N-of-one differential gene expression without control samples using a deep generative model.

Differential analysis of bulk RNA-seq data often suffers from lack of good controls. Here, we present a generative model that replaces controls, trained solely on healthy tissues. The unsupervised model learns a low-dimensional representation and can identify the closest normal representation for a given disease sample. This enables control-free, single-sample differential expression analysis. In breast cancer, we demonstrate how our approach selects marker genes and outperforms a state-of-the-art method. Furthermore, significant genes identified by the model are enriched in driver genes across cancers. Our results show that the in silico closest normal provides a more favorable comparison than control samples.

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.

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.

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.

Disease-associated mutations impacting BC-loop flexibility trigger long-range transthyretin tetramer destabilization and aggregation.

Hereditary transthyretin amyloidosis (ATTR) is an autosomal dominant disease characterized by the extracellular deposition of the transport protein transthyretin (TTR) as amyloid fibrils. Despite the progress achieved in recent years, understanding why different TTR residue substitutions lead to different clinical manifestations remains elusive. Here, we studied the molecular basis of disease-causing missense mutations affecting residues R34 and K35. R34G and K35T variants cause vitreous amyloidosis, whereas R34T and K35N mutations result in amyloid polyneuropathy and restrictive cardiomyopathy. All variants are more sensitive to pH-induced dissociation and amyloid formation than the wild-type (WT)-TTR counterpart, specifically in the variants deposited in the eyes amyloid formation occurs close to physiological pHs. Chemical denaturation experiments indicate that all the mutants are less stable than WT-TTR, with the vitreous amyloidosis variants, R34G and K35T, being highly destabilized. Sequence-induced stabilization of the dimer-dimer interface with T119M rendered tetramers containing R34G or K35T mutations resistant to pH-induced aggregation. Because R34 and K35 are among the residues more distant to the TTR interface, their impact in this region is therefore theorized to occur at long range. The crystal structures of double mutants, R34G/T119M and K35T/T119M, together with molecular dynamics simulations indicate that their strong destabilizing effect is initiated locally at the BC loop, increasing its flexibility in a mutation-dependent manner. Overall, the present findings help us to understand the sequence-dynamic-structural mechanistic details of TTR amyloid aggregation triggered by R34 and K35 variants and to link the degree of mutation-induced conformational flexibility to protein aggregation propensity.

Bcl-xL Dynamics under the Lens of Protein Structure Networks.

Understanding the finely orchestrated interactions leading to or preventing programmed cell death (apoptosis) is of utmost importance in cancer research because the failure of these systems could eventually lead to the onset of the disease. In this regard, the maintenance of a delicate balance between the promoters and inhibitors of mitochondrial apoptosis is crucial, as demonstrated by the interplay among the Bcl-2 family members. In particular, B-cell lymphoma extra-large (Bcl-xL) is a target of interest due to the forefront role of its dysfunctions in cancer development. Bcl-xL prevents apoptosis by binding both the pro-apoptotic BH3-only proteins, like PUMA, and the noncanonical partners, such as p53, at different sites. An allosteric communication between the BH3-only protein binding pocket and the p53 binding site, mediating the release of p53 from Bcl-xL upon PUMA binding, has been postulated and supported by nuclear magnetic resonance and other biophysical data. The molecular details of this mechanism, especially at the residue level, remain unclear. In this work, we investigated the distal communication between these two sites in Bcl-xL in its free state and when bound to PUMA. We also evaluated how missense mutations of Bcl-xL found in cancer samples might impair this communication and therefore the allosteric mechanism. We employed all-atom explicit solvent microsecond molecular dynamics simulations, analyzed through a Protein Structure Network approach and integrated with calculations of changes in free energies upon cancer-related mutations identified by genomics studies. We found a subset of candidate residues responsible for both maintaining protein stability and for conveying structural information between the two binding sites and hypothesized possible communication routes between specific residues at both sites.

The conformational and mutational landscape of the ubiquitin-like marker for autophagosome formation in cancer.

Macroautophagy/autophagy is a cellular process to recycle damaged cellular components, and its modulation can be exploited for disease treatments. A key autophagy player is the ubiquitin-like protein MAP1LC3B/LC3B. Mutations and changes in MAP1LC3B expression occur in cancer samples. However, the investigation of the effects of these mutations on MAP1LC3B protein structure is still missing. Despite many LC3B structures that have been solved, a comprehensive study, including dynamics, has not yet been undertaken. To address this knowledge gap, we assessed nine physical models for biomolecular simulations for their capabilities to describe the structural ensemble of MAP1LC3B. With the resulting MAP1LC3B structural ensembles, we characterized the impact of 26 missense mutations from pan-cancer studies with different approaches, and we experimentally validated our prediction for six variants using cellular assays. Our findings shed light on damaging or neutral mutations in MAP1LC3B, providing an atlas of its modifications in cancer. In particular, P32Q mutation was found detrimental for protein stability with a propensity to aggregation. In a broader context, our framework can be applied to assess the pathogenicity of protein mutations or to prioritize variants for experimental studies, allowing to comprehensively account for different aspects that mutational events alter in terms of protein structure and function.Abbreviations: ATG: autophagy-related; Cα: alpha carbon; CG: coarse-grained; CHARMM: Chemistry at Harvard macromolecular mechanics; CONAN: contact analysis; FUNDC1: FUN14 domain containing 1; FYCO1: FYVE and coiled-coil domain containing 1; GABARAP: GABA type A receptor-associated protein; GROMACS: Groningen machine for chemical simulations; HP: hydrophobic pocket; LIR: LC3 interacting region; MAP1LC3B/LC3B microtubule associated protein 1 light chain 3 B; MD: molecular dynamics; OPTN: optineurin; OSF: open software foundation; PE: phosphatidylethanolamine, PLEKHM1: pleckstrin homology domain-containing family M 1; PSN: protein structure network; PTM: post-translational modification; SA: structural alphabet; SLiM: short linear motif; SQSTM1/p62: sequestosome 1; WT: wild-type.

The PyInteraph Workflow for the Study of Interaction Networks From Protein Structural Ensembles.

PyInteraph is a software package designed for the analysis of structural communication from conformational ensembles, such as those derived from in silico simulations, under the formalism of protein structure networks. We demonstrate its usage for the calculation and analysis of intramolecular interaction networks derived from three different types of interactions, as well as with a more general protocol based on distances between centers of mass. We use the xPyder PyMOL plug-in to visualize such networks on the three-dimensional structure of the protein. We showcase our protocol on a molecular dynamics trajectory of the Cyclophilin A wild-type enzyme, a well-studied protein in which different allosteric mechanisms have been investigated.

Structure and Dynamics in the ATG8 Family From Experimental to Computational Techniques.

Autophagy is a conserved and essential intracellular mechanism for the removal of damaged components. Since autophagy deregulation is linked to different kinds of pathologies, it is fundamental to gain knowledge on the fine molecular and structural details related to the core proteins of the autophagy machinery. Among these, the family of human ATG8 proteins plays a central role in recruiting other proteins to the different membrane structures involved in the autophagic pathway. Several experimental structures are available for the members of the ATG8 family alone or in complex with their different biological partners, including disordered regions of proteins containing a short linear motif called LC3 interacting motif. Recently, the first structural details of the interaction of ATG8 proteins with biological membranes came into light. The availability of structural data for human ATG8 proteins has been paving the way for studies on their structure-function-dynamic relationship using biomolecular simulations. Experimental and computational structural biology can help to address several outstanding questions on the mechanism of human ATG8 proteins, including their specificity toward different interactors, their association with membranes, the heterogeneity of their conformational ensemble, and their regulation by post-translational modifications. We here summarize the main results collected so far and discuss the future perspectives within the field and the knowledge gaps. Our review can serve as a roadmap for future structural and dynamics studies of the ATG8 family members in health and disease.

Analyzing Biomolecular Ensembles.

Several techniques are available to generate conformational ensembles of proteins and other biomolecules either experimentally or computationally. These methods produce a large amount of data that need to be analyzed to identify structure-dynamics-function relationship. In this chapter, we will cover different tools to unveil the information hidden in conformational ensemble data and to guide toward the rationalization of the data. We included routinely used approaches such as dimensionality reduction, as well as new methods inspired by high-order statistics and graph theory.

The Mutational Landscape of the Oncogenic MZF1 SCAN Domain in Cancer.

SCAN domains in zinc-finger transcription factors are crucial mediators of protein-protein interactions. Up to 240 SCAN-domain encoding genes have been identified throughout the human genome. These include cancer-related genes, such as the myeloid zinc finger 1 (MZF1), an oncogenic transcription factor involved in the progression of many solid cancers. The mechanisms by which SCAN homo- and heterodimers assemble and how they alter the transcriptional activity of zinc-finger transcription factors in cancer and other diseases remain to be investigated. Here, we provide the first description of the conformational ensemble of the MZF1 SCAN domain cross-validated against NMR experimental data, which are probes of structure and dynamics on different timescales. We investigated the protein-protein interaction network of MZF1 and how it is perturbed in different cancer types by the analyses of high-throughput proteomics and RNASeq data. Collectively, we integrated many computational approaches, ranging from simple empirical energy functions to all-atom microsecond molecular dynamics simulations and network analyses to unravel the effects of cancer-related substitutions in relation to MZF1 structure and interactions.