Biology subjects

Colombo, G.

Publications and source records attributed to Colombo, G..

At least 19 recordsLinked to original sources

bioRxiv2026

Bioinf-Farma: supervised integration of epitope prediction and recombinant protein developability for automated vaccine candidate prioritization

Vaccine antigen discovery requires prioritizing protein candidates according to both immunogenic potential and recombinant expression feasibility. These properties are typically evaluated using separate computational tools, requiring researchers to integrate heterogeneous outputs through ad hoc workflows. Here, we present BIOINF-farma, a modular platform integrating epitope prediction and developability assessment for rational antigen selection within a unified environment. Candidates can be submitted as amino acid sequences or three-dimensional structures. When experimental structures are unavailable, BIOINF-farma automatically searches for models in AlphaFold DB or performs structure prediction using Boltz-2, ensuring a standardized structural representation for downstream analyses. Antigenicity is quantified by combining structure-based conformational epitope signals (MLCE/REBELOT-BEPPE) and sequence-based linear epitope propensity scores (BepiPred 3.0) into a protein-level Antigenicity Score, with a classification threshold optimized on a manually curated validation dataset. Developability is evaluated through two supervised Random Forest meta-learners that integrate three solubility predictors (DeepSoluE, SoluProt, Protein-Sol) and three thermal stability predictors (TemStaPro, ProLaTherm, BertThermo), whose outputs are combined into an Expression Efficiency Score (EES). By integrating complementary predictive signals, the meta-learning framework achieves greater accuracy and robustness than individual predictors while maintaining performance across a broad range of sequence identities. The Antigenicity Score effectively discriminates antigenic from non-antigenic proteins with a large effect size, whereas EES successfully distinguishes soluble from insoluble outcomes on an independent panel of recombinant proteins expressed in Escherichia coli. BIOINF-farma jointly assesses antigenicity and expression feasibility within a single framework. Its modular architecture facilitates the incorporation of future predictive methods, while its web-based interface makes the full pipeline accessible to users without programming expertise, supporting rapid candidate triage in vaccine research and emerging pathogen responses. Author SummaryVaccine development begins with a critical step: identifying, among the many proteins encoded in a pathogen genome, those most suitable as candidate antigens. A promising candidate must satisfy two requirements that are rarely evaluated together. It must be recognized by the immune system, so that vaccination elicits a protective response; and it must be amenable to recombinant production, since antigens that cannot be obtained in sufficient quantity and quality are of limited practical use. Current computational tools typically address only one of these aspects, and researchers must integrate their outputs manually, through procedures that are time-consuming and prone to inconsistency. We developed BIOINF-farma, an automated platform that brings these two assessments into a single analytical framework. Starting from a protein sequence or an experimental structure, the platform retrieves or predicts a three-dimensional model, evaluates the proteins antigenic potential by combining complementary epitope predictors, and estimates its expression feasibility by integrating multiple solubility and stability predictors through supervised machine learning. A web-based interface makes the full workflow available to experimental immunologists and vaccine developers without requiring computational expertise, supporting rational candidate prioritization in routine vaccine research and during emerging pathogen responses.

bioinformatics↗

bioRxiv2026

A tumor metabolism-angiogenesis-immune axis governs immunotherapy responses

Despite the clinical success of immunotherapy, long-lasting benefit remains restricted to a subset of patients. Tumor metabolic adaptation is emerging as a key factor limiting immunotherapy efficacy. We previously found that glycolysis-low tumor variants, compared to the parental glycolytic tumors, better respond to neoadjuvant CTLA-4 immune checkpoint blockade (ICB) therapy. Here, we investigated new rational modalities to restore immune sensitivity of glycolytic tumors by studying how lowering the tumor-cell glycolytic capacity reshapes the tumor microenvironment (TME) to favor long-lasting systemic anti-tumor responses upon immunotherapy. We found that lowering glycolysis in cancer cells through LDHA-knock down (KD) results in TME displaying normalized vasculature, reduced angiogenic markers, increased high endothelial venules (HEVs), and enhanced recirculation of CD8+ T cells both in and out of the tumor. By leveraging public transcriptomic data sets from human solid cancers, we confirmed that glycolysis positively correlates with neo-angiogenesis and inversely correlates with features of vascular normalization and immune cytolytic activity. Moreover, a tumor signature that incorporates glycolysis- and angiogenesis-related genes as positive features, and normal vasculature, HEV, and immune cytolytic activity genes as negative features predicted poor outcomes better than the individual features across most human solid tumor types in the TCGA. To determine the therapeutic implication of these interrelated processes, we asked if targeting the vasculature would restore immunotherapy responses in glycolytic tumors. We found that combining low-dose anti-VEGFR2 with CTLA-4 blockade induces tumor regressions and protection from metastases in glycolytic tumors. These therapeutic effects were associated with vasculature normalization and increased abundance of HEVs and concentrations of lymphangiogenic factors in the TME of glycolytic tumors. Moreover, anti-VEGFR2 with anti-CTLA-4 restored recirculation of anti-tumor CD8+ T cells in and out of the TME in glycolytic tumors, with specific increases in intratumoral recruitment and activation of cytolytic CD62LCD44CD8 T cells expressing VEGFR2 and low levels of CTLA-4, suggesting potential novel direct synergistic effects of anti-VEGFR2 and anti-CTLA-4 on CD8+ T cells. Conversely, this combination opposed the beneficial immune and vascular TME features of LDHA-KD tumors, indicating tumor-metabolic-dependent effects. Accordingly, we found that standard combined regimens of anti-VEGF and ICB therapy improve survival with respect to ICB alone in patients with glycolysis-high but not glycolysis-low tumors. Together, these findings indicate that tumor cell glycolysis "primes" the TME for aberrant vascular architecture and T-cell exclusion, and that modulating the tumor vasculature can unravel these mechanisms restoring immune responsiveness. This suggests that tailoring anti-angiogenic and immunotherapy combinations to the tumor glycolytic state and associated vasculature profiles may restore immune surveillance and overcome therapy resistance.

immunology↗

bioRxiv2026

Hierarchically engineered multi-enzyme nanoreactors for in vitro drug biosynthesis and pathway transplantation into cells

Even though most proteins evolved to function within multi-protein systems such as metabolic networks or signaling pathways, most technical protein applications are based on isolated proteins, for example therapeutic antibodies or industrial enzymes. Reliable methods for the stabilization, in vitro operation and intracellular delivery of multi-protein systems could unlock new applications in green biotechnology, diagnostics, and medical therapy. We demonstrate that the entire violacein biosynthesis pathway, consisting of up to six separately purified enzymes, can be infiltrated into a hierarchically etched MIL-101 (eMIL) metal organic framework. eMIL nanoreactors reshaped pathway dynamics and reaction flows, multiplied violacein yield in vitro and enabled pathway reuse, lyophilization and storage. Moreover, eMIL nanoreactors delivered the six-protein system into mammalian cells, where it produced violacein from cell-provided substrates and cofactors. These findings pave the way for the design of "smart" stimuli-responsive multi-enzyme nanoreactors for biotechnological and medical applications. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/704820v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@2ce26forg.highwire.dtl.DTLVardef@c5fe1eorg.highwire.dtl.DTLVardef@ae8e06org.highwire.dtl.DTLVardef@1ee4f24_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

Adaptive disorder as the hallmark of nanobodies antigen-binding loops

Nanobodies are antigen-binding proteins of great interest as diagnostics and therapeutics. Accurate and fast characterization of their complementarity-determining regions (CDRs) is crucial to uncover the principles guiding their design. Yet, this task remains challenging, as random recombination and somatic mutations generate highly diverse CDR sequences that escape motif-based or structure-prediction approaches currently used to identify them. To overcome this hurdle, we employed two independent strategies that converged on the same conclusion. At the sequence level, we developed a deep learning model to identify nanobody CDRs directly from the primary sequence. At the structural level, we applied an energy decomposition method, revealing CDRs as residues highly uncoupled to the rest of the fold. Explainability analyses showed the network captured intrinsic CDR properties, which notably aligned with these energy values. CDRs emerge as fuzzy regions capable of adopting diverse conformational ensembles, from which a preferred state is selected upon antigen binding. This finding supports a model where chaos in both sequence and structure appears adaptive and disorder emerges as the hallmark of nanobody CDRs. This work aims to advance the definition of rules for the design of antigen binding regions, paving the way for the next-generation immune diagnostics and therapeutics.

immunology↗

bioRxiv2025

Developmental excitation-inhibition imbalance permanently reprograms autism-relevant social brain circuits

An influential theory proposes that an imbalance between excitation and inhibition (E:I) plays a central role in the etiology of autism and related developmental disorders. However, controversy exists as to whether this imbalance is a direct causal mechanism for autism, or a compensatory response to other primary etiological factors. Using chemogenetic manipulations in neonatal mice, we show that a transient E:I imbalance during development is sufficient to permanently reprogram autism-relevant social brain circuits. Chemogenetically manipulated mice exhibit lifelong impairments in sociability, persistent dysregulation of multiple autism-risk synaptic genes, and sustained cortical hyperexcitability in adulthood. Importantly, these social impairments are robustly rescued by pharmacological inhibition of neuronal excitability. Developmental E:I imbalance also disrupts functional connectivity in social brain regions enriched for transcriptionally dysregulated genes, suggesting a convergence of transcriptional and circuit-level pathology. Finally, multivariate modelling shows that behavioral dysfunction in chemogenetically manipulated animals closely associates with disrupted connectivity between prefrontal and mesolimbic dopaminergic regions. Collectively, our findings reconcile conflicting theories in the field and point to activity-dependent transcriptional remodeling as a foundational mechanism by which transient E:I imbalance during development can cause lasting, autism-relevant circuit dysfunction.

neuroscience↗

bioRxiv2025

Molecular Basis and Cellular Effects of Janus-Class-Driven Cytoplasmic PYK2 Coacervates

Kinase activity is increasingly associated with biomolecular phase separation. Focal adhesion kinase (FAK) forms membrane-associated condensates with paxillin to promote adhesion. Here we show that its paralogue, proline-rich tyrosine kinase 2 (PYK2), undergoes phase separation via a distinct mechanism. PYK2 forms cytoplasmic condensates primarily driven by its kinase- FAT linker (KFL) region. Overexpression of PYK2 induces condensates enriched in its autophosphorylated form, which sequester paxillin from focal adhesions and impair cell adhesion. We uncover an autoregulatory mechanism involving the KFL, linking self-association, autophosphorylation, and condensation. Uncommon among known phase separation drivers, KFL condensation is phosphorylation-independent and its sequence belongs to the "Janus" class. Using a transformer-based protein language model, we identified non-homologous sequences with similar features, many from adhesion and cytoskeletal regulators. We validated the phase-separating potential of several of these sequences in cells. These findings reveal a novel mechanism linking phase separation with kinase activation, and demonstrate distinct condensation behavior in close homologues. Our results also highlight how protein concentration modulates condensate function, with implications for disease, and expand the landscape of phase separation drivers.

cell biology↗

bioRxiv2024

Point mutations of the mitochondrial chaperone TRAP1 affect its functions and pro-neoplastic activity.

The mitochondrial chaperone TRAP1 is a key regulator of cellular homeostasis and its activity has important implications in neurodegeneration, ischemia and cancer. Recent evidence has indicated that TRAP1 mutations are involved in several disorders, even though the structural basis for the impact of point mutations on TRAP1 functions has never been studied. By exploiting a modular structure-based framework and molecular dynamics simulations, we investigated the effect of five TRAP1 mutations on its structure and stability. Each mutation differentially impacts long-range interactions, intra and inter-protomer dynamics and ATPase activity. Changes in these parameters influence TRAP1 functions, as revealed by their effects on the activity of the TRAP1 interactor succinate dehydrogenase (SDH). In keeping with this, TRAP1 point mutations affect the growth and migration of aggressive sarcoma cells, and alter sensitivity to a selective TRAP1 inhibitor. Our work provides new insights on the structure-activity relationship of TRAP1, identifying crucial amino acid residues that regulate TRAP1 proteostatic functions and pro-neoplastic activity.

cancer biology↗

bioRxiv2024

Disulfide-mediated tetramerization of TRAP1 fosters its antioxidant and pro-neoplastic activities.

The mitochondrial chaperone TRAP1 exerts a protective function in cells exposed to diverse stress conditions in both physiological and pathological contexts. In cancer cells, it contributes to neoplastic progression ensuing metabolic rewiring and protection from oxidative insults. TRAP1 works as a homodimer, but recent evidence has indicated that it can form tetramers whose functional effects remain elusive. Here, we find that TRAP1 forms redox-sensitive tetramers via disulfide bonds involving two critical cysteine residues, C261 and C573. TRAP1 tetramerization is elicited by oxidative stress and abrogated upon expression of the double C261S/C573R mutant. In cancer contexts, the expression of the TRAP1 C261S/C573R mutant is unable to inhibit the activity of its client succinate dehydrogenase and to confer protection against oxidative insults, and it hampers invasiveness of aggressive sarcoma cells. Our data indicate that TRAP1 undergoes tetramerization in response to oxidative stress and identify C261 and C573 as critical for TRAP1 structural rearrangement and for functions.

cell biology↗

bioRxiv2024

Optic nerve crush does not induce retinal ganglion cell loss in the contralateral eye.

PurposeOptic nerve crush (ONC) is a model for studying optic nerve trauma. Unilateral ONC induces massive retinal ganglion cell (RGC) degeneration in the affected eye, leading to vision loss within a month. A common assumption has been that the non-injured contralateral eye is unaffected due to the minimal anatomical decussation of the RGC projections at the chiasm. Yet, recently, microglia, the brain-resident macrophages, have shown a responsive phenotype in the contralateral eye after ONC. Whether RGC loss accompanies this phenotype is still controversial. MethodsUsing the available RGCode algorithm and developing our own RGC-Quant deep-learning-based tool, we quantify RGCs total number and density across the entire retina after ONC. ResultsWe confirm a short-term microglia response in the contralateral eye after ONC, but this did not affect microglia number. Furthermore, we cannot confirm the previously reported RGC loss between naive and contralateral retinas five weeks after ONC induction across the commonly used Cx3cr1creERT2 and C57BL6/J mouse models. Neither sex nor the direct comparison of the RGC markers Brn3a and RBPMS, with Brn3a co-labeling, on average, 89% of the RBPMS+-cells, explained this discrepancy, suggesting that the early microglia-responsive phenotype does not have immediate consequences on the RGC number. ConclusionsOur results corroborate that unilateral optic nerve injury elicits a microglial response in the uninjured contralateral eye but without RGC loss. Therefore, the contralateral eye should be treated separately and not as an ONC control.

neuroscience↗

bioRxiv2024

How a pathogenic mutation impairs Hsp60 functional dynamics from monomeric to fully assembled states

Heat Shock Protein 60 kDa (Hsp60) is a mitochondrial chaperonin that cooperates with Hsp10 to drive the correct folding of client proteins. Monomers M of Hsp60 (featuring equatorial, intermediate, and apical domains) first assemble into 7-meric Single rings (S), then pairs of S interface equatorially to form 14-meric Double rings (D) that accommodate clients into their lumen. Recruitment of 7 Hsp10 molecules per pole turns D into a 28-meric Football-shaped complex (F). Sequential hydrolysis of ATP present in each Hsp60 unit of F finally drives client folding and F disassembly. Equatorial domain mutation V72I occurs in SPG13, a form of hereditary spastic paraplegia: while distal to the active site, this severely impairs the chaperone cycle and stability. To understand the molecular bases of this impairment we have run atomistic molecular dynamics (MD) simulations of M, S, D, and F for both WT and mutant Hsp60, with two catalytically relevant Hsp60 aspartates in D and F modelled in three different protonation states. Additionally, D in one protonation state was modelled post-hydrolysis (total production time: 36 {micro}s). By combining complementary experimental and computational approaches for the analysis of functional dynamics and allosteric mechanisms, we consistently find that mutation V72I significantly rewires allosteric routes present in WT Hsp60 across its complexes, from isolated M units right up to F, rigidifying them--as observed experimentally--by introducing a direct allosteric link between equatorial and apical Hsp60 domains that bypasses the ATP binding site (wherein we observe the alteration of mechanisms driving reactivity). Our results reveal a multiscale complexity of functional mechanisms for Hsp60 and its pathogenic mutant, and may lay the foundation for the design of experiments to fully understand both variants.

biochemistry↗

bioRxiv2023

Decrypting the languages of allostery in membrane-bound K-Ras4B using four complementary in silico approaches

Proteins are evolved molecular machines whose diverse biochemical functions can be dynamically regulated by allostery, through which even faraway protein residues can conformationally communicate. Allostery can express itself in different ways, akin to different "languages": allosteric control pathways predominating in an unperturbed protein are superseded by others as soon as a perturbation arises--e.g., a mutation--that alters its function (pathologically or not). Accurately modeling these often-unintuitive phenomena could therefore help explain functional changes in a specific protein whenever they are unclear. Unbiased molecular dynamics (MD) simulations are a possibility; however, since allostery can operate at longer timescales than those accessible by MD, simulations require integration with a reliable method able to, e.g., detect regions of incipient allosteric change or likely perturbation pathways. Several methods exist but are typically applied singularly: we argue their joint application, in a "multilingual" approach, could significantly enrich this picture. To prove this, we perform unbiased MD simulations ([~]100 {micro}s) of the widely studied, allosterically active oncotarget K-Ras4B, solvated and embedded in a phospholipid membrane, proceeding to decrypt its allostery using four showcase "languages": Distance Fluctuation analysis and the Shortest Path Map capture allosteric communication hotspots at equilibrium; Anisotropic Thermal Diffusion and Dynamical Non-Equilibrium MD simulations assess them once the GTP that oncogenically activates K-Ras4B is, respectively, either superheated or hydrolyzed. "Languages" provide a uniquely articulate, mutually coherent, experimentally consistent picture of allostery in K-Ras4B. At equilibrium, pathways stretch from the membrane-embedded hypervariable region all the way to the active site, touching known flexible allosteric "switches" and proposed pockets. Upon GTP cleavage/perturbation, known to inactivate K-Ras4B, allosteric signals most reverberate on switches and interfaces that recruit effector proteins. Our work highlights the benefits of integrating different allostery detection methods with unbiased MD.

biochemistry↗

bioRxiv2023

Echoes from the last Green Sahara: whole genome analysis of Fulani, a key population to unveil the genetic evolutionary history of Africa

BackgroundThe Sahelian Fulani are the largest nomadic pastoral ethnic group. Their origins are still largely unknown and their Eurasian genetic component is usually explained by recent admixture events with northern African groups. However, it has also been proposed that Fulani may be the descendants of ancient groups settled in the Sahara during its last Green phase (12000-5000 BP), as also suggested by Y chromosome results. ResultsWe produced 23 high-coverage (30 x) whole genomes from Fulani individuals from 8 Sahelian countries, plus 17 samples from other African groups and 3 Europeans as controls, for a total of 43 new whole genome sequences. These data have been compared with 814 published modern whole genomes and analyzed together with relevant published ancient individuals (for a total of > 1800 samples). These analyses showed that the non-sub-Saharan genetic ancestry component of Fulani cannot be only explained by recent admixture events, but it could be shaped at least in part by older events by events more ancient than previously reported, possibly tracing its origin to the last Green Sahara. ConclusionsAccording to our results, Fulani may be the descendants of Saharan cattle herders settled in that area during the last Green Sahara. The exact ancestry composition of such ghost Saharan population(s) cannot be completely unveiled from modern genomes only, but the joint analysis with the available African ancient samples suggested a similarity between ancient Saharans and Late Neolithic Moroccans.

genetics↗

bioRxiv2022

How Aberrant N-Glycosylation Can Alter Protein Functionality and Ligand Binding: an Atomistic View

Protein assembly defects due to enrichment of aberrant conformational variants of proteins are emerging as a new frontier in therapeutics design. Understanding, atomistically, structural elements that remodel the energy landscape of proteins, with the consequence of rewiring the conformational dynamics of proteins and pathologically perturbing functionally-oriented ensembles, is key for development of inhibitors. This is particularly relevant for molecular chaperones, hub proteins for the assembly of large multiprotein complexes, where enrichment of aberrant conformers can have a large impact on the cellular proteome, and in turn, on phenotypes. Here, we integrate computational and experimental tools to unveil how N-glycosylation of specific residues in glucose-regulated protein 94 (GRP94) modulates internal dynamics and alters the conformational fitness of regions fundamental for interaction with the nucleotide and synthetic ligands, and impacts substructures dedicated to recognition of interacting proteins. We show how N-glycosylation plays an active role in modulating the energy landscape of the protein, with specific glycosylation patterns determining specific functionally-oriented dynamic signatures. Our results provide support for leveraging the structural-dynamics knowledge on distinct glycosylation variants to design molecules targeting GRP94 disease-associated conformational states and assemblies. Since glycosylation is the most abundant form of post-translational modification, our results and mechanistic models can readily be transferred to other targets and contexts for cancers and other diseases.

biochemistry↗

bioRxiv2022

Sex, stress and UCP2: mitochondria illuminate microglial identity beyond the dichotomy

Balanced and dynamic mitochondrial networks are essential for cell survival and function. Mitochondrial networks remodel their connectivity, content and subcellular localization to support optimized energy production under conditions of increased stress. In vivo, stressors can arise from the environment, such as in neuronal injury, or from mutation-induced cellular dysfunction. Cells programmed to identify and respond to these stress signals, like microglia, rely on optimized mitochondrial function, however we know very little about mitochondrial networks of microglia in vivo or their adaptation to environmental or cellular stressors. Here, we define the mitochondrial networks of retinal microglia in physiological conditions in vivo and evaluate network alterations by taking advantage of a microglia-selective mitochondria-labeled mouse model. First, we demonstrate significant differences in the mitochondrial networks of microglia in vivo and in vitro. Then, we induced neuronal injury in the in vivo environment using optic nerve crush, where responsive microglia exhibit more fragmented mitochondrial networks with increased content and perinuclear localization, supporting a state of increased cellular stress. Surprisingly, when we selectively increase cellular stress by knocking out the mitochondria-associated gene uncoupling protein 2 (UCP2), only male UCP2KO microglia establish a hyperfused mitochondrial network after injury, indicating sex differences in microglial stress mitigation. Ovariectomy in UCP2KO females elicits a shift toward the male hyperfused mitochondrial phenotype suggesting that circulating estrogens are a contributing factor to the differences in microglial stress mitigation.

neuroscience↗

bioRxiv2022

Orexin action on the dopaminergic system modulates theta during REM sleep and wakefulness

Both dopaminergic (DA) and orexinergic (OX) systems establish brain-wide neuromodulatory circuits that profoundly influence brain states and behavioral outputs. To unravel their interactions, we inactivated OX-to-DA neurotransmission by selective disruption of HcrtR1/OxR1, or HcrtR2/OxR2, or both receptors, in DA neurons. Chronic loss of OXR2 in DA neurons (OxR2Dat-CKO mice) dramatically increased electrocorticographic (EcoG) theta rhythms in wakefulness and REM sleep. Episode duration and total times spent in active wakefulness and REMS were prolonged, and theta/fast-gamma wave coupling was enhanced in both states. Increased theta in OxR2DatCKO mice baseline wake was accompanied by diminished infra-theta and increased fast-gamma activities, i.e. the mice exhibited signs of constitutive electrocortical hyperarousal, albeit uncoupled with locomotor activity. These effects were not seen in OxR1-ablated dopaminergic mutants, which tended to show opposite phenotypes, resembling those caused by the loss of both receptors. Our data establish a clear, genetically-defined link between monosynaptic orexin-to-dopaminergic connectivity and the power of theta oscillations, with a differential role of OXR2 in cross-frequency wave coupling and attentional processes.

neuroscience↗

bioRxiv2021

Microglial MorphOMICs unravel region- and sex-dependent morphological phenotypes from postnatal development to degeneration

Microglia contribute to tissue homeostasis in physiological conditions with environmental cues influencing their ever-changing morphology. Strategies to identify these changes usually involve user-selected morphometric features, which, however, have proved ineffective in establishing a spectrum of context-dependent morphological phenotypes. Here, we have developed MorphOMICs, a topological data analysis approach to overcome feature-selection-based biases and biological variability. We extracted a spatially heterogeneous and sexually-dimorphic morphological phenotype for seven adult brain regions, with ovariectomized females forming their own distinct cluster. This sex-specific phenotype declines with maturation but increases over the disease trajectories in two neurodegeneration models, 5xFAD and CK-p25. Females show an earlier morphological shift in the immediately-affected brain regions. Finally, we demonstrate that both the primary- and the short terminal processes provide distinct insights to morphological phenotypes. MorphOMICs maps microglial morphology into a spectrum of cue-dependent phenotypes in a minimally-biased and semi-automatic way.

neuroscience↗

bioRxiv2021

SARS-CoV-2 Spike Protein Mutations and Escape from Antibodies: a Computational Model of Epitope Loss in Variants of Concern

The SARS-CoV-2 spike (S) protein is exposed on the viral surface and is the first point of contact between the virus and the host. For these reasons it represents the prime target for Covid-19 vaccines. In recent months, variants of this protein have started to emerge. Their ability to reduce or evade recognition by S-targeting antibodies poses a threat to immunological treatments and raises concerns for their consequences on vaccine efficacy. To develop a model able to predict the potential impact of S-protein mutations on antibody binding sites, we performed unbiased multi-microsecond molecular dynamics of several glycosylated S-protein variants and applied a straightforward structure-dynamics-energy based strategy to predict potential changes in immunogenic regions on each variant. We recover known epitopes on the reference D614G sequence. By comparing our results, obtained on isolated S-proteins in solution, to recently published data on antibody binding and reactivity in new S variants, we directly show that modifications in the S-protein consistently translate into the loss of potentially immunoreactive regions. Our findings can thus be qualitatively reconnected to the experimentally characterized decreased ability of some of the Abs elicited against the dominant S-sequence to recognize variants. While based on the study of SARS-CoV-2 Spike variants, our computational epitope-prediction strategy is portable and could be applied to study immunoreactivity in mutants of proteins of interest whose structures have been characterized, helping the development/selection of vaccines and antibodies able to control emerging variants.

biophysics↗

bioRxiv2021

Chimeric GPCRs mimic distinct signaling pathways and modulate microglia responses

G protein-coupled receptors (GPCRs) regulate multiple processes ranging from cell growth and immune responses to neuronal signal transmission. However, ligands for many GPCRs remain unknown, suffer from off-target effects or have poor bioavailability. Additional challenges exist to dissect cell type-specific responses when the same GPCR is expressed on different cells within the body. Here, we overcome these limitations by engineering DREADD-based GPCR chimeras that selectively bind their agonist clozapine-N-oxide (CNO) and mimic a GPCR-of-interest. We show that the chimeric DREADD-{beta}2-adrenergic receptor ({beta}2AR/ADRB2) triggers comparable responses to levalbuterol on second messenger and kinase activity, post-translational modifications, and protein-protein interactions. Moreover, we successfully recapitulate {beta}2AR-mediated filopodia formation in microglia, a {beta}2AR-expressing immune cell that can drive inflammation in the nervous system. To further dissect microglial inflammation, we compared DREADD-{beta}2AR with two additionally designed DREADD-based chimeras mimicking GPR65 and GPR109A/HCAR2, both enriched in microglia. DREADD-{beta}2AR and DREADD-GPR65 modulate the inflammatory response with a similar profile as endogenously expressed {beta}2AR, while DREADD-GPR109A had no impact. Our DREADD-based approach allows investigation of cell type-dependent signaling pathways and function without known endogenous ligands.

synthetic biology↗