Biology subjects

Leone, V.

Publications and source records attributed to Leone, V..

13 recordsLinked to original sources

bioRxiv2026

Structural modelling and functional analysis support lipid binding by the dimeric E. coli DedA protein YqjA

Maintenance of membrane homeostasis is essential for bacterial viability, yet the molecular functions of many membrane proteins involved in this process remain poorly understood. The widely distributed DedA superfamily of integral membrane proteins has been implicated in membrane homeostasis, with deletion of DedA genes resulting in sensitivities to temperature, pH and a range of antimicrobial compounds. Several bacterial DedA proteins have been linked to lipid transport, providing a potential connection between function and phenotype. However, a lack of direct functional and structural data means that the precise role of DedA proteins in bacterial membrane homeostasis remains unclear. Here, using analytical ultracentrifugation (AUC), we show that the Escherichia coli DedA protein YqjA exists predominantly as a dimer and, by combining structural modelling with site-specific cysteine crosslinking, we have identified the most likely dimer interface. Modelling of dimeric YqjA in the presence of lipids predicts an interfacial lipid-binding site located in close proximity to several conserved, functionally important residues. Consistent with lipid binding, addition of lipid substantially increased the thermal stability of YqjA. Furthermore, mutation of residues associated with the predicted binding site impaired YqjA function. Together, these findings provide new structural and functional insights into the DedA family and support a role for lipid binding in the activity of YqjA.

biochemistry↗

bioRxiv2024

A new class of binding protein-dependent solute transporter exemplified by the TAXI-GltS system from Bordetella pertussis

Tripartite ATP-dependent periplasmic (TRAP) transporters are widespread in prokaryotes, but absent in eukaryotes, and facilitate the uptake of a wide variety of substrates. TRAP transporters are composed of a substrate binding protein (SBP) and two unequally sized membrane components that exist as either separate proteins or are fused into a single polypeptide. Almost all TRAP SBPs exist as monomeric globular proteins that bind substrate and present it to the membrane component. Here, we describe the discovery and characterisation of a novel TRAP SBP from the TAXI subfamily with a previously unidentified architecture. BP0403 from the human pathogen Bordetella pertussis is a predicted lipoprotein composed of 3 distinct domains; an /{beta} globular domain with a unique fold, a long helical domain and a C-terminal TAXI SBP domain. Expression and purification of the full-length protein reveals that it forms a stable dimer. Structural modelling of the dimer interface and molecular weight analysis using size exclusion chromatography of the individual domains reveals that the interdomain helical region is solely responsible for dimerization. Differential scanning fluorimetry (DSF) and intrinsic tyrosine fluorescence reveal that BP0403 binds L-glutamate with nanomolar affinity. Unexpectedly, analysis of the genome context of BP0403 reveals the complete absence of characteristic genes for TRAP membrane components but co-localisation and translational coupling with gltS, encoding a Na+/glutamate symporter. In other bacteria, we identified fused BP0403-GltS homologues, strongly suggesting that this constitutes a completely novel SBP-dependent secondary active transporter. Structural comparisons suggest GltS operates by an elevator-type mechanism, like TRAP transporters; the association of an SBP with this class of secondary transporter is an emerging theme.

biochemistry↗

bioRxiv2024

Engineering de novo binder CAR-T cell therapies with generative AI

Chimeric antigen receptor T cell (CAR-T) therapies have revolutionized cancer treatment, with six CAR-T products currently in clinical use1-4. Despite their success, high resistance rates due to antigen escape remain a major challenge5,6. In silico design of de novo binders (DNBs) has the potential to accelerate the development of new binding domains for CAR-T, possibly enabling personalized therapies for cancer resistance7,8. Here, we show that DNBs can be used for CAR-T, targeting clinically relevant cancer antigens. Using a DNB against the epidermal growth factor receptor (EGFR), we demonstrate comparable cytotoxicity, cytokine secretion, long-term proliferation, and lysis of primary patient-derived cancer organoids with single-chain variable fragment (scFv)-based and DNB-based CAR-T cells. Moreover, we use generative artificial intelligence (AI) guided binder design with RFdiffusion9 to target the B cell maturation antigen (BCMA), a key antigen in multiple myeloma treatment10-17. We confirmed the activity of our AI-designed BCMA CAR-T in short- and long-term effector readouts, including a xenograft mouse model of multiple myeloma. Notably, our AI-guided CAR-T approach also successfully targets a mutated BCMA protein variant resistant to the clinically used bispecific antibody teclistamab. In sum, we demonstrate a proof-of-concept for engineering new, bespoke cellular immunotherapies targeting cancer resistance with the help of generative AI. This approach may further accelerate the development of new CAR-T therapies addressing cancer resistance.

bioengineering↗

bioRxiv2023

HMGB1 acts as an agent of host defense at the gut mucosal barrier

Mucosal barriers provide the first line of defense between internal body surfaces and microbial threats from the outside world.1 In the colon, the barrier consists of two layers of mucus and a single layer of tightly interconnected epithelial cells supported by connective tissue and immune cells.2 Microbes colonize the loose, outer layer of colonic mucus, but are essentially excluded from the tight, epithelial-associated layer by host defenses.3 The amount and composition of the mucus is calibrated based on microbial signals and loss of even a single component of this mixture can destabilize microbial biogeography and increase the risk of disease.4-7 However, the specific components of mucus, their molecular microbial targets, and how they work to contain the gut microbiota are still largely unknown. Here we show that high mobility group box 1 (HMGB1), the prototypical damage-associated molecular pattern molecule (DAMP), acts as an agent of host mucosal defense in the colon. HMGB1 in colonic mucus targets an evolutionarily conserved amino acid sequence found in bacterial adhesins, including the well-characterized Enterobacteriaceae adhesin FimH. HMGB1 aggregates bacteria and blocks adhesin-carbohydrate interactions, inhibiting invasion through colonic mucus and adhesion to host cells. Exposure to HMGB1 also suppresses bacterial expression of FimH. In ulcerative colitis, HMGB1 mucosal defense is compromised, leading to tissue-adherent bacteria expressing FimH. Our results demonstrate a new, physiologic role for extracellular HMGB1 that refines its functions as a DAMP to include direct, virulence limiting effects on bacteria. The amino acid sequence targeted by HMGB1 appears to be broadly utilized by bacterial adhesins, critical for virulence, and differentially expressed by bacteria in commensal versus pathogenic states. These characteristics suggest that this amino acid sequence is a novel microbial virulence determinant and could be used to develop new approaches to diagnosis and treatment of bacterial disease that precisely identify and target virulent microbes.

microbiology↗

bioRxiv2022

Autoregulation of a trimeric transporter involves the cytoplasmic domains of both adjacent subunits

Membrane transporters mediate the passage of molecules across membranes and are essential for cellular function. While the transmembrane region of these proteins is responsible for substrate transport, often the cytoplasmic regions are required for modulating their activity. However, it can be difficult to obtain atomic-resolution descriptions of these autoregulatory domains by classical structural biology techniques if they lack a single, defined structure, as they may not be resolved or may be truncated or modified to facilitate crystallization. The betaine permease, BetP, a homotrimer, is a prominent and well-studied example of a membrane protein whose autoregulation depends on cytoplasmic N- and C-terminal segments. These domains sense and transduce changes in K+ concentration and in lipid bilayer properties caused by osmotic stress. However, structural data for these terminal domains is incomplete, which hinders a clear description of the molecular mechanism of autoregulation. Here we used {micro}s-scale molecular simulations of the BetP trimer to compare reported conformations of the 45 amino-acid long C-terminal tails. The simulations provide support for the idea that the conformation derived from EM data represents a stable global orientation of the C-terminal segment under downregulating conditions. The simulations also allow a detailed molecular description of the C-terminal tail dynamics as well as its interactions with lipids, potassium ions, and the cytoplasmic surface of neighboring transporter subunits. Nevertheless, they do not provide information on the N-terminal segment, whose structure was not resolved by the structural studies. We therefore examined the possible interactions of the N-terminal tail by generating de novo models of its structure in the context of the EM-derived structure. The resultant full-length models of the BetP trimer provide a molecular framework for the arrangement of the terminal domains in the downregulated protein. In this framework, each C-terminal tail contacts the neighboring protomer in the clockwise direction (viewed from the cytoplasm), while the N-terminal tails only contact the protomer in the counterclockwise direction. This framework indicates an intricate interplay between the three protomers of BetP and, specifically, a multi-directionality that may facilitate autoregulation of betaine transport.

biochemistry↗

bioRxiv2022

Osmotic stress response in BetP: How lipids and K+ team up to overcome downregulation

The trimeric betaine symporter BetP senses an osmotic upshift via its osmosensory C-terminal domain and responds rapidly with a steep increase in transport rate. Full activation requires both an elevated internal K+ concentration and a yet unknown membrane stimulus. Moreover, the molecular mechanisms of stress sensing and upregulation remain unclear. Here, we show that K+ binding to BetP in vivo is highly cooperative. Using X-ray crystallography, we identify four putative K+ interaction sites at the C-terminal domains of BetP. Single particle CryoEM on BetP reconstituted in amphipols (AMP-BetP), in the absence of K+, revealed that the three C-terminal domains are oriented symmetrically near the membrane surface, representing a new downregulated state. The presence of K+ resulted in asymmetric partial unfolding of the C-terminal domains, which was assigned as an intermediate between the downregulated state and the conformation observed in crystal structures. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy in 2D crystals of BetP reveal glutamate/aspartate and tyrosine responses to K+, in agreement with the identified K+ interaction sites, as well as specific unfolding events in the C-terminal domain upon activation. A rearrangement of the relative protomer orientations confers upregulation through key structural elements involved in the alternating access of BetP affecting sodium and betaine binding affinities. Although K+-specific regulation is unique to BetP we discuss unfolding/refolding of sensory domains as a unifying element in hyperosmotic stress response of osmoregulated transporters.

biophysics↗

bioRxiv2020

Chloride-dependent conformational changes in the GlyT1 glycine transporter

The human GlyT1 glycine transporter requires chloride for its function. However, the mechanism by which Cl- exerts its influence is unknown. To examine the role that Cl- plays in the transport cycle, we measured the effect of Cl- on both glycine binding and conformational changes. The ability of glycine to displace the high-affinity radioligand [3H]CHIBA-3007 required Na+ and was potentiated over 1000-fold by Cl-. We generated GlyT1b mutants containing reactive cysteine residues in either the extracellular or cytoplasmic permeation pathways and measured changes in the reactivity of those cysteine residues as indicators of conformational changes in response to ions and substrate. Na+ increased accessibility in the extracellular pathway and decreased it in the cytoplasmic pathway, consistent with stabilizing an outward-open conformation as observed in other members of this transporter family. In the presence of Na+, both glycine and Cl- independently shifted the conformation of GlyT1b toward an outward-closed conformation. Together, Na+, glycine and Cl- stabilized an inward-open conformation of GlyT1b. We then examined whether Cl- acts by interacting with a conserved glutamine to allow formation of an ion pair that stabilizes the closed state of the extracellular pathway. Molecular dynamics simulations of a GlyT1 homologue indicated that this ion pair is formed more frequently as that pathway closes. Mutation of the glutamine blocked the effect of Cl-, and substituting it with glutamate or lysine resulted in outward- or inward-facing transporter conformations, respectively. These results provide novel and unexpected insight into the role of Cl- in this family of transporters.

biochemistry↗

bioRxiv2020

High fat diet disrupts diurnal interactions between REG3g and small intestinal gut microbes resulting in metabolic dysfunction

Gut microbial diurnal oscillations are important diet-dependent drivers of host circadian rhythms and metabolism that ensure optimal energy balance. Yet, the interplay between diet, microbes, and host factors that sustain intestinal oscillations is complex and poorly understood. Here, we report the host C-type lectin antimicrobial peptide Reg3{gamma} works with key ileal microbes to orchestrate these interactions in a bi-directional manner, independent from the intestinal core circadian clock. High fat diet diminishes physiologically relevant microbial oscillators essential for host metabolic homeostasis, resulting in arrhythmic host Reg3{gamma} expression and increased abundance and oscillation of Reg3{gamma}-independent gut microbes. This illustrates a transkingdom co-evolved biological rhythm involving reciprocating, sensor-effector signals between key host and microbial components that ultimately drive metabolism, but are also heavily influenced by diet. Restoring the gut microbiotas capacity to sense and transduce dietary signals mediated by specific host factors such as Reg3{gamma} could be harnessed to improve metabolic dysfunction.

microbiology↗

bioRxiv2020

Metagenomic alterations in gut microbiota precede and predict onset of colitis in the IL10 gene-deficient murine model

Background & AimsInflammatory bowel diseases (IBD) are chronic inflammatory disorders where predictive biomarkers for the disease development and clinical course are sorely needed for development of prevention and early intervention strategies that can be implemented to improve clinical outcomes. Since gut microbiome alterations can reflect and/or contribute to impending host health changes, we examined whether gut microbiota metagenomic profiles would provide more robust measures for predicting disease outcomes in colitis-prone hosts. MethodsUsing the IL-10 gene-deficient (IL-10 KO) murine model where early life dysbiosis from antibiotic (cefoperozone, CPZ) treated dams vertically-transferred to pups increases risk for colitis later in life, we investigated temporal metagenomic profiles in the gut microbiota of post-weaning offspring and determined their relationship to eventual clinical outcomes. ResultsCompared to controls, offspring acquiring maternal CPZ-induced dysbiosis exhibited a restructuring of intestinal microbial membership both in bacteriome and mycobiome that were associated with alterations in specific functional subsystems. Furthermore, among IL-10 KO offspring from CPZ-treated dams, several functional subsystems, particularly nitrogen metabolism, diverged between mice that developed spontaneous colitis (CPZ-colitis) versus those that did not (CPZ-no-colitis) at a time point prior to eventual clinical outcome. ConclusionsOur findings provide support that functional metagenomic profiling of gut microbes has potential and promise meriting further study for development of tools to assess risk and manage human IBD. SynopsisCurrently, predictive markers for the development and course of inflammatory bowel diseases (IBD) are not available. This study supports the notion that gut microbiome metagenomic profiles could be developed into a useful tool to assess risk and manage human IBD.

microbiology↗

bioRxiv2020

Development of early-stage type 1 diabetes in germ-free interleukin-10 deficient mice

Several experimental models demonstrate a role for gut microbiota in the progression of type 1 diabetes (T1D) in genetically prone hosts. While the association between disturbances in gut microbiota, or microbial dysbiosis, and complex immune diseases such as inflammatory bowel diseases (IBD) are well established, less is known about its role in T1D pathogenesis. In IBD-prone interleukin-10 deficient (IL-10 KO) mice, the absence of gut microbiota under germ-free (GF) conditions prevents IBD development. However, in aged GF IL-10 KO mice (>6-months of age), polyuria and pancreatic lymphocytic infiltration resembling T1D lesions was observed. Approximately 50% of male and female mice above 6-months of age develop pancreatic immune cell infiltration, as compared to none in conventionally-raised and fecal microbiota transplanted (FMT) IL-10 KO counterparts. Immunofluorescence staining of islet infiltrates was positive for adaptive and innate immunological markers, including lymphoid and myeloid cell markers, which typically characterize autoimmune T1D lesions. A subset of GF IL-10 KO mice was also positive for insulin autoantibodies (IAA), but the majority of mice did not become diabetic. Our findings of early stage lymphocytic infiltrates in the pancreas and IAA in the absence of overt diabetes in GF IL-10 KO mice embody the early stages of T1D pathogenesis. As such, we propose that the presence of gut microbiota play a protective role against immune infiltration in the pancreas of genetically prone hosts. Moreover, our model provides an opportunity to better understand the role of the microbiota in the early stages of immune pathogenesis and perhaps conceive the development of microbe-mediated prophylactic strategies to treat or even prevent T1D.

physiology↗

bioRxiv2020

Early-life microbial intervention reduces colitis risk promoted by antibiotic-induced gut dysbiosis

Perturbations in the early life gut microbiome are associated with increased risk to complex immune disorder like inflammatory bowel diseases. We previously showed maternal antibiotic-induced gut dysbiosis vertically passed to offspring increases experimental colitis risk in IL-10 gene deficient (IL-10-/-) mice. While this could arise from emergence of pathobionts or loss/lack of essential microbes needed for appropriate immunological education, our findings suggest the latter. A dominant Bacteroides strain absent following antibiotic-induced perturbation was cultivated from murine fecal samples. Addition of this strain into mice with antibiotic-induced dysbiosis significantly promoted immune tolerance and reduced incidence of colitis in IL-10-/- mice, but only if engrafted early in life, and not during adulthood. Thus, key members of the gut microbiome are essential for development of immune tolerance to commensal microbes in early life and their addition in presence of gut dysbiosis during this period can reduce colitis risk in genetically prone hosts. HighlightsO_LISpecific gut microbes promote early life immune tolerance to key commensal microbes C_LIO_LILoss of early life keystone microbes increases colitis risk in genetically prone hosts C_LIO_LIEmergence of absent commensal microbes late in life worsened colitis outcome C_LIO_LIEarly life exposure to a missing keystone Bacteroides strain reduced colitis risk C_LI

microbiology↗

bioRxiv2019

Phenylpropionc acid produced by gut microbiota alleviates acetaminophen-induced hepatotoxicity

ABSTRACTAcetaminophen (APAP) overdose causes hepatic injury and is major contributor to acute liver injury cases. To investigate potential roles of gut microbiota in APAP-induced liver injury, C57BL/6 mice from Jackson (JAX) or Taconic (TAC) were challenged with APAP. TAC mice were more susceptible to APAP toxicity, and this disappeared upon co-housing of JAX and TAC mice. When the cecum contents from JAX and TAC mice were transplanted to germ-free mice, the mice that received TAC gut microbiota exhibited more significant hepatotoxicity after APAP administration. Non-targeted metabolomic analysis using portal vein serum and liver tissue of the mice led to identification of 19 metabolites the levels of which are associated with JAX or TAC gut microbiota. A gut bacteria-derived metabolite phenylpropionic acid (PPA) levels in cecum contents and blood were higher in mice harboring JAX gut microbiota. PPA supplementation in drinking water alleviated APAP-induced hepatotoxicity in TAC mice. This was accompanied by reduced hepatic protein levels of cytochrome P450 (CYP) 2E1, the enzyme responsible for APAP bioactivation to a toxic metabolite. This illustrates a gut microbe-liver interaction mediated by a gut bacteria-derived metabolite in modulating drug-induced liver injury.Competing Interest StatementThe authors have declared no competing interest.View Full Text

pharmacology and toxicology↗

bioRxiv2018

Assignment of the resting state of the secondary active transporter BetP by integrating spectroscopic measurements and molecular simulations

The glycine betaine symporter BetP regulates the osmotic stress response of Corynebacterium glutamicum, a soil bacterium used extensively in biotechnology. Although BetP is a homotrimer, biochemical studies have shown that each protomer is able to transport its substrate independently. Crystallographic structures of BetP have been determined in several conformations, seemingly capturing outward-open, inward-open and occluded states, both loaded with the substrate and in the apo form. However, it has been challenging to establish a correspondence between each of these structures and specific states in the mechanism of the transporter under more physiological conditions. To this end, we examined the dynamics of spin-labelled BetP using pulsed electron-electron double resonance (PELDOR) under different stimuli. We then carried out molecular simulations of structures of the BetP monomer to interpret the PELDOR data, using the enhanced-sampling methodology EBMetaD (1), whereby the dynamics of the protein are minimally biased so as to reproduce the experimental data. Comparison of the magnitude of the biasing work required for different input structures permitted us to assign them to specific states of the transport cycle under each of the experimental conditions. In particular, this analysis showed that BetP adopts inward-facing conformations in the presence of excess sodium, and a mixture of states when betaine is added. These studies better delineate the major conformations adopted by BetP in its transport cycle, and therefore provide important insights into its mechanism. More broadly, we illustrate how integrative simulations can aid interpretation of ambiguous structural and spectroscopic data on membrane proteins.\n\nAbbreviations

biophysics↗