Biology subjects

Trovato, F.

Publications and source records attributed to Trovato, F..

6 recordsLinked to original sources

bioRxiv2025

Interactions with lipid membrane modulate the conformational dynamics and energy landscape of Tumor Necrosis Factor

Tumor Necrosis Factor (TNF) is a trimeric cytokine that exists in soluble (sTNF) and membrane-bound (mTNF) forms, both of which regulate immune responses through interactions with their cognate receptors. sTNF has been the subject of extensive biophysical investigations, leading to a mechanistic model in which a symmetric arrangement of the trimer promotes receptor signaling, whereas asymmetric conformations inhibit this function. In contrast, the structural and energetic landscape of mTNF remains largely under-explored. Here, we combined multi-microsecond unbiased molecular dynamics and Metadynamics-Adaptive Biasing Force simulations to characterize mTNF embedded in a physiologically relevant lipid bilayer. We show that in the absence of membrane engagement, the extracellular domain (ECD) dynamically samples multiple asymmetric conformations similar to those observed in sTNF. The association of the ECD with the membrane, mediated primarily by the basic residues R78-R82, R107, R108, R120, and R207, restricts conformational heterogeneity and stabilizes the symmetric state. By quantifying the energetic effects of ECD-membrane association, we demonstrate that the symmetric state in the membrane-bound ECD is stabilized over asymmetric conformations to a greater extent than in sTNF. This energetic effect, together with the spatial confinement imposed by the lipid bilayer, may explain the previously reported reduction in affinity of certain biologics for mTNF. In conclusion, our work elucidates how the membrane influences the structure, dynamics, and energetics of TNF. These mechanistic insights could guide future efforts to design mTNF-selective inhibitors that account for both membrane constraints and the energetic modulation of the ECD conformational landscape.

biophysics↗

bioRxiv2025

Computational Modeling of Ternary Complex Formation and Targeted Protein Degradation Mediated by Molecular Glues

The rational design of molecular glue degraders is challenging because glue-mediated protein degradation depends on a complex interplay of molecular mechanisms: an effective molecular glue must simultaneously bring together two distinct proteins to form an interface that is both stable and accessible for ubiquitination by E3 ligases. We present GlueMap, a computational platform for discovering and optimizing molecular glues (MGs) through integrative structural and pharmacodynamic modeling. GlueMap combines molecular dynamics (MD) simulations and machine learning (ML) to model targeted protein degradation (TPD) mediated by molecular glues. We validated GlueMap by applying it to two case studies: DDB1-dependent degradation of CDK12 and CRBN-dependent degradation of GSPT1. In the DDB1-CDK12 system, GlueMap revealed strong correlations between ternary-complex stability, quantified through rigorous free-energy calculations, and experimental degradation efficiency. Notable outliers underscored the importance of incorporating conformational dynamics within assembled Cullin-RING ligase complexes. Using a supervised variational autoencoder combined with attention-based regression, GlueMap achieved high predictive accuracy for degradation potency from structural features. For CRBN-GSPT1, GlueMap reproduced known ternary-complex crystal structures and identified previously unrecognized protein-protein interactions (PPIs) that may guide the design of next-generation glues. Hierarchical virtual screening of more than 12 billion compounds successfully recovered known degraders and uncovered nine new potent MGs with DC50 values below 50 nM. Across both systems, GlueMap outperformed Boltz-1 and Boltz-2, state-of-the-art structure-prediction models that failed to reproduce the CDK12-DDB1 crystal structure and captured substantially less PPI diversity. By integrating structural, thermodynamic, and ubiquitination metrics, GlueMap establishes a multi-criteria framework for rational molecular glue design. These results demonstrate that GlueMap provides both mechanistic insight and practical acceleration of molecular glue discovery and optimization across diverse therapeutic targets.

bioinformatics↗

bioRxiv2024

A sequential event-responsive fluorescent reporter for inducible Cre and Flp combinatorial recombination

Current technologies for precise genetic manipulation of cells often utilize site-specific recombinases, enabling the creation of conditional transgenic models for studying gene functions in specific tissues and at defined time windows. The advent of drug-inducible recombinases has further enhanced this field by allowing temporal control of gene expression. However, detecting gene expression patterns when multiple recombinases are used is challenging, particularly when visualizing combinatorial maps and their temporal profile. To address this, we developed Rubik, a fluorescent genetic reporter specifically designed to monitor the action of Cre and Flp recombinases by expressing different fluorophores depending on the temporal sequence of recombination events. Rubik offers four alternative fluorescent configurations: blue for Cre recombination, green for Flp recombination, yellow for Cre followed by Flp recombination, and red for Flp followed by Cre recombination. Additionally, Rubik integrates light-gated ion channels, allowing optogenetic excitation or inhibition of electrically-excitable cells based on the temporal sequence of recombination events. Moreover, to add temporal control over Rubik recombination, we optimized a trimethoprim-inducible FlpO recombinase (FlpO-DD) to use in combination with the tamoxifen-inducible Cre recombinase (ERT2CreERT2). We validated Rubik by generating a stable knock-in HeLa cell line using CRISPR-Cas9 and single-cell sorting. Our results confirmed the effective functioning of Rubik with Cre and Flp recombinases and showed the ability to induce selective recombination via either tamoxifen or trimethoprim. This system holds potential for neuroscience research, particularly in generating knock-in mice expressing Rubik, which could be instrumental in precisely defining the roles of specific neuronal populations based on Cre and Flp activity.

molecular biology↗

bioRxiv2021

Universal protein misfolding intermediates can bypass the proteostasis network and remain soluble and non-functional

Misfolded protein conformations with decreased functionality can bypass the proteostasis machinery and remain soluble in vivo. This is an unexpected phenomenon as several cellular quality control mechanisms have evolved to rid cells of misfolded proteins. Three questions, then, are: how is it structurally possible for long-lived, soluble, misfolded proteins to bypass the proteostasis machinery and processes? How widespread are these soluble, misfolded states across the proteome? And how long do they persist for? Here, we address these questions using coarse-grain molecular dynamics simulations of the synthesis, termination, and post-translational dynamics of a representative set of cytosolic E. coli proteins. We predict that half of all proteins exhibit subpopulations of misfolded conformations that are likely to bypass molecular chaperones, avoid aggregation, and not be rapidly degraded. These misfolded states may persist for months or longer for some proteins. Structurally characterizing these misfolded states, we observe they have a large amount of native structure, but also contain localized misfolded regions from non-native changes in entanglement, in which a protein segment threads through a loop formed by another portion of the protein that is not found in the native state. The surface properties of these misfolded states are native like, suggesting they may bypass the proteostasis machinery and its regulatory processes to remain soluble, while their entanglements make these states long-lived kinetic traps, as disentanglement requires unfolding of already folded portions of the protein. In terms of function, we predict that one-third of proteins have subpopulations that misfold into less-functional states that have structurally perturbed functional sites yet remain soluble. Data from limited-proteolysis mass spectrometry experiments, which interrogate the misfolded conformations populated by proteins upon unfolding and refolding, are consistent with the structural changes seen in the entangled states of glycerol-3-phosphate dehydrogenase upon misfolding. These results provide an explanation for how proteins can misfold into soluble conformations with reduced functionality that can bypass cellular quality controls, and indicate, unexpectedly, this may be a wide-spread phenomenon in proteomes. Such entanglements are observed in many native structures, suggesting the non-native entanglements we observe are plausible. More broadly, these near-native entangled structures suggest a hypothesis for how synonymous mutations can modulate downstream protein structure and function, with these mutations partitioning nascent proteins between these kinetically trapped states.

biochemistry↗

bioRxiv2019

A Cre-amplifier to generate and detect genetic mosaics in vivo

Cre-Lox manipulation is the gold standard for cell-specific expression or knockout of selected genes. However, it is not unusual to deal with conditions of low Cre expression or transient activation, which can often go undetected by conventional Cre-reporters. We designed Beatrix, a general-purpose tool specifically devised to amplify weak Cre recombinase activity, and we used it to develop a powerful approach for the in vivo generation and detection of sparse mosaics of mutant and wild type cells.

neuroscience↗

bioRxiv2018

Domain topology, stability, and translation speed determine mechanical force generation on the ribosome

The concomitant folding of a nascent protein domain with its synthesis can generate mechanical forces that act on the ribosome and alter translation speed. Such changes in speed can affect the structure and function of the newly synthesized protein as well as cellular phenotype. The domain properties that govern force generation have yet to be identified and understood, and the influence of translation speed is unknown as all reported measurements have been carried out on arrested ribosomes. Here, using coarse-grained molecular simulations and statistical mechanical modeling of protein synthesis, we demonstrate that force generation is determined by a domains stability and topology, as well as translation speed. The statistical mechanical models we create predict how force profiles depend on these properties. These results indicate that force measurements on arrested ribosomes will not always accurately reflect what happens in a cell, especially for slow-folding domains, and suggest the possibility that certain domain properties may be enriched or depleted across the structural proteome of organisms through evolutionary selection pressures to modulate protein synthesis speed and post-translational protein behavior.\n\nSignificance StatementMechanochemistry, the influence of molecular-scale mechanical forces on chemical processes, can occur on actively translating ribosomes through the force-generating actions of motor proteins and the co-translational folding of domains. Such forces are transmitted to the ribosomes catalytic core and alter rates of protein synthesis; representing a form of mechanical allosteric communication. These changes in translation-elongation kinetics are biologically important because they can influence protein structure, function, and localization within a cell. Many fundamental questions are unresolved concerning the properties of protein domains that determine mechanical force generation, the effect of translation speed on this force, and exactly how, at the molecular level, force is generated. In this study we answer these questions using cutting-edge molecular simulations and statistical mechanical modeling.

biophysics↗