant models of membranes for MD simulations. Similar applications easily result in a massive amount of simulation data where the bottleneck becomes the analysis of the data. In this context, we developed LipidDyn, a Python-based pipeline to streamline the analyses of MD simulations of membranes of different compositions. Once the simulations are collected, LipidDyn provides average properties and time series for several membrane properties such as area per lipid, thickness, order parameters, diffusion motions, lipid density, and lipid enrichment/depletion. The calculations exploit parallelization, and the pipeline includes graphical outputs in a publication-ready form. We applied LipidDyn to different case studies to illustrate its potential, including membranes from cellular compartments and transmembrane protein domains. LipidDyn is available free of charge under the GNU General Public License from https://github.com/ELELAB/LipidDyn.

Structural Details of BH3 Motifs and BH3-Mediated Interactions: an Updated Perspective.

Apoptosis is a mechanism of programmed cell death crucial in organism development, maintenance of tissue homeostasis, and several pathogenic processes. The B cell lymphoma 2 (BCL2) protein family lies at the core of the apoptotic process, and the delicate balance between its pro- and anti-apoptotic members ultimately decides the cell fate. BCL2 proteins can bind with each other and several other biological partners through the BCL2 homology domain 3 (BH3), which has been also classified as a possible Short Linear Motif and whose distinctive features remain elusive even after decades of studies. Here, we aim to provide an updated overview of the structural features characterizing BH3s and BH3-mediated interactions (with a focus on human proteins), elaborating on the plasticity of BCL2 proteins and the motif properties. We also discussed the implication of these findings for the discovery of interactors of the BH3-binding groove of BCL2 proteins and the design of mimetics for therapeutic purposes.

Cancer-related Mutations with Local or Long-range Effects on an Allosteric Loop of p53.

The tumor protein 53 (p53) is involved in transcription-dependent and independent processes. Several p53 variants related to cancer have been found to impact protein stability. Other variants, on the contrary, might have little impact on structural stability and have local or long-range effects on the p53 interactome. Our group previously identified a loop in the DNA binding domain (DBD) of p53 (residues 207-213) which can recruit different interactors. Experimental structures of p53 in complex with other proteins strengthen the importance of this interface for protein-protein interactions. We here characterized with structure-based approaches somatic and germline variants of p53 which could have a marginal effect in terms of stability and act locally or allosterically on the region 207-213 with consequences on the cytosolic functions of this protein. To this goal, we studied 1132 variants in the p53 DBD with structure-based approaches, accounting also for protein dynamics. We focused on variants predicted with marginal effects on structural stability. We then investigated each of these variants for their impact on DNA binding, dimerization of the p53 DBD, and intramolecular contacts with the 207-213 region. Furthermore, we identified variants that could modulate long-range the conformation of the region 207-213 using a coarse-grain model for allostery and all-atom molecular dynamics simulations. Our predictions have been further validated using enhanced sampling methods for 15 variants. The methodologies used in this study could be more broadly applied to other p53 variants or cases where conformational changes of loop regions are essential in the function of disease-related proteins.