1181 Citations
Melanin binding of small molecule drugs can lead to targeted disposition to the pigmented tissues and prolonged pharmacological responses in the eye Melanin binding of drugs in vitro correlates with in vivo binding but current workflows for binding affinity require multiple slow steps and analytical method development and or they may result in high data variability We developed tangential flow filtration-based methodology to produce size-specific fractions of water-soluble melanin nanoparticles MNPs reducing production time from - days to just a few hours and yielding MNPs with enhanced fluorescence signal Improved MNPs enabled modifications to a previously published microscale thermophoresis-based melanin ... More
Melanin binding of small molecule drugs can lead to targeted disposition to the pigmented tissues and prolonged pharmacological responses in the eye. Melanin binding of drugs in vitro correlates with in vivo binding, but current workflows for binding affinity require multiple slow steps, and analytical method development, and/or they may result in high data variability. We developed tangential flow filtration-based methodology to produce size-specific fractions of water-soluble melanin nanoparticles (MNPs), reducing production time from 2-3 days to just a few hours and yielding MNPs with enhanced fluorescence signal. Improved MNPs enabled modifications to a previously published microscale thermophoresis-based melanin binding protocol, shifting analytical focus toward thermophoretic behavior, reducing data variability, and improving reproducibility. The process was tested with nine compounds with varying melanin binding affinities, and the results were consistent with literature, confirming the ability of the method to differentiate compounds based on melanin binding. Fast and reliable workflow will be useful in screening binding affinity for therapeutics and new drug candidates to melanin thereby facilitating ocular drug discovery and construction of predictive pharmacokinetic simulation models. Less
SummaryBACH is a transcriptional regulator that modulates various cytoprotective pathways Among these pathways BACH regulates the cellular oxidative stress responses by suppressing the expression of cytoprotective genes Dysregulated BACH activity has been implicated in a range of pathologies including chronic inflammatory diseases fibrosis and cancer making it a promising therapeutic target However BACH remains an underexploited drug target with limited pharmacological inhibitors available We have developed a novel luciferase-based reporter cell line enabling quantitative high-throughput assessment of BACH inhibition Using this platform we rigorously screened two small-molecule libraries with compounds and identified four structurally distinct compounds that robustly inhibit BACH ... More
SummaryBACH1 is a transcriptional regulator that modulates various cytoprotective pathways. Among these pathways BACH1 regulates the cellular oxidative stress responses by suppressing the expression of cytoprotective genes. Dysregulated BACH1 activity has been implicated in a range of pathologies, including chronic inflammatory diseases, fibrosis, and cancer, making it a promising therapeutic target. However, BACH1 remains an underexploited drug target, with limited pharmacological inhibitors available. We have developed a novel luciferase-based reporter cell line enabling quantitative, high-throughput assessment of BACH1 inhibition. Using this platform, we rigorously screened two small-molecule libraries with 2,046 compounds and identified four structurally distinct compounds that robustly inhibit BACH1 function. Notably, these compounds simultaneously activate transcription factor NRF2, suggesting the potential for a broader modulation of oxidative stress pathways.Importantly, we demonstrate that commonly used 2D migration assays may fail to detect phenotypes consistent with BACH1 inhibition, resulting in false negatives. In contrast, we establish that 3D invasion assays more robustly capture anti-invasive effects of BACH1 functional inhibition. Using this 3D system, we validate the identified compounds as potent suppressors of lung cancer cell invasion in vitro.This study delivers a novel screening platform for BACH1-targeted drug discovery, and challenges current in vitro standards by establishing 3D invasion assays as a more accurate functional readout for BACH1-targeting compounds. Additionally, it identifies new dual functional BACH1 inhibitors/NRF2 activators, offering novel chemical scaffolds for the development of anti-metastatic therapies and potentially treatments for diseases driven by oxidative stress and inflammation. Less
The precise and selective transport of protons across cellular membranes relies on the dynamic formation and dissipation of hydrogen-bonding networks involving water molecules protein sidechains and backbone carbonyls As in aqueous solution protons are conducted over long distances along chains of hydrogen-bonded water molecules within narrow protein pores To engineer proton-conductive pathways therefore we must explicitly account for the dynamic behavior of these networks In previous work we showed that incorporation of polar Gln residues into hydrophobic pores drives formation of transient single-file water wires that enable proton-selective transport Here we sought to enhance conduction by introducing targeted Ile-to-Ser substitutions ... More
The precise and selective transport of protons across cellular membranes relies on the dynamic formation and dissipation of hydrogen-bonding networks involving water molecules, protein sidechains, and backbone carbonyls. As in aqueous solution, protons are conducted over long distances along chains of hydrogen-bonded water molecules within narrow protein pores. To engineer proton-conductive pathways, therefore, we must explicitly account for the dynamic behavior of these networks. In previous work, we showed that incorporation of polar Gln residues into hydrophobic pores drives formation of transient, single-file water wires that enable proton-selective transport. Here, we sought to enhance conduction by introducing targeted Ile-to-Ser substitutions to extend connectivity across the pore. We find that the position of Ser relative to Gln modulates sidechain dynamics and, in turn, channel hydration. Although increased polarity reduces hydrophobic length and enhances hydration, these effects alone do not explain the observed conduction rates. Instead, asymmetry in the arrangement and dynamics of polar sidechains emerges as a key determinant of proton conductivity. Together, these results demonstrate that proton conduction is governed not only by pore polarity and hydration, but also by the dynamic and asymmetric organization of hydrogen-bonding networks. This work establishes design principles for engineering proton-selective channels and reveals how asymmetry enables efficient proton transport across biological membranes. Less
The evolution of transcription factor TF DNA-binding specificity is a major driver of gene regulatory innovation Unlike most TFs which diversify through gene duplication and neofunctionalization the plant-specific LEAFY LFY TF evolved novel binding specificities without extensive duplication Here we combine experimental structural determination and biochemical assays to reveal how LFY s dimerization and DNA-binding preferences shifted during the water-to-land transition We present crystal structures of the LFY DNA-binding domain DBD from the hornwort Nothoceros aenigmaticus and the alga Interfilum paradoxum bound to DNA demonstrating two distinct dimerization mechanisms one mediated by direct protein-protein interactions and another driven by DNA-mediated ... More
The evolution of transcription factor (TF) DNA-binding specificity is a major driver of gene regulatory innovation. Unlike most TFs, which diversify through gene duplication and neofunctionalization, the plant-specific LEAFY (LFY) TF evolved novel binding specificities without extensive duplication. Here, we combine experimental structural determination and biochemical assays to reveal how LFY’s dimerization and DNA-binding preferences shifted during the water-to-land transition. We present crystal structures of the LFY DNA-binding domain (DBD) from the hornwort Nothoceros aenigmaticus and the alga Interfilum paradoxum bound to DNA, demonstrating two distinct dimerization mechanisms: one mediated by direct protein-protein interactions and another driven by DNA-mediated cooperativity. In the ancestral state, LFY likely bound DNA as a dimer through DNA-mediated cooperativity, with protein-protein dimerization emerging later, enforcing new DNA-binding preferences. Our findings support a revised evolutionary scenario for LFY, highlighting the dynamic interplay between protein-DNA and protein-protein interactions as key drivers of TF binding specificity. This work deepens our understanding of how structural adaptations in TFs underpin evolutionary transitions in gene regulation. Less
The richness of our somatosensory experience is reflected in the functional diversity of somatic sensory neurons Single-cell RNA sequencing of sensory neurons has revealed a molecular basis for such diversity However sensory neuron diversity has yet to be captured at the level of the proteome Here we combined electrophysiology with deep visual proteomics to quantify over proteins from phenotypically-defined sensory neurons in mice and identified proteomic markers of sensory neuron subtypes Comparative analysis revealed both concordance and meaningful divergence between transcriptomes and proteomes We further show that up to proteins can be quantified from one-fourth of a single neuron demonstrating ... More
The richness of our somatosensory experience is reflected in the functional diversity of somatic sensory neurons. Single-cell RNA sequencing of sensory neurons has revealed a molecular basis for such diversity1,2,3. However, sensory neuron diversity has yet to be captured at the level of the proteome. Here, we combined electrophysiology with deep visual proteomics 4 to quantify over 6000 proteins from phenotypically-defined sensory neurons in mice and identified proteomic markers of sensory neuron subtypes. Comparative analysis revealed both concordance and meaningful divergence between transcriptomes and proteomes. We further show that up to 3000 proteins can be quantified from one-fourth of a single neuron, demonstrating subset-specific protein signatures. In culture, nociceptive neurons can be acutely sensitized to mechanical stimuli by nerve growth factor (NGF) which normally drives inflammatory pain in vivo5. Indeed, overnight exposure of peptidergic nociceptors to NGF and a protein kinase C (PKC) activator produced functional sensitization associated with proteome changes. Functional knockdown experiments identified the up-regulated B3GNT2 enzyme as a potential effector of nociceptor sensitization. In summary, we present a high-resolution proteomic resource linking molecular identity to function, enabling the discovery of mechanisms underlying somatic sensation and pain sensitization. Less
Constant changes in SARS-CoV- in human populations as well as potential future spillovers from animal coronaviruses have provided the impetus for the development of additional direct-acting antivirals We describe herein the discovery of a new class of broad-spectrum inhibitors of coronavirus C-like protease CLpro a cysteine protease essential for viral replication and a validated drug target that incorporate in their structure a -oxazaphospholidin- -one scaffold Inhibitors and were found to have EC values of and nM against SARS-CoV- CLpro respectively and CC values M These compounds also potently inhibited MERS-CoV CLpro IC nM and nM respectively Importantly several of the ... More
Constant changes in SARS-CoV-2 in human populations as well as potential future spillovers from animal coronaviruses have provided the impetus for the development of additional direct-acting antivirals. We describe herein the discovery of a new class of broad-spectrum inhibitors of coronavirus 3C-like protease (3CLpro), a cysteine protease essential for viral replication and a validated drug target, that incorporate in their structure a 1,3,2-oxazaphospholidin-3-one scaffold. Inhibitors 1 and 2 were found to have EC50 values of 60 and 50 nM against SARS-CoV-2 3CLpro, respectively, and CC50 values >100 µM. These compounds also potently inhibited MERS-CoV 3CLpro (IC50 120 nM and 90 nM, respectively). Importantly, several of the synthesized compounds inhibited recombinant human cathepsin L with IC50 values in the low nM to sub-nM range. Thus, the compounds can potentially exhibit high antiviral potency by abrogating viral entry via the inhibition of cathepsin L and viral replication by inhibition of 3CLpro. High resolution cocrystal structures were determined to elucidate the mechanism of action, identify the molecular determinants associated with binding, and to inform the optimization process. Less
Virtual reality VR devices are increasingly being utilized within operating theaters and intensive care units where appropriate sanitation is vital to ensure that patients do not unnecessarily acquire hospital-associated infections The morphology of VR devices in conjunction with the variety of materials and internal components provides challenges to their repurposing This study aimed to evaluate the microorganisms remaining on VR headsets following sanitation by laboratory staff in a medical education anatomy teaching facility The external components and internal facial interface were swabbed and separately cultured on four AGAR plates Horse Blood Nutrient bile Esculin and Mannitol Salt Colonies were counted ... More
Virtual reality (VR) devices are increasingly being utilized within operating theaters and intensive care units where appropriate sanitation is vital to ensure that patients do not unnecessarily acquire hospital-associated infections. The morphology of VR devices in conjunction with the variety of materials and internal components provides challenges to their repurposing. This study aimed to evaluate the microorganisms remaining on VR headsets following sanitation by laboratory staff in a medical education anatomy teaching facility. The external components and internal facial interface were swabbed and separately cultured on four AGAR plates (Horse Blood, Nutrient, bile Esculin, and Mannitol Salt). Colonies were counted, sampled, pooled and subsequently processed for shotgun metagenomic sequencing. A higher number of colonies were present on surfaces closest to the eyes and facial interface compared to the external components. Metagenomic analysis identified 27 pathogenic bacteria including 4 “ESKAPE” pathogens (Enterobacter sp., Staphylococcus aureus, Klebsiella spp. and, Escherichia coli) and numerous organisms associated with ocular infections. A broad range of antimicrobial resistance genes were identified conveying resistance to Methicillin, Aminoglycosides, Macrolides, Tetracyclines, and Polymixins. Further research is required to ensure that current sanitization practices of VR head mounted displays are appropriate within high-risk hospital settings. Less
The PD- PD-L immune checkpoint is a pivotal target for cancer immunotherapy Monoclonal antibodies mAbs targeting the PD- PD-L interaction have achieved clinical success but face limitations including high production costs suboptimal tumor penetration and potential immunogenicity To address these challenges we present the DNA-linked Inhibitor Antibody Assay DIANA a robust high-throughput screening platform optimized for identifying and characterizing low-molecular-weight inhibitors of human PD-L DIANA integrates competitive binding with qPCR detection enabling single-well determination of dissociation constants Kd and rapid screening of thousands of compounds The assay was validated using three FDA-approved mAbs atezolizumab avelumab and durvalumab the PD-L -binding ... More
The PD-1/PD-L1 immune checkpoint is a pivotal target for cancer immunotherapy. Monoclonal antibodies (mAbs) targeting the PD-1/PD-L1 interaction have achieved clinical success but face limitations, including high production costs, suboptimal tumor penetration, and potential immunogenicity. To address these challenges, we present the DNA-linked Inhibitor Antibody Assay (DIANA)─a robust, high-throughput screening platform optimized for identifying and characterizing low-molecular-weight inhibitors of human PD-L1. DIANA integrates competitive binding with qPCR detection, enabling single-well determination of dissociation constants (Kd) and rapid screening of thousands of compounds. The assay was validated using three FDA-approved mAbs (atezolizumab, avelumab, and durvalumab), the PD-L1-binding macrocyclic peptide WL12, and the native PD-1 receptor, yielding Kd values consistent with the literature. DIANA demonstrated a broad dynamic range spanning more than 4 orders of magnitude, excellent robustness (Z′-factor = 0.94), and high tolerance to DMSO (up to 10%). We applied DIANA to screen two libraries: a 5,280-compound in-house library (pooled format) and a 1,298-compound commercial peptidomimetic library (individual format). While very weak initial hits were detected, none were confirmed in follow-up manual (non-HTS) experiments or in an orthogonal cell-based assay. Nonetheless, DIANA’s sensitivity, scalability, and minimal sample requirements establish it as a powerful tool for accelerating the discovery of next-generation PD-1/PD-L1 inhibitors and overcoming key limitations of conventional screening methods. Less
Structural analyses of Trichomonas vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK)
Trichomonas vaginalis causes trichomoniasis the most common non-viral sexually transmitted disease in humans T vaginalis pyrophosphate-dependent phosphofructokinase TvPPi-PFK is a putative target for rational structure-based drug discovery given its absence in mammals and its importance for parasite survival TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose- -phosphate using pyrophosphate PPi as the phosphoryl donor This reversible reaction catalyzed by TvPPi-PFK is the first committed step in glycolysis Its reverse reaction is vital for gluconeogenesis in T vaginalis The purification crystallization structure determination and preliminary structure-functional analyses of three crystal structures of TvPPi-PFK are presented All three structures ... More
Trichomonas vaginalis causes trichomoniasis, the most common non-viral sexually transmitted disease in humans. T. vaginalis pyrophosphate-dependent phosphofructokinase (TvPPi-PFK) is a putative target for rational, structure-based drug discovery, given its absence in mammals and its importance for parasite survival. TvPPi-PFK is a cytosolic enzyme that catalyzes the phosphorylation of fructose-6-phosphate using pyrophosphate (PPi) as the phosphoryl donor. This reversible reaction, catalyzed by TvPPi-PFK, is the first committed step in glycolysis. Its reverse reaction is vital for gluconeogenesis in T. vaginalis. The purification, crystallization, structure determination, and preliminary structure-functional analyses of three crystal structures of TvPPi-PFK are presented. All three structures organize as tetramers with the conserved motifs essential for pyrophosphate binding and PPi-PFK catalytic activity. Comparative analysis with structural neighbors from other organisms demonstrated that despite sharing <29% sequence identity, TvPPi-PFK’s protomer shares overall topology with both PPi- and ATP-dependent PFKs. Mass photometry confirmed that TvPPi-PFK formed tetramers under near-physiological conditions. Unexpectedly, TvPPi-PFK crystals dephosphorylate ATP to AMP during soaking. In all three structures, either ATP or AMP is bound at the enzyme’s dimer interface, typical of ATP-PFKs, but a novel finding for PPi-PFKs. Furthermore, a sugar phosphate binding site was observed in proximity to the ATP-binding site. Thus, the three reported TvPPi-PFK structures validate its established PPi-dependent activity while revealing previously unreported ATP and sugar phosphate binding. This study also lays a foundation for future research into putative ATP-dependent activity of TvPPi-PFK and for evaluating known phosphofructokinase inhibitors as potential therapeutics for trichomoniasis. These findings expand our understanding of PFK superfamily diversity and support the continued exploration of TvPPi-PFK as a drug target for trichomoniasis Less
The ToxRS system belongs to a family of co-component transmembrane transcription regulators that act as sensors of environmental cues and regulate virulence gene expression in several bacterial pathogens These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors In the enteric pathogens Vibrio parahaemolyticus and Vibrio cholerae the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans ToxR is a DNA-binding regulator associated in the periplasm with ToxS a protein of poorly understood function ToxS modulates the activity of its binding partner ToxR in the presence ... More
The ToxRS system belongs to a family of co-component transmembrane transcription regulators that act as sensors of environmental cues and regulate virulence gene expression in several bacterial pathogens. These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors. In the enteric pathogens Vibrio parahaemolyticus and Vibrio cholerae, the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans. ToxR is a DNA-binding regulator associated in the periplasm with ToxS, a protein of poorly understood function. ToxS modulates the activity of its binding partner ToxR in the presence of bile salts, antimicrobial cholesterol metabolites secreted into the gut. To date, the molecular mechanism underlying this regulation remains unclear. We present crystal structures of the V. parahaemolyticus ToxS periplasmic domain (ToxSp) with and without the bile salt glycocholate. ToxSp forms an 8-stranded broken β-barrel with a central α-helix and is structurally homologous to a group of chaperone proteins. ToxSp has a highly conserved hydrophobic core that stabilizes the β-barrel fold, while the binding pocket tolerates substantial variation, consistent with binding hydrophobic ligands. Strikingly, we discovered that Vp-ToxSp binds three molecules of glycocholate and the presence of this bile salt leads to the formation a strand-swapped ToxS homodimer. Finally, modeling two ToxR periplasmic domains in complex with the glycocholate-bound ToxSp homodimer provides a structure-based model for bile salt-mediated heterotetramerization of the ToxRS system. Overall, our study addresses a major longstanding question in the field of Vibrio virulence regulation providing a scenario that could apply to other pathogens that utilize these membrane-bound family transcriptional regulators. Less
Nucleic acid nanoparticles NANPs fabricated by using DNA origami are an emerging delivery vector for nucleic acid therapeutics Despite their advantages over other nanomaterials that include controlled spatial presentation of targeting ligands such as lipids and sugars understanding their cell targeting and uptake mechanisms remains limited Here we investigated NANP cellular targeting uptake and delivery of small interfering RNAs siRNAs to liver and neuronal cell models in vitro Using a rational design approach we targeted NANPs to two clinically validated receptors the asialoglycoprotein receptor ASGPR and the low-density lipoprotein receptor LDLR respectively using GalNAc and lipidation We systematically evaluated how ... More
Nucleic acid nanoparticles (NANPs) fabricated by using DNA origami are an emerging delivery vector for nucleic acid therapeutics. Despite their advantages over other nanomaterials that include controlled spatial presentation of targeting ligands such as lipids and sugars, understanding their cell targeting and uptake mechanisms remains limited. Here, we investigated NANP cellular targeting, uptake, and delivery of small interfering RNAs (siRNAs) to liver and neuronal cell models in vitro. Using a rational design approach, we targeted NANPs to two clinically validated receptors, the asialoglycoprotein receptor (ASGPR) and the low-density lipoprotein receptor (LDLR), respectively, using GalNAc and lipidation. We systematically evaluated how the ligand valency, interligand spacing, linker length, and ligand chemistry affected NANP association with on- and off-target liver cell types, revealing the relative roles of the biomolecular corona, receptor engagement, and endocytosis in these targeting strategies. We found that lipidation enhanced NANP uptake into HepG2 cells, a model cell line for hepatocytes, by promoting apolipoprotein recruitment, LDLR engagement, and clathrin-mediated endocytosis and also increased association with nonparenchymal cells. HepG2 uptake was further improved by conjugating NANPs to lipids with higher valency provided that lipids were adequately displayed away from the surface of NANP edges with more lipophilic lipids yielding greater cell association. We then benchmarked the potential for NANPs to deliver siRNAs to HepG2 cells in comparison with lipid nanoparticle and conjugate technologies and explored lipid functionalization as a strategy for nonhepatic NANP targeting to model neuronal cells. Overall, this study advances the foundational understanding of how clinically relevant targeting ligands mediate NANP interactions with both on- and off-target liver cell types in vitro, offering insights into potential design criteria for nucleic acid therapeutic delivery. Less
Recoverin is a key calcium sensor that controls the desensitization of the visual rhodopsin by GRK Previous studies have traditionally been conducted on bovine protein bRec while data on human ortholog hRec remain scarce Here we combine X-ray crystallography Xray absorption spectroscopy XANES quantum mechanical calculations molecular dynamics and functional assays to provide an integrated characterization of hRec The Ca -bound hRec structure was solved at showing that unlike bRec hRec interacts with ROS membranes at physiologically relevant submicromolar Ca levels due to a speciesspecific charge distribution that might influence membrane interactions Both recoverins form a set of Ca Zn ... More
Recoverin is a key calcium sensor that controls the desensitization of the visual rhodopsin
by GRK1. Previous studies have traditionally been conducted on bovine protein (bRec), while
data on human ortholog (hRec) remain scarce. Here, we combine X-ray crystallography, Xray absorption spectroscopy (XANES), quantum mechanical calculations, molecular
dynamics, and functional assays to provide an integrated characterization of hRec. The
2Ca2+-bound hRec structure was solved at 1.60 Å, showing that, unlike bRec, hRec interacts
with ROS membranes at physiologically relevant submicromolar Ca2+ levels, due to a speciesspecific charge distribution that might influence membrane interactions. Both recoverins
form a set of Ca2+/Zn2+-bound conformers with improved functional performance. X-ray
crystallography (1.85 Å) and XANES revealed a specific tetrahedral Zn2+ site in 1Ca2+-bound
hRec, the first such site reported in the NCS family. In 1Ca2+-bound hRec, zinc promotes the
formation of active state, whereas in 2Ca2+-state of bRec, it significantly enhances GRK1
binding, as the latter can complement the Zn2+ coordination. These data refine our
understanding of recoverin function in humans and highlight its role as a key link between
calcium and zinc signaling in mammalian photoreceptors under normal and pathological
conditions. Less
by GRK1. Previous studies have traditionally been conducted on bovine protein (bRec), while
data on human ortholog (hRec) remain scarce. Here, we combine X-ray crystallography, Xray absorption spectroscopy (XANES), quantum mechanical calculations, molecular
dynamics, and functional assays to provide an integrated characterization of hRec. The
2Ca2+-bound hRec structure was solved at 1.60 Å, showing that, unlike bRec, hRec interacts
with ROS membranes at physiologically relevant submicromolar Ca2+ levels, due to a speciesspecific charge distribution that might influence membrane interactions. Both recoverins
form a set of Ca2+/Zn2+-bound conformers with improved functional performance. X-ray
crystallography (1.85 Å) and XANES revealed a specific tetrahedral Zn2+ site in 1Ca2+-bound
hRec, the first such site reported in the NCS family. In 1Ca2+-bound hRec, zinc promotes the
formation of active state, whereas in 2Ca2+-state of bRec, it significantly enhances GRK1
binding, as the latter can complement the Zn2+ coordination. These data refine our
understanding of recoverin function in humans and highlight its role as a key link between
calcium and zinc signaling in mammalian photoreceptors under normal and pathological
conditions. Less
AXIS: a Lab-in-the-Loop machine learning approach for automated detection of macromolecular crystals
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design Nowadays the use of robotic systems for crystal growth and diffraction analysis is widespread and high-throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field However the identification of crystals is still largely carried out through manual inspection sometimes involving tens of thousands of images which represents a bottleneck in an otherwise highly automated process Here we describe AXIS an AI-based Crystal Identification System combining the DINOv computer vision model state-of-the-art transfer learning and MARCO the largest crystallization dataset available to date for ... More
Macromolecular crystallography provides mechanistic understanding of biological processes and can be applied in drug design. Nowadays, the use of robotic systems for crystal growth and diffraction analysis is widespread and high-throughput protein-to-structure pipelines for ligand and fragment screening are revolutionizing the field. However, the identification of crystals is still largely carried out through manual inspection, sometimes involving tens of thousands of images, which represents a bottleneck in an otherwise highly automated process. Here we describe AXIS, an AI-based Crystal Identification System combining the DINOv2 computer vision model, state-of-the-art transfer learning and MARCO, the largest crystallization dataset available to date, for automated crystal detection. AXIS can operate with both visible and UV light images and integrates a Lab-in-the-Loop approach combining ML and expert inputs for iterative learning and specialization. AXIS enables automated annotation of large crystallization image datasets with performance and accuracy comparable to that of human experts, and the Lab-in-the-Loop approach introduced here enables efficient adaptation to local conditions, facilitating widespread application, which has been a major limitation to date. AXIS can help to correct human errors in image annotation and removes critical bottlenecks, particularly in the context of extensive crystallization screens or high-throughput applications like fragment and ligand screening, unlocking the potential for higher levels of automation that are key in both fundamental and translational research. Less
Antisense oligonucleotides ASOs are promising therapeutics but safety concerns such as liver toxicity and off-target OffT effects necessitate thorough evaluation during the compound selection process This study leverages time course global proteomics and transcriptomics to assess ASO-induced changes in vitro comparing liver toxic versus non-liver toxic ASOs The research confirms that ASOs perturb different cellular pathways at both RNA and protein levels effectively discriminating between liver toxic and non-liver toxic ASOs Contrary to expectations protein level reduction isn t delayed relative to ASO-induced RNA reduction highlighting the importance of understanding RNA and protein level relationships in specific model systems Furthermore ... More
Antisense oligonucleotides (ASOs) are promising therapeutics, but safety concerns such as liver toxicity and off-target (OffT) effects necessitate thorough evaluation during the compound selection process. This study leverages time course global proteomics and transcriptomics to assess ASO-induced changes in vitro, comparing liver toxic versus non-liver toxic ASOs. The research confirms that ASOs perturb different cellular pathways at both RNA and protein levels, effectively discriminating between liver toxic and non-liver toxic ASOs. Contrary to expectations, protein level reduction isn’t delayed relative to ASO-induced RNA reduction, highlighting the importance of understanding RNA and protein level relationships in specific model systems. Furthermore, many OffT effects observed at the RNA level do not directly translate to corresponding protein level changes. These findings suggest that current RNA-focused OffT assessment strategies capture predicted OffTs but could benefit from protein level studies that could potentially de-risk oligonucleotide drug (OND) candidates with seemingly problematic OffT profiles at the RNA level. The study underscores the value of global proteomics as a complement to RNAseq in ASO drug development, refining safety assessment and improving candidate selection. Less
Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane OM provides an intrinsic barrier to many antimicrobials Key to this barrier function is the asymmetric structure of the OM with phospholipids constituting the inner leaflet and lipopolysaccharides the outer leaflet Although the mechanism of phospholipid transport between the inner membrane IM and OM remains poorly understood recent studies implicate TamB YhdP and YdbH as functionally redundant proteins mediating this process in Escherichia coli Accordingly collective loss of these three paralogs is lethal and any one of them is sufficient for growth YdbH is anchored to the ... More
Gram-negative bacteria pose a threat to global healthcare mainly because their outer membrane (OM) provides an intrinsic barrier to many antimicrobials. Key to this barrier function is the asymmetric structure of the OM, with phospholipids constituting the inner leaflet and lipopolysaccharides the outer leaflet. Although the mechanism of phospholipid transport between the inner membrane (IM) and OM remains poorly understood, recent studies implicate TamB, YhdP, and YdbH as functionally redundant proteins mediating this process in Escherichia coli. Accordingly, collective loss of these three paralogs is lethal and any one of them is sufficient for growth. YdbH is anchored to the IM and its periplasmic repeating β-sheet groove domain interacts with the OM lipoprotein YnbE via β-strand augmentation to form an intermembrane bridge. Additionally, YnbE multimerizes, and the periplasmic protein YdbL is proposed to modulate YnbE multimerization to facilitate its stacking on the C-terminus of YdbH. Here, we demonstrate that excess YdbL specifically inhibits the function of the YdbH-YnbE complex since overexpression of ydbL causes lethality in the ΔyhdP ΔtamB double mutant but the presence of both ydbH and ynbE in trans abrogates this lethality. We resolve high-resolution structural data for YdbL and ascertain its interaction site with the YnbE C-terminal α-helix, with residues mediating this interface highly conserved and critical for YdbL function. Finally, we show that YdbL is protected from degradation by the protease DegP when complexed with YnbE. Overall, our data supports a model in which YdbL ensures proper YdbH-YnbE intermembrane bridge formation by directly interacting with YnbE. Less
UreE is a nickel chaperone that is required for the safe and efficient delivery of nickel to the active site of the metalloenzyme urease which is a key virulence factor of the urinary-tract pathogen Proteus mirabilis We investigated the structural features of P mirabilis UreE PmUreE using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma mass spectrometry Here we report a resolution crystal structure of homodimeric PmUreE and show that it has the capacity to bind five Ni II ions per dimer Truncation of the histidine-rich C-terminus reduced the nickel-binding capacity by two Ni II ions per ... More
UreE is a nickel chaperone that is required for the safe and efficient delivery of nickel to the active site of the metalloenzyme urease, which is a key virulence factor of the urinary-tract pathogen Proteus mirabilis. We investigated the structural features of P. mirabilis UreE (PmUreE) using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma mass spectrometry. Here, we report a 2.0 Å resolution crystal structure of homodimeric PmUreE and show that it has the capacity to bind five Ni(II) ions per dimer. Truncation of the histidine-rich C-terminus reduced the nickel-binding capacity by two Ni(II) ions per dimer, and comparison with homologous UreE structures allowed the assignment of putative nickel-binding sites within the PmUreE structure. These findings increase our understanding of how PmUreE binds nickel and ultimately prevents this toxic metal from causing significant cellular damage in P. mirabilis. Less
The Smart-seq family of methods represents the gold standard for high-sensitivity full-length single-cell RNA sequencing Despite iterative improvements fundamental challenges remain the generation of non-specific PCR products that limit sensitivity the inability to capture precise Transcription End Sites TES and the insidious generation of phantom UMIs artificial molecular barcodes created during PCR that systematically inflate molecular counts Here we present ESPeR-seq a novel architecture that resolves these barriers To enable precise stranded TES capture we developed an Omega-dT primer that bypasses synthetic poly-T tracts restoring high-quality sequencing directly at transcript termini To eliminate both PCR background and phantom UMIs we ... More
The Smart-seq family of methods represents the gold standard for high-sensitivity, full-length single-cell RNA sequencing. Despite iterative improvements, fundamental challenges remain: the generation of non-specific PCR products that limit sensitivity, the inability to capture precise Transcription End Sites (TES), and the insidious generation of “phantom UMIs”—artificial molecular barcodes created during PCR that systematically inflate molecular counts. Here, we present ESPeR-seq, a novel architecture that resolves these barriers. To enable precise, stranded TES capture, we developed an “Omega-dT” primer that bypasses synthetic poly-T tracts, restoring high-quality sequencing directly at transcript termini. To eliminate both PCR background and phantom UMIs, we implemented a biochemical “multi-lock” mechanism utilizing uracil-containing TSOs and a uracil-intolerant DNA polymerase. We validate this approach using the logQ-slope, a novel metric that sensitively diagnoses UMI fidelity. Benchmarking reveals that while state-of-the-art methods still exhibit signs of UMI inflation, ESPeR-seq strictly prevents it. Furthermore, the strandedness and precise end-delineation provided by TSO and dT reads support robust de novo gene model reconstruction, enabling the discovery of novel multi-exon genes, unannotated 3’ UTR extensions, and candidate eRNAs across aggregated single-cell populations. Thus, ESPeR-seq establishes a robust framework for absolute quantitative accuracy and full-length isoform resolution. Less
Mass spectrometry-based proteomics increasingly demands platforms that combine quantitative rigor with the discovery capabilities of accurate mass systems Here we present the ZenoTOF system a compact mass spectrometry system that integrates enhanced ion capture and transmission optics with an optical detection system Zeno trap-enhanced MS MS electron-activated dissociation and scanning quadrupole data-independent acquisition ZT Scan DIA We show that ZT Scan DIA outperforms conventional variable-window DIA Zeno SWATH DIA in both identifications and quantitative reproducibility and demonstrate the platform s versatility across proteomics applications thousands of protein groups from bulk samples at up to samples per day single-cell proteomics yielding ... More
Mass spectrometry-based proteomics increasingly demands platforms that combine quantitative rigor with the discovery capabilities of accurate mass systems. Here we present the ZenoTOF 8600 system, a compact mass spectrometry system that integrates enhanced ion capture and transmission optics with an optical detection system, Zeno trap-enhanced MS/MS, electron-activated dissociation, and scanning quadrupole data-independent acquisition (ZT Scan DIA). We show that ZT Scan DIA outperforms conventional variable-window DIA (Zeno SWATH DIA) in both identifications and quantitative reproducibility, and demonstrate the platform’s versatility across proteomics applications: thousands of protein groups from bulk samples at up to 500 samples per day, single-cell proteomics yielding up to 4,700 proteins, accurate ratio recovery in mixed-species quantitative benchmarks, low-attomole targeted quantitation, and detection of disease-relevant phosphorylation in a Parkinson’s disease cellular model using complementary CID and EAD fragmentation. The instrument’s compact footprint makes it attractive for settings where both analytical breadth and operational robustness are required. Less
The chikungunya virus CHIKV outbreak imposes a significant burden on healthcare systems and raises an urgent need for effective antiviral therapies So far there are no specific drugs against CHIKV A CHIKV macrodomain is critical for virulence and counteracts the host immune response representing a promising antiviral drug target Here we describe small molecule inhibitors targeting the CHIKV macrodomain Compound MDOLL- was identified through a high-throughput screening using a fluorescence resonance energy transfer FRET -based assay and its inhibitory activity was validated through multiple orthogonal assays Compound has a dual thiobarbiturate-indole scaffold and exhibits an IC of M X-ray crystallography ... More
The chikungunya virus (CHIKV) outbreak imposes a significant burden on healthcare systems and raises an urgent need for effective antiviral therapies. So far there are no specific drugs against CHIKV. A CHIKV macrodomain is critical for virulence and counteracts the host immune response, representing a promising antiviral drug target. Here, we describe small molecule inhibitors targeting the CHIKV macrodomain. Compound 1 (MDOLL-0273) was identified through a high-throughput screening using a fluorescence resonance energy transfer (FRET)-based assay, and its inhibitory activity was validated through multiple orthogonal assays. Compound 1 has a dual thiobarbiturate-indole scaffold and exhibits an IC50 of 8.9 µM. X-ray crystallography revealed that the inhibitor occupies an adenine binding site of the macrodomain and extends into a novel cryptic pocket. Notably, the inhibitor shows high selectivity for the CHIKV macrodomain over a panel of human and viral ADP-ribosyl binding and hydrolyzing proteins. Structure-activity relationship studies and medicinal chemistry efforts provide a promising starting point for further hit optimization. Less
The advancement of single-crystal structural analysis has emerged as a pivotal technology surpassing spectroscopic methods in revealing the intricate structural details of organic small molecules including crystal packing and stereochemical configurations It plays a critical role across scientific domains such as chemistry biology agronomy and medicine Traditional single-crystal X-ray diffraction SCXRD has always been restricted by its stringent requirements on the physical state size and quality of crystals This review discusses the arsenal of equipment and theoretical techniques for obtaining single-crystal structures including SCXRD PXRD CSP and more recently Micro-ED It further explores the significant crystal growth techniques based on ... More
The advancement of single-crystal structural analysis has emerged as a pivotal technology surpassing spectroscopic methods in revealing the intricate structural details of organic small molecules, including crystal packing and stereochemical configurations. It plays a critical role across scientific domains such as chemistry, biology, agronomy, and medicine. Traditional single-crystal X-ray diffraction (SCXRD) has always been restricted by its stringent requirements on the physical state, size, and quality of crystals. This review discusses the arsenal of equipment and theoretical techniques for obtaining single-crystal structures, including SCXRD, PXRD & CSP, and more recently, Micro-ED. It further explores the significant crystal growth techniques based on three foundational methods: solution-based crystallization, melt crystallization, and sublimation crystallization. Detailed discussion is provided on the crystallizability of molecules and the refinement of crystal growth methods. Specifically, for crystallizable analytes, a combination of crystal growth enhancement techniques and high-throughput technologies (under-oil) can compensate for poor crystallinity, small size, and defects under normal conditions. For molecules inherently resistant to crystallization, a “crystallization chaperone”, such as a MOFs as a crystalline sponge or tetraaryladamantane as a cocrystallization chaperone, can determine absolute configurations. Looking ahead, this review emphasizes the potential of artificial intelligence and machine learning approaches for crystal growth and structural prediction. The development of integrated analysis strategies combining SCXRD, PXRD, and Micro-ED is identified as a future trend for providing comprehensive structural insights. This review highlights the significance of advancements in single-crystal structural analysis techniques, paving the way for groundbreaking innovations in molecular design and materials science, and predicts a bright future for the field with new technologies. Less
Safe and effective vaccines against co-circulating mosquito-borne orthoflaviviruses such as Zika virus ZikV and the four serotypes of Dengue virus DenV - must elicit broadly neutralizing antibodies bnAbs to prevent the risk of enhancement of infection by non-neutralizing antibodies We recently discovered new orthoflavivirus-directed bnAbs including F S which neutralizes DenV - and ZikV with comparable or superior potency to the previously characterized E dimer epitope EDE bnAbs Here we used cryoEM and X-ray crystallography to understand the basis of cross-neutralization of F S at the molecular level We obtained a cryoEM structure of F S Fab bound to a ... More
Safe and effective vaccines against co-circulating mosquito-borne orthoflaviviruses such as Zika virus (ZikV) and the four serotypes of Dengue virus (DenV1-4) must elicit broadly neutralizing antibodies (bnAbs) to prevent the risk of enhancement of infection by non-neutralizing antibodies. We recently discovered new orthoflavivirus-directed bnAbs, including F25.S02, which neutralizes DenV1-4 and ZikV with comparable or superior potency to the previously characterized E dimer epitope (EDE) bnAbs. Here, we used cryoEM and X-ray crystallography to understand the basis of cross-neutralization of F25.S02 at the molecular level. We obtained a ~ 4.2 Å cryoEM structure of F25.S02 Fab bound to a stabilized DenV3 soluble E protein dimer and a 2.3 Å crystal structure of F25.S02 Fab bound to ZikV soluble E protein dimer. Like previously described EDE1 bnAbs, the structural epitope of F25.S02 is at the E dimer interface, encompassing predominantly conserved regions in domain II, including the fusion loop. However, unlike EDE1 bnAbs, F25.S02 binding is almost entirely dependent on the heavy chain and is shifted slightly away from the dimer symmetry axis. Our findings emphasize the importance of this cross-neutralizing site of vulnerability for DenV and ZikV that can facilitate rational design of vaccines and therapeutics. Less
-Hemolysin Ahly is a major Staphylococcus aureus virulence determinant implicated in tissue injury and immune dysregulation antibody inhibitors have reached clinical trials but alternatives with improved ease of manufacture and tissue penetration are desirable Here we demonstrate that phage-derived bicyclic peptides can serve as compact chemically tractable Ahly neutralisers Using TATB-scaffolded M phage libraries we identified WNP-motif containing bicyclic binders with a lead hit of Peptide KD nM and progressed the lead by iterative affinity maturation to Peptide KD nM and by incorporation of strategically chosen non-canonical amino acids to yield Peptide KD nM A co-crystal structure with AhlyH A ... More
α-Hemolysin (Ahly) is a major Staphylococcus aureus virulence determinant implicated in tissue injury and immune dysregulation; antibody inhibitors have reached clinical trials but alternatives with improved ease of manufacture and tissue penetration are desirable. Here we demonstrate that phage-derived bicyclic peptides can serve as compact, chemically tractable Ahly neutralisers. Using TATB-scaffolded M13 phage libraries we identified WNP-motif containing bicyclic binders, with a lead hit of Peptide 14 (KD = 1792nM) and progressed the lead by iterative affinity maturation to Peptide 20 (KD = 609 nM) and by incorporation of strategically chosen non-canonical amino acids to yield Peptide 88 (KD = 96 nM). A 2.2 Å co-crystal structure with AhlyH35A locates the binding footprint on the rim domain and explains the critical role of the WNP motif in target engagement. Functional assays show that the Peptide 88 blocks Ahly mediated hemolysis, inhibits Ahly driven ADAM10 activation, and elucidate its inhibitory mechanism of preventing Ahly binding to human A549 epithelial cells. Peptide 88 protects A549 cells from recombinant toxin and attenuates cytotoxicity in S. aureus co-culture experiments, whilst showing no toxicity to A549 cells. Bicyclic peptides thus represent a new and promising anti-virulence modality: small, synthetically accessible molecules that mimic antibody recognition, with therapeutic potential against S. aureus infections. Less
Imidazolium LipidBrick cationic lipid nanoparticles LNPs provide a pH-independent alternative to conventional ionizable systems for nucleic acid delivery Through a high-throughput screen of formulations spanning eight imidazolium cores three helper lipids and varying PEG densities we found that more than half of the library outperformed the clinical ionizable benchmark ALC- in multiple representative mammalian cell types Top-performing candidates showed robust cellular uptake efficient endosomal escape and strong transgene expression both in vitro and following intramuscular administration A lead formulation C LNP incorporating an imidazolium lipid core bearing a hydroxyethyl substituent with mol DOPE achieved comparable intramuscular luciferase expression and antibody ... More
Imidazolium LipidBrick® cationic lipid nanoparticles (LNPs) provide a pH-independent alternative to conventional ionizable systems for nucleic acid delivery. Through a high-throughput screen of 1,944 formulations spanning eight imidazolium cores, three helper lipids, and varying PEG densities, we found that more than half of the library outperformed the clinical ionizable benchmark ALC-0315 in multiple representative mammalian cell types. Top-performing candidates showed robust cellular uptake, efficient endosomal escape, and strong transgene expression both in vitro and following intramuscular administration. A lead formulation (C3 LNP), incorporating an imidazolium lipid core bearing a hydroxyethyl substituent, with 30 mol% DOPE, achieved comparable intramuscular luciferase expression and antibody titers to ALC-0315, while eliciting ∼ 3-fold stronger ovalbumin-specific IFN-γ+ T-cell responses and maintaining low cytotoxicity. Machine-learning analysis of the dataset further distilled transferable design rules to inform future formulation strategies. Collectively, these findings establish cationic LipidBrick® LNPs as a versatile platform for mRNA delivery, offering a generalizable framework for the high-throughput discovery of ionization-independent systems that effectively prime adaptive immune responses. Less
Tankyrases are poly-ADP-ribosyltransferases that orchestrate numerous biological processes involved in disease Their established regulatory roles particularly within the WNT -catenin pathway have driven notable drug discovery efforts aimed at inhibiting their catalytic activity Targeting tankyrases interaction with proteins through their ARC domains represents an alternative strategy to be explored as a therapeutic approach against specific protein-protein interactions In this article we employed a pre-established FRET-based assay to screen the EU-OPENSCREEN libraries for identification of ARC inhibitors We discovered a series of pyrrolone-based compounds and we synthesized compound S ARCher- which binds selectively to ARC with a potency of M NMR ... More
Tankyrases are poly-ADP-ribosyltransferases that orchestrate numerous biological processes involved in disease. Their established regulatory roles, particularly within the WNT/β-catenin pathway, have driven notable drug discovery efforts aimed at inhibiting their catalytic activity. Targeting tankyrases’ interaction with proteins through their ARC domains represents an alternative strategy to be explored as a therapeutic approach against specific protein-protein interactions. In this article, we employed a pre-established FRET-based assay to screen the EU-OPENSCREEN libraries for identification of ARC4 inhibitors. We discovered a series of pyrrolone-based compounds, and we synthesized compound S8 (ARCher-142), which binds selectively to ARC4 with a potency of 8 μM. NMR analysis and X-ray crystallography allowed us to identify the binding site and to rationalize the observed selectivity. Despite binding exclusively to ARC4, the inhibitor can attenuate the WNT/β-catenin signaling pathway in cells. Our work demonstrates that targeting single ARC domains is possible, offering an inhibition approach tailored to tankyrase ARC4. Less
Complement and pathogenic antibodies act independently and together to mediate the pathology of many autoimmune diseases To address these drivers of disease we generated a monoclonal antibody mAb CSL that binds and inhibits both complement and the neonatal Fc fragment crystallizable receptor FcRn The fragment antigen binding Fab portion of CSL was engineered to bind both human C huC zymogen and the active fragment huC b to inhibit the classical and lectin complement pathways in vitro and C b deposition on primary lung endothelial cells using a -dimensional microvascular model system Engineering of a triple amino acid mutation YPY motif ... More
Complement and pathogenic antibodies act independently and together to mediate the pathology of many autoimmune diseases. To address these drivers of disease, we generated a monoclonal antibody (mAb), CSL305, that binds and inhibits both complement and the neonatal Fc (fragment crystallizable) receptor FcRn. The fragment antigen binding (Fab) portion of CSL305 was engineered to bind both human C2 (huC2) zymogen and the active fragment huC2b to inhibit the classical and lectin complement pathways in vitro, and C3b deposition on primary lung endothelial cells using a 3-dimensional microvascular model system. Engineering of a triple amino acid mutation (“YPY” motif) into the Fc region of CSL305 increased its affinity to FcRn at both acidic and neutral pH, allowing it to also act as a potent FcRn antagonist. Intracellular trafficking experiments demonstrated that CSL305, but not the wild-type (WT) mAb lacking the YPY motif, was able to block immunoglobulin G (IgG) recycling in vitro. The generation of a high resolution 2.6Å crystal structure of CSL305 Fab region bound to huC2b showed that the epitope lies directly over the huC2b catalytic triad, providing evidence of its complement mechanism of action as a neutralising mAb. Early pharmacokinetic (PK)/pharmacodynamic (PD) studies using CSL305 in cynomolgus monkeys demonstrated both complement inhibition and FcRn antagonism in vivo, with reductions in complement classical pathway activity and endogenous IgG observed following single intravenous (IV) administration. CSL305 thus represents a dual-functional mAb as a potential therapeutic candidate. Less
Improving health and quality of life in our society is a key focus of drug development Methods for drug discovery are being optimized in multiple ways to reduce costs and timelines Crystallographic fragment screening CFS is increasingly being employed as an early screening method in drug discovery projects Here we demonstrate that selecting the optimal protein crystal form can significantly impact hit rates Two CFS campaigns are carried out against the two crystal forms of the SARS-CoV- main protease using the same fragment library and an almost identical experimental setup Although both crystal forms exhibit similar diffraction properties the observed ... More
Improving health and quality of life in our society is a key focus of drug development. Methods for drug discovery are being optimized in multiple ways to reduce costs and timelines. Crystallographic fragment screening (CFS) is increasingly being employed as an early screening method in drug discovery projects. Here, we demonstrate that selecting the optimal protein crystal form can significantly impact hit rates. Two CFS campaigns are carried out against the two crystal forms of the SARS-CoV-2 main protease, using the same fragment library and an almost identical experimental setup. Although both crystal forms exhibit similar diffraction properties, the observed hit rates in the two campaigns differ significantly. A hit rate of 3% is determined for the monoclinic crystals, while a hit rate of 16% is observed for the orthorhombic crystals. These findings are consistent with the more open molecular packing in the orthorhombic crystals, where the solvent channels leading to the active sites are approximately twice the size of those in the monoclinic crystal form. Our results highlight the critical importance of the crystal form in a crystallographic screening, identifying it as one of the most important parameters to optimize when preparing a CFS campaign. Less
Light Oxygen Voltage LOV domains are important widespread receptors of blue light that also found applications in optogenetics and imaging While LOV domains from mesophiles are relatively well characterized their counterparts from thermophilic microorganisms remain understudied Here we express two constructs of a LOV domain belonging to a histidine kinase from Meiothermus ruber MrLOV and MrLOVe and show that they are photoactive with recovery time values of and min respectively and thermostable Crystal structures reveal that MrLOV which lacks helices A and J forms a parallel dimer whereas MrLOVe is a tetramer organized as an antiparallel dimer of two parallel ... More
Light Oxygen Voltage (LOV) domains are important widespread receptors of blue light that also found applications in optogenetics and imaging. While LOV domains from mesophiles are relatively well characterized, their counterparts from thermophilic microorganisms remain understudied. Here, we express two constructs of a LOV domain belonging to a histidine kinase from Meiothermus ruber, MrLOV and MrLOVe, and show that they are photoactive, with recovery time values of 21 and 27 min, respectively, and thermostable. Crystal structures reveal that MrLOV, which lacks helices A’α and Jα, forms a parallel dimer, whereas MrLOVe is a tetramer organized as an antiparallel dimer of two parallel dimers interacting via helices Jα. One MrLOVe dimer is symmetric, and the other is asymmetric, with conformational differences mirroring activation-related changes in other LOV domains. Our data provide the structural basis for understanding and engineering of thermophilic LOVs and pave the way for development of thermostable and photostable LOV-derived optogenetic tools and flavin-based fluorescent proteins. Less
Background and objectives The PWWP domain of lens epithelium-derived growth factor p LEDGF p mediates chromatin engagement through recognition of histone H lysine di- and trimethylation H K me and nucleosomal DNA LEDGF p plays a role in multiple human diseases In particular its interaction with HIV- integrase enables viral genome integration However the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets Here we report the generation of high-affinity nanobodies Nbs to investigate the structure and function of this domain Methods Camelids were immunized with recombinant LEDGF PWWP domain and immune ... More
Background and objectives: The PWWP domain of lens epithelium-derived growth factor p75 (LEDGF/p75) mediates chromatin engagement through recognition of histone H3 lysine 36 di- and trimethylation (H3K36me2/3) and nucleosomal DNA. LEDGF/p75 plays a role in multiple human diseases. In particular, its interaction with HIV-1 integrase enables viral genome integration. However, the LEDGF PWWP domain remains difficult to target with small molecules as it lacks optimally shaped binding pockets. Here we report the generation of high-affinity nanobodies (Nbs) to investigate the structure and function of this domain. Methods: Camelids were immunized with recombinant LEDGF PWWP domain, and immune phage display libraries were screened for affinity. Selected Nbs were recombinantly expressed in E. coli and purified. Their interaction with the PWWP domain of LEDGF and its close homolog HRP-2 was characterized using size-exclusion chromatography and surface plasmon resonance. Structural characterization of the Nbs was performed by X-ray crystallography. Functional effects on chromatin engagement were evaluated using the AlphaScreen assay. Results: Nine sequence-distinct Nbs were identified, seven of which were confirmed to bind the LEDGF PWWP domain with nanomolar affinities. Five Nbs also bound the HRP-2 domain, consistent with conserved functional surfaces, while two showed reduced affinity. Crystal structures of two Nbs (NbC03 and NbH10) confirmed canonical immunoglobulin folds, while the latter additionally revealed a domain-swapped dimer. Moreover, NbH10 dose-dependently inhibited the interaction between full-length LEDGF/p75 and H3K36me3-modified nucleosomes in vitro. Conclusions: This work establishes a validated panel of Nbs targeting the LEDGF PWWP domain and demonstrates their ability to functionally disrupt the LEDGF-chromatin interaction. These Nbs serve as valuable tools towards functional studies and structure-based drug design. Less
Gene expression is governed by dynamic switches between repressive and activating transcriptional states Among the molecules mediating these transitions chromatin readers and transcription factors play pivotal roles However how they assemble with regulatory machineries to enable crosstalk between gene repression and activation remains unknown Here we use an integrative structural dynamics approach combining cryo-EM crosslinking mass spectrometry fragment-resolved protein interactome mapping and crystallography to show how the dual-role chromatin reader Cti and transcription factors Ash and Ume engage the Sin deacetylase complex a major regulatory hub in eukaryotes We find that Cti competes with Ash to drive its dynamic recruitment ... More
Gene expression is governed by dynamic switches between repressive and activating transcriptional states1,2. Among the molecules mediating these transitions, chromatin readers and transcription factors play pivotal roles3,4. However, how they assemble with regulatory machineries to enable crosstalk between gene repression and activation remains unknown. Here, we use an integrative structural dynamics approach – combining cryo-EM, crosslinking mass spectrometry, fragment-resolved protein interactome mapping and crystallography – to show how the dual-role chromatin reader Cti6 and transcription factors Ash1 and Ume6 engage the Sin3 deacetylase complex, a major regulatory hub in eukaryotes5. We find that Cti6 competes with Ash1 to drive its dynamic recruitment to a shared peripheral module, while Ume6 engages the Sin3 scaffold through a defined, minimal interface. Using high-throughput mutational scanning, we reveal deleterious and gain-of-function mutations in Sin3, identifying evolutionarily conserved residues essential for anchoring transcription factors. Together, these results provide structural and functional insights into how dual-role regulators engage the central Sin3 complex, revealing subtle assembly principles that may facilitate crosstalk between gene repression and activation. They also establish an integrative multidisciplinary framework to dissect the dynamics of macromolecular assemblies across biological systems. Less
Filament-forming proteins such as TasA Bacillus subtilis and camelysins CalY CalY Bacillus cereus pose a particular challenge for structural analysis due to their strong tendency to self-association and their polydispersity which severely limits their ability to crystallize or to be a target for NMR-spectroscopy To address this it is necessary to modify the amino acid sequence to prevent filamentation Engineering a series of N- and C-terminal truncated variants by removing flexible parts is often key to success N-terminal extensions are also a powerful tool for obtaining crystals of fiber-forming proteins
Epstein-Barr virus EBV causes infectious mononucleosis and contributes to neurodegenerative disorders and malignancies particularly in immune-compromised hosts Transplant patients face high risk of post-transplant lymphoproliferative disease a life-threatening EBV-driven lymphoma There are no EBV-specific vaccines or treatments however neutralizing antibodies against EBV glycoproteins may offer utility as therapeutic agents EBV entry into B cells involves gp which binds complement receptors and gp which engages HLA class II to trigger fusion Most existing monoclonal antibodies mAbs against these antigens are non-human limiting clinical use Using a transgenic mouse model we generate two gp and eight gp genetically human neutralizing mAbs that ... More
Epstein-Barr virus (EBV) causes infectious mononucleosis and contributes to neurodegenerative disorders and malignancies, particularly in immune-compromised hosts. Transplant patients face high risk of post-transplant lymphoproliferative disease, a life-threatening EBV-driven lymphoma. There are no EBV-specific vaccines or treatments; however, neutralizing antibodies against EBV glycoproteins may offer utility as therapeutic agents. EBV entry into B cells involves gp350, which binds complement receptors, and gp42, which engages HLA class II to trigger fusion. Most existing monoclonal antibodies (mAbs) against these antigens are non-human, limiting clinical use. Using a transgenic mouse model, we generate two gp350 and eight gp42 genetically human neutralizing mAbs that block receptor binding. Structural analyses reveal extended sites of vulnerability relevant to vaccine development. Delivery of a gp42 mAb protects humanized mice from EBV challenge, while a gp350 mAb provides partial protection. These mAbs highlight the utility of transgenic mice to produce therapeutic mAbs for preventing EBV-driven disease. Less
HECT E ligases regulate many cellular processes yet how they recognise their substrates and synthesise specific types of poly-ubiquitin chains is still incompletely understood HECTD a member of the other HECT family is implicated in the regulation of inflammation apoptosis and infection and highly expressed in several cancers These functions are largely attributed to its ligase activity and modification of diverse substrates with different types of ubiquitin chains We present a detailed analysis of the ligase activity of HECTD including its ubiquitin linkage preferences oligomeric state and substrate ubiquitination Using cryo-EM we provide the full-length structures of HECTD in both ... More
HECT E3 ligases regulate many cellular processes, yet how they recognise their substrates and synthesise specific types of poly-ubiquitin chains is still incompletely understood. HECTD3, a member of the “other HECT” family, is implicated in the regulation of inflammation, apoptosis, and infection and highly expressed in several cancers. These functions are largely attributed to its ligase activity and modification of diverse substrates with different types of ubiquitin chains. We present a detailed analysis of the ligase activity of HECTD3, including its ubiquitin linkage preferences, oligomeric state and substrate ubiquitination. Using cryo-EM, we provide the full-length structures of HECTD3 in both apo and ubiquitin-loaded forms, revealing key insights into its domain organisation, including discovery of a distinct fold of the N-terminal region, and mechanistic features. Some of these are shared with other HECT ligases, while others are unique to HECTD3 and contribute to differences in its catalytic mechanisms and functional diversity. Less
Autosomal recessive congenital ichthyosis ARCI refers to a group of rare highly debilitating skin disorders that significantly impair patients quality of life and lack any effective treatment options Here we report clinically relevant in situ correction of the most common ARCI-causing mutation TGM c - A G a splice-site aberration in human disease models Targeted skin barrier modulation followed by topical application of the cytosine base editor eTd packaged into lipid nanoparticles yielded functional restoration of of wild-type transglutaminase activity in skin tissue Toxicity studies and comprehensive off-target analysis demonstrated an excellent safety profile even after repeated application without systemic ... More
Autosomal recessive congenital ichthyosis (ARCI) refers to a group of rare, highly debilitating skin disorders that significantly impair patients’ quality of life and lack any effective treatment options. Here, we report clinically relevant in situ correction of the most common ARCI-causing mutation, TGM1 c.877-2A>G, a splice-site aberration, in human disease models. Targeted skin barrier modulation followed by topical application of the cytosine base editor eTd packaged into lipid nanoparticles yielded functional restoration of ∼30% of wild-type transglutaminase 1 activity in skin tissue. Toxicity studies and comprehensive off-target analysis demonstrated an excellent safety profile even after repeated application, without systemic distribution of the lipid nanoparticles or the genetic cargo as determined via highly sensitive methods, including desorption electrospray ionization (DESI) metabolic imaging. This study presents comprehensive preclinical data on the feasibility of in situ gene correction of genodermatoses-causing mutations, showcasing its therapeutic potential and paving the way for curative next-generation treatments for severe genetic skin diseases. Less
Transformation of agro-industrial products into value-added products such as prebiotic oligosaccharides is a key element of the emerging bioeconomy Here we characterized a new GH glucuronoxylanase from Bacillus pumilus BpXyn A for its potential in producing xylooligosaccharides XOS BpXyn A showed tolerance to ethanol and NaCl and released both linear and branched XOS containing MeGlcA at the penultimate nonreducing end residue Its X-ray structure determined at resolution revealed high similarity to other glucuronoxylanases Furthermore BpXyn A achieved higher xylan conversion yields from corn cob and Eucalyptus sawdust than Ruminococcus champanellensisRcXyn A Finally fermentation assays showed that Bifidobacterium adolescentis metabolized neutral ... More
Transformation of agro-industrial products into value-added products, such as prebiotic oligosaccharides, is a key element of the emerging bioeconomy. Here, we characterized a new GH30_8 glucuronoxylanase from Bacillus pumilus (BpXyn30_8A) for its potential in producing xylooligosaccharides (XOS). BpXyn30_8A showed tolerance to ethanol and NaCl and released both linear and branched XOS containing MeGlcA at the penultimate nonreducing end residue. Its X-ray structure, determined at 2.16 Å resolution, revealed high similarity to other glucuronoxylanases. Furthermore, BpXyn30_8A achieved higher xylan conversion yields from corn cob and Eucalyptus sawdust than Ruminococcus champanellensisRcXyn30A. Finally, fermentation assays showed that Bifidobacterium adolescentis metabolized neutral XOS to acetate and lactate, whereas acidic XOS were poorly utilized. These results highlight the potential of BpXyn30_8A as a valuable enzyme for the green transformation of plant biomass into prebiotic oligosaccharides with promising applications in human and animal nutrition, health, and biotechnology. Less
Stable carbon and nitrogen isotope ratios are widely used in the life sciences to investigate diet trophic interactions and metabolic fluxes but conventional isotope ratio mass spectrometry requires milligram-scale samples limiting its applicability to small or rare biological specimens Fourier Transform Isotopic Ratio Mass Spectrometry FT IsoR MS enables amino acid resolved isotope analysis in a proteomics-compatible workflow and has previously been demonstrated at the microgram scale Here we assess the lower sample limit of FT IsoR MS by integrating it with single-cell proteomics style sample preparation Using human HeLa cells cultured in C-glucose enriched and control media we show ... More
Stable carbon and nitrogen isotope ratios are widely used in the life sciences to investigate diet, trophic interactions, and metabolic fluxes, but conventional isotope ratio mass spectrometry requires milligram-scale samples, limiting its applicability to small or rare biological specimens. Fourier Transform Isotopic Ratio Mass Spectrometry (FT IsoR MS) enables amino acid–resolved isotope analysis in a proteomics-compatible workflow and has previously been demonstrated at the microgram scale. Here, we assess the lower sample limit of FT IsoR MS by integrating it with single-cell proteomics–style sample preparation. Using human HeLa cells cultured in 13C-glucose–enriched and control media, we show that reliable relative δ13C measurements can be obtained from as few as 50 cells, corresponding to <10 ng of total protein, with a precision of approximately ±9‰. The observed amino acid–specific labeling patterns are metabolically coherent and consistent with bulk measurements, while smaller cell numbers (≤10 cells) do not yield statistically robust results. These findings establish the practical sensitivity threshold of FT IsoR MS at the low-nanogram level and demonstrate its suitability for isotope-resolved analyses of small cell populations, micro-organoids, and other low-input biological samples, thereby extending stable isotope analysis toward single-cell–scale applications. Less
Mitochondrial crista junctions CJs operate as regulated gateways into the cristae microenvironment whose protein metabolite and ion compositions are finely tuned for mitochondrial function The Mic -Mic complex of the mitochondrial contact site and cristae organizing system MICOS complex was suggested to span across CJs and act as a diffusion barrier but little is known of how its dynamic architecture facilitates this task To address this open question we determined the crystal structure of an amino-terminal dimeric helical bundle of human Mic These and previous structural and biochemical data were harnessed in molecular dynamic MD simulations to develop a dynamic ... More
Mitochondrial crista junctions (CJs) operate as regulated gateways into the cristae microenvironment, whose protein, metabolite, and ion compositions are finely tuned for mitochondrial function. The Mic60-Mic19 complex of the mitochondrial contact site and cristae organizing system (MICOS) complex was suggested to span across CJs and act as a diffusion barrier, but little is known of how its dynamic architecture facilitates this task. To address this open question, we determined the crystal structure of an amino-terminal dimeric helical bundle of human Mic60. These and previous structural and biochemical data were harnessed in molecular dynamic (MD) simulations to develop a dynamic model of the human tetrameric Mic60-Mic19 subcomplex in the CJ environment, to validate its architecture using in organello cross-linking data and to computationally characterize its function as a diffusion barrier. Our integrative structural biology approach enables the functional investigation of flexible, multidomain protein complexes which escape conventional structural biology methods. Less
Natural resistance-associated macrophage proteins Nramps are divalent transition metal transporters found in most organisms typically coupling metal uptake to proton co-transport How this coupling evolved however remains unclear We present structural functional and evolutionary analyses of a clade B Nramp from the gut bacterium Bacteroides fragilis BfraNramp Phylogenetic reconstruction positions clade B as the most basal group of canonical Nramps retaining conserved metal-binding motifs while lacking most residues that form the canonical proton pathway We show that BfraNramp efficiently transports Mn and Cd with high apparent affinity but without proton co-transport or dependence on membrane potential or pH Structures of ... More
Natural resistance-associated macrophage proteins (Nramps) are divalent transition metal transporters found in most organisms, typically coupling metal uptake to proton co-transport. How this coupling evolved, however, remains unclear. We present structural, functional, and evolutionary analyses of a clade B Nramp from the gut bacterium Bacteroides fragilis (BfraNramp). Phylogenetic reconstruction positions clade B as the most basal group of canonical Nramps, retaining conserved metal-binding motifs while lacking most residues that form the canonical proton pathway. We show that BfraNramp efficiently transports Mn²⁺ and Cd²⁺ with high apparent affinity but without proton co-transport or dependence on membrane potential or pH. Structures of metal-free and Mn²⁺-bound BfraNramp reveal an inward-open conformation and a distinct metal coordination geometry involving a conserved glutamate on transmembrane helix 3. Together, these results identify clade B Nramps as proton-independent transition metal uniporters and suggest that proton coupling emerged later in Nramp evolution, following establishment of the metal-binding site. Less
The recent discovery of the isonitrile biosynthetic enzyme ScoE expanded the catalytic repertoire of the Fe II KG-dependent dioxygenase enzyme family ScoE synthesizes an isonitrile functional group from a glycyl-fatty acid adduct with both the isonitrile nitrogen and carbon atoms coming from the glycyl moiety This challenging chemistry cannot be performed in a single step Instead the mechanism appears to require two half reactions each involving KG cleavage to generate a highly reactive iron-oxygen species Here we report sixteen crystal structures that provide snapshots along the reaction trajectory of Rv a ScoE homolog from Mycobacterium tuberculosis These structures which are ... More
The recent discovery of the isonitrile biosynthetic enzyme ScoE expanded the catalytic repertoire of the Fe(II)/αKG-dependent dioxygenase enzyme family. ScoE synthesizes an isonitrile functional group from a glycyl-fatty acid adduct, with both the isonitrile nitrogen and carbon atoms coming from the glycyl moiety. This challenging chemistry cannot be performed in a single step. Instead, the mechanism appears to require two half reactions, each involving αKG cleavage to generate a highly reactive iron-oxygen species. Here, we report sixteen crystal structures that provide snapshots along the reaction trajectory of Rv0097, a ScoE homolog from Mycobacterium tuberculosis. These structures, which are both of wild-type and Rv0097 variants, include a substrate 3-((carboxymethyl)amino)decanoic acid (CADA)-bound structure, an αKG-bound structure, and a structure with both CADA and αKG bound. These structural data reveal how Rv0097 employs conformational rearrangements to protect the unstable CADA-reaction intermediate that is formed in the first half reaction while swapping out αKG cleavage products for a second molecule of αKG. Additionally, these structures, together with data from site-directed mutagenesis, provide insight into Rv0097’s preference for substrates with long alkyl chains, potentially facilitating efforts to re-engineer ScoE/Rv0097 to synthesize isonitrile functional groups on a wider range of small molecules. Less
Functional testing of cytotoxic lymphocytes is essential for research and quality control QC but most assays require freshly prepared target cells and extensive handling A ready-to-thaw no-wash flow cytometry based cytotoxicity assay was developed using pre-labeled K targets cryopreserved in STEM-CELLBANKER EX SCB as suitably sized aliquots SCB tolerability was evaluated in K NK- and primary natural killer NK cells post-cryopreservation label stability of CellTrace Violet CTV and carboxyfluorescein succinimidyl ester CFSE was assessed freezing and thawing conditions were optimized and wash versus no-wash workflows were compared using viability-based and absolute-count readouts across effector-to-target E T ratios with NK donors ... More
Functional testing of cytotoxic lymphocytes is essential for research and quality control (QC), but most assays require freshly prepared target cells and extensive handling. A ready-to-thaw, no-wash, flow cytometry–based cytotoxicity assay was developed using pre-labeled K562 targets cryopreserved in STEM-CELLBANKER® EX (SCB) as suitably sized aliquots. SCB tolerability was evaluated in K562, NK-92, and primary natural killer (NK) cells; post-cryopreservation label stability of CellTrace™ Violet (CTV) and carboxyfluorescein succinimidyl ester (CFSE) was assessed; freezing and thawing conditions were optimized; and wash versus no-wash workflows were compared using viability-based and absolute-count readouts, across effector-to-target (E:T) ratios with NK donors and NK-92 cells. Effector viability remained high at SCB concentrations up to 10%, and 5% SCB was selected for assay design. After cryopreservation, CTV labeling remained stable over the tested storage period, whereas CFSE showed substantial signal loss. Warm-medium thawing performed comparably to water-bath thawing, and the consolidated protocol (SCB plus fetal calf serum and thermal buffering) maintained high post-thaw target viability and recovery. In killing assays, lysis increased with increasing E:T ratios; omission of the post-thaw wash had minimal impact, and 5% SCB did not impair cytotoxic function. This ready-to-thaw workflow reduces hands-on time and sample manipulation, while improving standardization for reproducible results and enabling high-throughput functional testing and QC. Less
TIR Toll interleukin- receptor domains are found in proteins involved in immunity pathways in organisms ranging from humans and plant to bacteria Bacterial TIR domain-containing proteins have been shown to contribute to pathogenicity and anti-viral activity of bacteria During bacterial infection some TIR domain-containing proteins act as virulence factors to inhibit immune responses by interfering with Toll-like receptor signalling Other bacterial TIR domain-containing proteins are involved in bacterial anti-viral defence Many of TIR domain-containing proteins have been shown to have NAD cleavage activity and relevant to host cell death and bacterial anti-phage defence system In addition some TIR domain-containing proteins ... More
TIR (Toll/interleukin-1 receptor) domains are found in proteins involved in immunity pathways in organisms ranging from humans and plant to bacteria. Bacterial TIR domain-containing proteins have been shown to contribute to pathogenicity and anti-viral activity of bacteria. During bacterial infection, some TIR domain-containing proteins act as virulence factors to inhibit immune responses by interfering with Toll-like receptor signalling. Other bacterial TIR domain-containing proteins are involved in bacterial anti-viral defence. Many of TIR domain-containing proteins have been shown to have NAD+ cleavage activity and relevant to host cell death and bacterial anti-phage defence system. In addition, some TIR domain-containing proteins have been reported that has DNA binding activity. Here, we report our studies on two bacterial TIR domain-containing proteins: AbTir and PumA. AbTir (Acinetobacter baumannii TIR domain-containing) is one of the few bacterial proteins that has been reported to produce a variant of cyclic ADPR (ADP ribose) after NAD+ cleavage. Previous study in our lab determined the crystal structure of AbTir TIR domain in its monomeric form and the chemical structure of the cyclic ADPR it produces (termed 2’cADPR). However, we could not find the significant NAD+ binding pocked in the AbTirTIR crystal structure. PumA is a TIR domain-containing protein from the multi-drug resistant pathogen Pseudomonas aeruginosa PA7 is essential for its virulence. Study shows that PumA can block host’s immune signalling pathway
In Chapter 2, we demonstrated that PumA, like AbTir, has NAD⁺ cleavage activity and forms filaments upon 3AD binding. We used nanobodies to stabilize PumA and successfully obtained several PumA:nanobody complex crystals. NADase assays showed that some nanobodies significantly inhibit its enzymatic activity, offering potential inhibitory tools.
In Chapter 3, we extended the structural analysis of PumA. We solved the crystal structure of a PumA mutant and characterized several nanobody-bound complexes. Cryo-EM and MicroED efforts revealed filament formation in the presence of 3AD, but bundling limited resolution. Nanobodies again showed inhibitory effects on NADase activity.
In Chapter 4, we investigated DNA binding by bacterial TIR proteins. We found that AbTir lacks DNA-binding activity, while PumA and its homologs from other pathogens bind DNA and form filaments upon DNA interaction. Cryo-EM confirmed DNA-induced filament formation by PumA, suggesting a role in nucleic acid sensing.
Overall, my project shows bacterial TIR domain-containing proteins assemblies in bacterial virulence and antiviral defense, and to identify potential small-molecule inhibitors targeting these mechanisms. Less
In Chapter 2, we demonstrated that PumA, like AbTir, has NAD⁺ cleavage activity and forms filaments upon 3AD binding. We used nanobodies to stabilize PumA and successfully obtained several PumA:nanobody complex crystals. NADase assays showed that some nanobodies significantly inhibit its enzymatic activity, offering potential inhibitory tools.
In Chapter 3, we extended the structural analysis of PumA. We solved the crystal structure of a PumA mutant and characterized several nanobody-bound complexes. Cryo-EM and MicroED efforts revealed filament formation in the presence of 3AD, but bundling limited resolution. Nanobodies again showed inhibitory effects on NADase activity.
In Chapter 4, we investigated DNA binding by bacterial TIR proteins. We found that AbTir lacks DNA-binding activity, while PumA and its homologs from other pathogens bind DNA and form filaments upon DNA interaction. Cryo-EM confirmed DNA-induced filament formation by PumA, suggesting a role in nucleic acid sensing.
Overall, my project shows bacterial TIR domain-containing proteins assemblies in bacterial virulence and antiviral defense, and to identify potential small-molecule inhibitors targeting these mechanisms. Less
Insulin resistance drives cardiometabolic disease yet its molecular signatures and tissue origins remain incompletely characterized and scalable assessment methods are lacking Here we apply Multi-Workflow Proteomics on plasma from individuals spanning the metabolic spectrum defined by hyperinsulinemic euglycemic clamp derived insulin sensitivity We identify proteins associated with insulin sensitivity revealing contributions from liver adipose tissue and immune cells alongside underappreciated roles for brain and heart An exercise intervention demonstrated these signatures are modifiable We developed a model combining proteins including IGFBP LEP GDF PON and LDLR with clinical variables sex HbA c TG HDL ratio that estimates hyperinsulinemic euglycemic clamp-derived ... More
Insulin resistance drives cardiometabolic disease, yet its molecular signatures and tissue origins remain incompletely characterized, and scalable assessment methods are lacking. Here, we apply Multi-Workflow Proteomics on plasma from 161 individuals spanning the metabolic spectrum defined by hyperinsulinemic–euglycemic clamp–derived insulin sensitivity. We identify 488 proteins associated with insulin sensitivity, revealing contributions from liver, adipose tissue, and immune cells alongside underappreciated roles for brain and heart. An exercise intervention demonstrated these signatures are modifiable. We developed a model combining 13 proteins, including IGFBP1, LEP, GDF15, PON3, and LDLR, with clinical variables (sex, HbA1c, TG/HDL ratio) that estimates hyperinsulinemic–euglycemic clamp-derived insulin sensitivity (R² = 0.73). Applied to ~20,000 UK Biobank participants, estimated insulin sensitivity outperformed TG/HDL in predicting type 2 diabetes (c-index 0.86 vs. 0.71) and other cardiometabolic outcomes, including obesity, cardiovascular disease, and chronic kidney disease. This proteomic atlas enables scalable insulin resistance assessment and precision risk stratification. Less
Conversion of prothrombin to thrombin occurs in the final step of the blood coagulation cascade and depends on association of the serine protease factor f Xa and the cofactor fVa on activated cell surfaces to form the prothrombinase complex Prothrombinase cleaves prothrombin at two sites in a processive manner -times faster than fXa on its own How fVa confers rapid and processive cleavage of prothrombin is an enzymatical mystery with profound consequence We created a variant of fXa that binds to fVa with high affinity in the absence of phospholipids that preserves the activity of wild-type prothrombinase and recently reported ... More
Conversion of prothrombin to thrombin occurs in the final step of the blood coagulation cascade and depends on association of the serine protease, factor (f) Xa, and the cofactor fVa on activated cell surfaces to form the prothrombinase complex. Prothrombinase cleaves prothrombin at two sites in a processive manner ∼500,000-times faster than fXa on its own. How fVa confers rapid and processive cleavage of prothrombin is an enzymatical mystery with profound consequence. We created a variant of fXa that binds to fVa with high affinity in the absence of phospholipids that preserves the activity of wild-type prothrombinase, and recently reported on the cryo-EM structure of the complex. It revealed an extensive interface between the two proteins, including a critical interaction between the first acidic region C-terminal to the A2 domain of fVa (the N-terminal portion of the a2-loop) with the heparin binding site of fXa. Here we present the cryo-EM structures of prothrombinase bound to prothrombin and the intermediate meizothrombin, both to 3.1 Å resolution. The prothrombin complex revealed a surprising interaction between the second acidic region of the a2-loop with exosite I of prothrombin, accounting for 70% of the total buried surface area. Cleavage at Arg320 triggers the zymogen-to-protease conformational change in meizothrombin which alters all domain-domain and fVa interactions, and results in the presentation of the second cleavage site (Arg271) for processing. Together, these structures reveal a remarkable enzymatic mechanism that depends on the active participation of the substrate itself, and introduce the new paradigm of substrate allostery. Less
Liver-enriched antimicrobial peptide LEAP can act as a natural antagonist or inverse agonist of the growth hormone secretagogue receptor also known as the ghrelin receptor In our previous studies palm-LEAP a truncated palmitoylated analogue of LEAP proved highly stable and exerted an acute anorexigenic effect Ghrelin the endogenous agonist of the ghrelin receptor and the anorexigenic hormone leptin have opposing effects on appetite We hypothesized that leptin supplementation combined with long-term antagonism of the ghrelin receptor using palm-LEAP might have a beneficial and potentially additive effect on obesity-related metabolic stress in leptin-deficient ob ob mice influencing diabetes liver steatosis impaired ... More
Liver-enriched antimicrobial peptide 2 (LEAP2) can act as a natural antagonist or inverse agonist of the growth hormone secretagogue receptor, also known as the ghrelin receptor. In our previous studies, palm-LEAP2(1–14), a truncated palmitoylated analogue of LEAP2, proved highly stable and exerted an acute anorexigenic effect. Ghrelin, the endogenous agonist of the ghrelin receptor, and the anorexigenic hormone leptin have opposing effects on appetite. We hypothesized that leptin supplementation combined with long-term antagonism of the ghrelin receptor using palm-LEAP2(1–14) might have a beneficial and potentially additive effect on obesity-related metabolic stress in leptin-deficient ob/ob mice, influencing diabetes, liver steatosis, impaired locomotor activity, and hypothermia.
Here, we demonstrated a clear additive effect of combined leptin and palm-LEAP2(1–14) treatment, which increased locomotor activity, reduced plasma cholesterol, liver steatosis, and mRNA expression of the metabolic stress marker Fgf21, and upregulated hypothalamic Pomc gene expression – outcomes not achieved by either treatment alone. Additionally, leptin, palm-LEAP2(1–14), and both treatments combined each reduced cumulative food intake and circulating FGF21 levels. Only leptin reduced body weight, elevated rectal temperature, increased Ucp1 and Pgc1 mRNA expression in brown adipose tissue, and attenuated hyperinsulinemia, indicating that the weight-reducing and antidiabetic effects were attributable to leptin – not palm-LEAP2(1–14). Liver metabolic stress was further alleviated by decreases in plasma FGF21 and hepatic expression of the lipogenic enzymes.
Our study demonstrated the beneficial effects of palm-LEAP2(1–14) on obesity-induced metabolic stress, which were further attenuated when palm-LEAP2(1–14) was coadministered with leptin in ob/ob mice. Less
Here, we demonstrated a clear additive effect of combined leptin and palm-LEAP2(1–14) treatment, which increased locomotor activity, reduced plasma cholesterol, liver steatosis, and mRNA expression of the metabolic stress marker Fgf21, and upregulated hypothalamic Pomc gene expression – outcomes not achieved by either treatment alone. Additionally, leptin, palm-LEAP2(1–14), and both treatments combined each reduced cumulative food intake and circulating FGF21 levels. Only leptin reduced body weight, elevated rectal temperature, increased Ucp1 and Pgc1 mRNA expression in brown adipose tissue, and attenuated hyperinsulinemia, indicating that the weight-reducing and antidiabetic effects were attributable to leptin – not palm-LEAP2(1–14). Liver metabolic stress was further alleviated by decreases in plasma FGF21 and hepatic expression of the lipogenic enzymes.
Our study demonstrated the beneficial effects of palm-LEAP2(1–14) on obesity-induced metabolic stress, which were further attenuated when palm-LEAP2(1–14) was coadministered with leptin in ob/ob mice. Less
Quantitative proteomics relies on accurate selection of fragment ions for quantification yet most current algorithms apply simple strategies such as median intensity or single quality filters Modern data-independent acquisition DIA searches generate rich features such as fragment ion correlations retention time and many others that could be leveraged to assess fragment quality We introduce QuantSelect a novel strategy to select optimal fragments by systematically integrating these features via self-supervised deep learning QuantSelect uses a regularized weighted-variance loss on intensity traces normalized via our directLFQ algorithm This allows learning a fragment quality score without ground truth labels enabling on-the-fly training on ... More
Quantitative proteomics relies on accurate selection of fragment ions for quantification, yet most current algorithms apply simple strategies such as median intensity or single quality filters. Modern data-independent acquisition (DIA) searches generate rich features such as fragment ion correlations, retention time and many others that could be leveraged to assess fragment quality. We introduce QuantSelect, a novel strategy to select optimal fragments by systematically integrating these features via self-supervised deep learning. QuantSelect uses a regularized, weighted-variance loss on intensity traces normalized via our directLFQ algorithm. This allows learning a fragment quality score without ground truth labels, enabling on-the-fly training on label-free DIA datasets. Integrated within our alphaDIA pipeline, QuantSelect significantly improves quantitative accuracy and in some cases substantially corrects protein intensity estimation. Sensitivity in differential expression improved by 68% in a mixed-species benchmarking dataset and by 18% in single-cell data. QuantSelect provides a practical framework for data-driven fragment selection that improves accuracy, precision and downstream inference in DIA proteomics. Less
The ToxRS system is a member of a two-protein transmembrane transcriptional regulator family of proteins that act as critical environmental stress sensors and regulate virulence gene expression in some bacterial pathogens These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors In Vibrio parahaemolyticus and Vibrio cholerae the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans ToxS has been shown to modulate the activity of its binding partner ToxR by binding bile salts antimicrobial cholesterol metabolites secreted into the human gut However the molecular mechanism ... More
The ToxRS system is a member of a two-protein transmembrane transcriptional regulator family of proteins that act as critical environmental stress sensors and regulate virulence gene expression in some bacterial pathogens. These systems are thought to operate by sensing environmental stimuli and transmitting signals through periplasmic domains to activate DNA-binding transcription factors. In Vibrio parahaemolyticus and Vibrio cholerae, the ToxRS system regulates virulence factors responsible for severe gastrointestinal symptoms in humans. ToxS has been shown to modulate the activity of its binding partner ToxR by binding bile salts, antimicrobial cholesterol metabolites secreted into the human gut. However, the molecular mechanism underlying this regulation is unclear. Here, we present the crystal structures of the V. parahaemolyticus ToxS periplasmic domain (ToxSp) with and without the bile salt glycocholate. ToxSp forms an 8-stranded broken β-barrel with a central α-helix and is structurally homologous to a group of chaperone proteins. Notably, the glycocholate-bound ToxSp structure forms a strand-swapped homodimer containing three bound glycocholate molecules. Modeling two ToxR periplasmic domains in complex with the glycocholate-bound ToxSp dimer provides a structure-based model for bile salt activation of the ToxRS system and suggests that ToxRS homologs may be regulated in a similar manner across diverse bacterial species. Less
Iron homeostasis in various pathogenic bacteria is regulated by bacterioferritins Bfr which function to store Fe and release Fe as needed for metabolic processes The Bfr structure consists of kDa subunits in which dimer pairs bind a heme molecule and are assembled into a highly symmetrical -meric spherical structure with an internal core diameter of approximately Release of iron is facilitated by the binding of a kDa Fe- S ferredoxin Bfd to specific sites on the surface of Bfr which transfers electrons to the core thereby reducing the stored Fe to Fe for mobilization The crystal structures of Bfr from ... More
Iron homeostasis in various pathogenic bacteria is regulated by bacterioferritins (Bfr) which function to store Fe3+ and release Fe2+ as needed for metabolic processes. The Bfr structure consists of 18 kDa subunits in which dimer pairs bind a heme molecule and are assembled into a highly symmetrical 24-meric spherical structure with an internal core diameter of approximately 80 Å. Release of iron is facilitated by the binding of a 7 kDa [2Fe-2S] ferredoxin (Bfd) to specific sites on the surface of Bfr which transfers electrons to the core thereby reducing the stored Fe3+ to Fe2+ for mobilization. The crystal structures of Bfr from Brucella abortus (Ba) in the apo and iron bound forms are presented and compared with those from Acinetobacter baumannii (Ab) and Pseudomonas aeruginosa (Pa). Additionally, models of the Bfr:Bfd complexes for Ba and Ab are provided and compared with the Pa complex. Finally, compounds known to target the Bfr:Bfd interaction in Pa were docked to the Ba and Ab structures which provided insight regarding the potential binding mode and inhibitory mechanism. Less
Achieving high-resolution spatial tissue proteomes requires careful balancing and integration of optimized sample processing chromatography and MS acquisition Here we present an advanced cellenONE protocol for loss-reduced tissue processing and compare all Evosep ONE Whisper Zoom gradients and samples per day along with three common DIA acquisition schemes on a timsUltra AIP mass spectrometer We found that tissue type was as important as gradient length and sample amount in determining proteome coverage Moreover the benefit of increased tissue sampling was gradient- and dynamic range-dependent Analyzing mouse liver a high dynamic range tissue over tenfold more tissue sampling led to only ... More
Achieving high-resolution spatial tissue proteomes requires careful balancing and integration of optimized sample processing, chromatography, and MS acquisition. Here, we present an advanced cellenONE protocol for loss-reduced tissue processing and compare all Evosep ONE Whisper Zoom gradients (20, 40, 80, and 120 samples per day), along with three common DIA acquisition schemes on a timsUltra AIP mass spectrometer. We found that tissue type was as important as gradient length and sample amount in determining proteome coverage. Moreover, the benefit of increased tissue sampling was gradient- and dynamic range-dependent. Analyzing mouse liver, a high dynamic range tissue, over tenfold more tissue sampling led to only ~30% gain in protein identification for short gradients (120 SPD and 80 SPD). However, even the lowest tested tissue amount (0.04 nL, 40,000 µm3) yielded 3,200 reproducibly quantified proteins for the 120 SPD method. Longer gradients (40 SPD and 20 SPD) instead significantly benefited from more tissue sampling, quantifying over 7,500 proteins from 0.5 nL of tonsil T-cell niches. Finally, we applied our workflow to a rare squamous cell carcinoma of the oral cavity, uncovering disease-associated pathways and region-specific protein level changes. Our study demonstrates that more than 100 high-quality spatial tissue proteomes can be prepared and acquired daily, laying a strong foundation for cohort-size spatial tissue proteomics in translational research. Less
Obtaining high-quality single crystals containing charged molecules from aqueous solutions can sometimes be challenging In this publication we studied the crystallization of polycationic porphyrins Crystals of three polycationic porphyrins -tetrakis -methylpyridin- -ium- -yl porphyrin - -cyanophenyl - -tris -methylpyridin- -ium- -yl porphyrin and - -carboxy phenyl - -tris -methylpyridin- -ium- -yl porphyrin were obtained with different anions We started initially with nano-crystallization' screening in order to identify promising combinations of the cations with suitable anions followed by optimizing the ratio of anions to cations Both steps yielded crystals leading to the determination of one porphyrin-containing crystal structure from each step ... More
Obtaining high-quality single crystals containing charged molecules from aqueous solutions can sometimes be challenging. In this publication, we studied the crystallization of polycationic porphyrins. Crystals of three polycationic porphyrins, [5,10,15,20-tetrakis(1-methylpyridin-1-ium-4-yl)porphyrin, 5-(4-cyanophenyl)-10,15,20-tris(1-methylpyridin-1-ium-4-yl)porphyrin and 5-(4-carboxyphenyl)-10,15,20-tris(1-methylpyridin-1-ium-4-yl)porphyrin], were obtained with different anions. We started initially with `nano-crystallization' screening in order to identify promising combinations of the cations with suitable anions, followed by optimizing the ratio of anions to cations. Both steps yielded crystals, leading to the determination of one porphyrin-containing crystal structure from each step. In the next step, five different organic solvents were trialled as additives in an aqueous hanging-drop crystallization setup, which led to the determination of three more crystal structures. It was shown that it was possible to improve the quality of the crystals without the organic additive being incorporated into the crystal structure. Finally, crystals of one porphyrin salt were obtained by gel crystallization. These crystals were higher quality than the crystal grown by vapour diffusion, leading to a high-quality and higher-resolution X-ray structure determination. This is the first porphyrin structure to be determined from a crystal grown by gel crystallizaton. Less
UreE is a nickel chaperone required for the safe and efficient delivery of nickel to the active site of the metalloenzyme urease a key virulence factor of the urinary tract pathogen Proteus mirabilis We investigated the structural features of P mirabilis UreE using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma-mass spectrometry Here we report a crystal structure of homodimeric PmUreE and show it has capacity to bind five nickel ions per dimer Truncation of the histidine-rich C-terminus reduced nickel binding capacity by two nickel ions per dimer and comparison with homologous UreE structures allowed the assignment ... More
UreE is a nickel chaperone required for the safe and efficient delivery of nickel to the active site of the metalloenzyme, urease; a key virulence factor of the urinary tract pathogen, Proteus mirabilis. We investigated the structural features of P. mirabilis UreE using protein X-ray crystallography and its nickel-binding capacity by inductively coupled plasma-mass spectrometry. Here, we report a 2.0 Å crystal structure of homodimeric PmUreE and show it has capacity to bind five nickel ions per dimer. Truncation of the histidine-rich C-terminus reduced nickel binding capacity by two nickel ions per dimer and comparison with homologous UreE structures allowed the assignment of putative nickel binding sites within the PmUreE structure. These findings increase our understanding of how PmUreE binds nickel and ultimately prevents this toxic metal from causing significant cellular damage in P. mirabilis. Less
To ensure safe long-term use topical products should be investigated to understand how they interact with the resident skin microbiota to mitigate potential risk Sunscreens are essential for protecting skin from UV damage but their effects on skin-resident microbes have not been well characterized We examined the impact of two sunscreen formulations containing titanium dioxide or zinc oxide on both cultured skin bacteria and the skin microbiomes of human volunteers No loss of viability was observed after a h exposure to either sunscreen in cultures of Staphylococcus epidermidis Staphylococcus capitis Staphylococcus hominis Micrococcus luteus and Corynebacterium tuberculostearicum The effects of ... More
To ensure safe, long-term use, topical products should be investigated to understand how they interact with the resident skin microbiota to mitigate potential risk. Sunscreens are essential for protecting skin from UV damage, but their effects on skin-resident microbes have not been well characterized. We examined the impact of two sunscreen formulations (containing titanium dioxide or zinc oxide) on both cultured skin bacteria and the skin microbiomes of human volunteers. No loss of viability was observed after a 2 h exposure to either sunscreen in cultures of Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hominis, Micrococcus luteus, and Corynebacterium tuberculostearicum. The effects of the sunscreens were then studied across the skin microbiomes of 20 human participants. Skin swabs were collected before application and at 1, 6, and 24 h afterward. DNA was extracted and sequenced at the 16S rRNA V4 region, and sequences were denoised and taxonomically assigned using the nf-core/ampliseq pipeline. Across all time points, alpha diversity (Shannon index, Friedman test) and beta diversity (permutational multivariate analysis of variance) remained stable, with no significant differences in beta dispersion. Differential abundance analysis revealed minor fluctuations in some low-abundance genera, identified as likely transient due to their low prevalence, but overall resident community composition was not significantly altered. These findings suggest that short-term sunscreen application does not disrupt the skin microbiome, supporting their safe use from a microbial standpoint. Outcomes from both in vitro and in vivo experimentation point to the compositional resilience of the skin microbiota to sunscreens. Less