1191 Citations
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
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
Human T-cell Leukemia Virus type HTLV- is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology especially when compared to the closely related virus HIV A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host Importantly the first in class anti-capsid drug approved lenacapavir can provide long-acting pre-exposure prophylaxis Such a property would provide a means to prevent the transmission of HTLV- but its capsid has not previously ... More
Human T-cell Leukemia Virus type 1 (HTLV-1) is an untreatable retrovirus that causes lethal malignancies and degenerative inflammatory conditions. Effective treatments have been delayed by substantial gaps in our knowledge of the fundamental virology, especially when compared to the closely related virus, HIV. A recently developed and highly effective anti-HIV strategy is to target the virus with drugs that interfere with capsid integrity and interactions with the host. Importantly, the first in class anti-capsid drug approved, lenacapavir, can provide long-acting pre-exposure prophylaxis. Such a property would provide a means to prevent the transmission of HTLV-1, but its capsid has not previously been considered as a drug target. Here we describe the first high-resolution crystal structures of the HTLV-1 capsid protein, define essential lattice interfaces, and identify a previously unknown ligand-binding pocket. We show that this pocket is essential for virus infectivity, providing a potential target for future anti-capsid drug development. Less
Prophages dormant bacteriophage genomes integrated within the bacterial chromosome play pivotal roles in shaping microbial communities when awakened Our current understanding of prophage activation is largely shaped by a narrow set of traditional DNA-damaging inducers such as mitomycin C and ciprofloxacin which trigger the bacterial SOS response This study employed high-throughput screening of compounds to identify novel prophage inducers using model lambdoid prophage HK We identified multiple new inducers across diverse pharmacological classes including dietary supplements and therapeutics Despite the variety in compounds all acted through SOS-dependent pathways However bleomycin an antineoplastic antibiotic demonstrated broad-spectrum and potent prophage induction exceeding ... More
Prophages, dormant bacteriophage genomes integrated within the bacterial chromosome, play pivotal roles in shaping microbial communities when awakened. Our current understanding of prophage activation is largely shaped by a narrow set of traditional DNA-damaging inducers, such as mitomycin C and ciprofloxacin, which trigger the bacterial SOS response. This study employed high-throughput screening of 3,921 compounds to identify novel prophage inducers using model lambdoid prophage HK97. We identified multiple new inducers across diverse pharmacological classes, including dietary supplements and therapeutics. Despite the variety in compounds, all acted through SOS-dependent pathways. However, bleomycin, an antineoplastic antibiotic, demonstrated broad-spectrum and potent prophage induction exceeding standard inducers, with activity validated across multiple phage-host pairings. These findings expand the repertoire of prophage inducers into commonly ingested xenobiotics and introduce bleomycin as a powerful, cost-effective tool for prophage research.
Around 75% of bacteria carry within them dormant viruses (prophages), which can awaken when the bacterium is stressed, killing the bacterium. Historically, this has been done using DNA-damaging antibiotics, but increasingly, more such signals have been discovered. Here, through a high-throughput screen, we identify phage-waking activity in several commonly consumed compounds, such as the SSRI Prozac, as well as a new DNA-damaging agent that is much more effective in waking phages than the previous gold standard. Less
Around 75% of bacteria carry within them dormant viruses (prophages), which can awaken when the bacterium is stressed, killing the bacterium. Historically, this has been done using DNA-damaging antibiotics, but increasingly, more such signals have been discovered. Here, through a high-throughput screen, we identify phage-waking activity in several commonly consumed compounds, such as the SSRI Prozac, as well as a new DNA-damaging agent that is much more effective in waking phages than the previous gold standard. Less
Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy StayGold a novel dimeric GFP recently monomerized through sequence engineering addresses this challenge with its high photostability There is now a focus on producing different colored StayGold derivatives to facilitate concurrent tagging of multiple targets The unnatural amino acid -aminotyrosine has previously been shown to redshift superfolder GFP upon incorporation into its chromophore via genetic code expansion Here we apply the same strategy to redshift StayGold through substitution of tyrosine- with -aminotyrosine The resultant red fluorescent protein StayRose shows an excitation wavelength maximum of nm and an ... More
Photobleaching of fluorescent proteins often limits the acquisition of high-quality images in microscopy. StayGold, a novel dimeric GFP recently monomerized through sequence engineering, addresses this challenge with its high photostability. There is now a focus on producing different colored StayGold derivatives to facilitate concurrent tagging of multiple targets. The unnatural amino acid 3-aminotyrosine has previously been shown to redshift superfolder GFP upon incorporation into its chromophore via genetic code expansion. Here, we apply the same strategy to redshift StayGold through substitution of tyrosine-58 with 3-aminotyrosine. The resultant red fluorescent protein, StayRose, shows an excitation wavelength maximum of 530 nm and an emission wavelength maximum of 588 nm. Importantly, the monomeric mStayRose retains the favorable photostability in vivo in Escherichia coli and zebrafish embryos. A high-resolution crystal structure of StayRose confirms the modified structure of the amino chromophore within an unperturbed 3D fold. Although reliant on genetic code expansion, StayRose provides an important step toward developing redshifted StayGold derivatives. Less
Heliorhodopsins HeRs the third rhodopsin family are characterized by inverted membrane topology and confinement to monoderm organisms yet their biological meaning has so far remained a mystery We report the first crystal structure of a eukaryotic HeR supported by structural modeling and comparative analyses across all domains of life A conserved carotenoid-binding site reminiscent of secondary antennae in some microbial rhodopsins is identified and found to be common among HeRs We show that inverted topology allows recruitment of exogenous xanthophylls inaccessible in diderm cells explaining HeRs distinctive orientation and distribution These findings reveal a previously unrecognized light-harvesting mechanism of HeRs ... More
Heliorhodopsins (HeRs), the third rhodopsin family, are characterized by inverted membrane topology and confinement to monoderm organisms, yet their biological meaning has so far remained a mystery. We report the first crystal structure of a eukaryotic HeR, supported by structural modeling and comparative analyses across all domains of life. A conserved carotenoid-binding site, reminiscent of secondary antennae in some microbial rhodopsins, is identified and found to be common among HeRs. We show that inverted topology allows recruitment of exogenous xanthophylls, inaccessible in diderm cells, explaining HeRs’ distinctive orientation and distribution. These findings reveal a previously unrecognized light-harvesting mechanism of HeRs, expand the known repertoire of microbial phototrophy, and suggest evolutionary constraints linking membrane topology to environmental metabolite accessibility. Less
The composition of the primordial genetic material remains uncertain Studies of duplex structure and stability and of nonenzymatic template copying chemistry provide insight into the viability of potentially primordial genetic polymers Recent work suggests that - deoxyribo-purine nucleotides may have been generated together with ribonucleotides on the early Earth Since DNA RNA duplexes are known to be less stable than RNA RNA duplexes we have examined the impact of dA dI and dG substitutions on RNA structure and nonenzymatic template copying We find that single -deoxyribo-purine substitutions reduce RNA duplex stability as expected Crystallographic studies show that such substitutions lead ... More
The composition of the primordial genetic material remains uncertain. Studies of duplex structure and stability, and of nonenzymatic template copying chemistry, provide insight into the viability of potentially primordial genetic polymers. Recent work suggests that 2′- deoxyribo-purine nucleotides may have been generated together with ribonucleotides on the early Earth. Since DNA/RNA duplexes are known to be less stable than RNA/RNA duplexes, we have examined the impact of dA, dI, and dG substitutions on RNA structure and nonenzymatic template copying. We find that single 2′-deoxyribo-purine substitutions reduce RNA duplex stability, as expected. Crystallographic studies show that such substitutions lead to minimal structural changes but point to diminished solvation as a likely reason for duplex destabilization. Kinetic studies show that dI and dG substrates exhibit slightly weaker template binding and slower rates of template-directed primer extension than the corresponding ribo-purine substrates. In contrast, dA substrates exhibit much slower reaction kinetics but higher template affinity than rA substrates. Our results suggest that a mixed RNA/DNA primordial genetic polymer would have suffered from moderately slower rates of template copying, but that this could have been offset by an advantage due to more facile strand separation or exchange. Less
C domains are ubiquitous membrane-binding modules of residues in eukaryotes that are often associated with proteins involved in membrane trafficking and lipid modification The genome of Trichomonas vaginalis the most common non-viral sexually transmitted human pathogen encodes eight genes that contain a N-terminal C module linked to a XYPPX-repeat domain of more than four XYPPX repeats C -XYPPX While the function of the XYPPX-repeat domain remains unknown its multiple association with C domains in T vaginalis suggests it is important The C domain from one of these C -XYPPX-repeat proteins Tv-C - was structurally and physically characterized using X-ray crystallography ... More
C2 domains are ubiquitous membrane-binding modules of ∼130 residues in eukaryotes that are often associated with proteins involved in membrane trafficking and lipid modification. The genome of Trichomonas vaginalis, the most common, non-viral, sexually transmitted human pathogen, encodes eight genes that contain a N-terminal C2 module linked to a XYPPX-repeat domain of more than four XYPPX repeats (C2-XYPPX). While the function of the XYPPX-repeat domain remains unknown, its multiple association with C2 domains in T. vaginalis suggests it is important. The C2 domain from one of these C2-XYPPX-repeat proteins, Tv-C2-1, was structurally and physically characterized using X-ray crystallography and NMR spectroscopy. The crystal structure for Tv-C2-1 shows that this domain shares a fold common to all C2 domains, a compact Greek-key motif composed of eight anti-parallel β-strands in the type-2 topology. An NMR chemical shift perturbation study with Ca2+ showed that Tv-C2-1 bound two Ca2+ atoms primarily via two loops (loop-1 and loop-3) on the predicted calcium binding face of the protein with Kds of 58.0 ± 0.1 μM and 232 ± 6 μM. Estimations of the overall rotational correlation time, τc, in the apo (11.1 ns) and Ca2+-bound (9.2 ns) state suggests the protein becomes more compact upon Ca2+ binding, consistent with a decrease in dynamics in loop-3 and marginally in loop-1 suggested by amide 15N heteronuclear steady-state {1H}-15N NOEs. Showing Tv-C2-1 binds calcium and adopts a compact Greek-key motif structure, two primary features of C2 domains, suggests understanding the function of the XYPPX-repeat domain may be warranted. Less
Spermine a pivotal player in biomolecular condensation and diverse cellular processes has emerged as a focus of investigation in aging neurodegeneration and other diseases Despite its significance the mechanistic details of spermine remain incompletely understood Here we describe the distinct modulation by spermine on Alzheimer s Tau and Parkinson s -synuclein elucidating their condensation behaviors in vitro and in vivo Using biophysical techniques including time-resolved SAXS and NMR we trace electrostatically driven transitions from atomic-scale conformational changes to mesoscopic structures Notably spermine extends lifespan ameliorates movement deficits and restores mitochondrial function in C elegans models expressing Tau and -synuclein Acting ... More
Spermine, a pivotal player in biomolecular condensation and diverse cellular processes, has emerged as a focus of investigation in aging, neurodegeneration, and other diseases. Despite its significance, the mechanistic details of spermine remain incompletely understood. Here, we describe the distinct modulation by spermine on Alzheimer’s Tau and Parkinson’s α-synuclein, elucidating their condensation behaviors in vitro and in vivo. Using biophysical techniques including time-resolved SAXS and NMR, we trace electrostatically driven transitions from atomic-scale conformational changes to mesoscopic structures. Notably, spermine extends lifespan, ameliorates movement deficits, and restores mitochondrial function in C. elegans models expressing Tau and α-synuclein. Acting as a molecular glue, spermine orchestrates in vivo condensation of α-synuclein, influences condensate mobility, and promotes degradation via autophagy, specifically through autophagosome expansion. This study unveils the interplay between spermine, protein condensation, and functional outcomes, advancing our understanding of neurodegenerative diseases and paving the way for therapeutic development. Less
Plasma and cerebrospinal fluid are complementary sources of biomarkers for neurodegenerative diseases The wide dynamic range of protein abundances particularly in plasma hampers detection of low-abundance proteins Depletion of high-abundance proteins and efficient enzymatic digestion can improve proteome coverage but must be carefully optimized for reproducibility throughput and cost-efficiency for use in large-scale clinical proteomic studies We developed a scalable sample preparation workflow for plasma and cerebrospinal fluid CSF that integrates depletion of high-abundance proteins optimized digestion using Lys-C and trypsin and compatibility with both label-free and tandem mass tag TMTpro -based quantification We systematically evaluated protein depletion and enzyme ... More
Plasma and cerebrospinal fluid are complementary sources of biomarkers for
neurodegenerative diseases. The wide dynamic range of protein abundances, particularly in
plasma, hampers detection of low-abundance proteins. Depletion of high-abundance proteins
and efficient enzymatic digestion can improve proteome coverage but must be carefully
optimized for reproducibility, throughput, and cost-efficiency for use in large-scale clinical
proteomic studies.
We developed a scalable sample preparation workflow for plasma and cerebrospinal fluid (CSF)
that integrates depletion of high-abundance proteins, optimized digestion using Lys-C and
trypsin, and compatibility with both label-free and tandem mass tag (TMTpro)-based
quantification. We systematically evaluated protein depletion and enzyme digestion conditions,
and the effect of deoxycholate on digestion, monitoring the number of detectable proteins and
the quantitation precision. Less
neurodegenerative diseases. The wide dynamic range of protein abundances, particularly in
plasma, hampers detection of low-abundance proteins. Depletion of high-abundance proteins
and efficient enzymatic digestion can improve proteome coverage but must be carefully
optimized for reproducibility, throughput, and cost-efficiency for use in large-scale clinical
proteomic studies.
We developed a scalable sample preparation workflow for plasma and cerebrospinal fluid (CSF)
that integrates depletion of high-abundance proteins, optimized digestion using Lys-C and
trypsin, and compatibility with both label-free and tandem mass tag (TMTpro)-based
quantification. We systematically evaluated protein depletion and enzyme digestion conditions,
and the effect of deoxycholate on digestion, monitoring the number of detectable proteins and
the quantitation precision. Less
The Maternal-to-Zygotic transition MZT is a reprograming process encompassing zygotic genome activation ZGA and the clearance of maternally-provided mRNAs While some factors regulating MZT have been identified there are thousands of maternal RNAs whose function has not been ascribed yet Here we have performed a proof-of-principle CRISPR-RfxCas d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT Bckdk mRNA knockdown caused epiboly defects ZGA deregulation H K ac reduction and a partial impairment of miR- processing Phospho-proteomic analysis revealed that Phf Baf a a chromatin remodeling factor is ... More
The Maternal-to-Zygotic transition (MZT) is a reprograming process encompassing zygotic genome activation (ZGA) and the clearance of maternally-provided mRNAs. While some factors regulating MZT have been identified, there are thousands of maternal RNAs whose function has not been ascribed yet. Here, we have performed a proof-of-principle CRISPR-RfxCas13d maternal screening targeting mRNAs encoding protein kinases and phosphatases in zebrafish and identified Bckdk as a novel post-translational regulator of MZT. Bckdk mRNA knockdown caused epiboly defects, ZGA deregulation, H3K27ac reduction and a partial impairment of miR-430 processing. Phospho-proteomic analysis revealed that Phf10/Baf45a, a chromatin remodeling factor, is less phosphorylated upon Bckdk depletion. Further, phf10 mRNA knockdown also altered ZGA and Phf10 constitutively phosphorylated rescued the developmental defects observed after bckdk mRNA depletion. Altogether, our results demonstrate the competence of CRISPR-RfxCas13d screenings to uncover new regulators of early vertebrate development and shed light on the post-translational control of MZT mediated by protein phosphorylation. Less
High-grade serous ovarian cancer HGSOC is a devastating disease that is frequently detected at an advanced and incurable stage Advances in ultrasensitive mass spectrometry-based spatial proteomics have provided a unique opportunity to uncover early molecular events in tumorigenesis and common dysregulated pathways with high therapeutic potential Here we present a comprehensive proteomic analysis of serous tubal intraepithelial carcinoma STIC the HGSOC precursor lesion covering more than proteins from ultralow input archival tissue We discovered that STICs and concurrent invasive carcinomas were indistinguishable at the global proteome level revealing a similar level of phenotypic and molecular heterogeneity Using cell-type resolved tissue ... More
High-grade serous ovarian cancer (HGSOC) is a devastating disease that is frequently detected at an advanced and incurable stage. Advances in ultrasensitive mass spectrometry-based spatial proteomics have provided a unique opportunity to uncover early molecular events in tumorigenesis and common dysregulated pathways with high therapeutic potential. Here, we present a comprehensive proteomic analysis of serous tubal intraepithelial carcinoma (STIC), the HGSOC precursor lesion, covering more than 10,000 proteins from ultralow input archival tissue. We discovered that STICs and concurrent invasive carcinomas were indistinguishable at the global proteome level, revealing a similar level of phenotypic and molecular heterogeneity. Using cell-type resolved tissue proteomics, we revealed strong cell-of-origin signatures preserved in STICs and invasive tumors and identified early dysregulated pathways of therapeutic relevance. These include proliferation and DNA damage repair signatures, as well as onco-metabolic changes, such as increased cholesterol biosynthesis. Finally, we uncovered substantial remodeling of the co-evolving tumor microenvironment, affecting approximately one-third of the stromal proteome, and derived a common signature associated with progressive immunosuppression and ECM restructuring. In summary, our study highlights the power of spatially resolved quantitative proteomics to dissect the molecular underpinnings of early carcinogenesis and provides a rich proteomic resource for future biomarker and drug target discovery. Less
The increase in multidrug-resistant bacteria underscores an urgent need for additional antibiotics Here we integrate small-molecule high-throughput screening with a deep-learning-based virtual screening approach to uncover new antibacterial compounds We screen million small molecules against a sensitized Escherichia coli strain yielding thousands of hits We use these data to train a deep learning model GNEprop to predict antibacterial activity retrospectively validating robustness with respect to out-of-distribution generalization and activity cliff prediction Virtual screening of over billion synthetically accessible compounds identifies potential candidates of which exhibit antibacterial activity on the same strain illustrating a -fold improved hit rate over the high-throughput ... More
The increase in multidrug-resistant bacteria underscores an urgent need for additional antibiotics. Here, we integrate small-molecule high-throughput screening with a deep-learning-based virtual screening approach to uncover new antibacterial compounds. We screen ~2 million small molecules against a sensitized Escherichia coli strain, yielding thousands of hits. We use these data to train a deep learning model, GNEprop, to predict antibacterial activity, retrospectively validating robustness with respect to out-of-distribution generalization and activity cliff prediction. Virtual screening of over 1.4 billion synthetically accessible compounds identifies potential candidates, of which 82 exhibit antibacterial activity on the same strain, illustrating a 90-fold improved hit rate over the high-throughput screening experiment used for training. Many newly identified compounds exhibit high dissimilarity to known antibiotics, potency beyond the training bacterial strain and selectivity. Biological characterization identifies specific, validated targets, indicating promising avenues for further exploration in antibiotic discovery. Less
The ribosome is a universally conserved and essential protein complex but its biogenesis in mammals is more complex than in single-celled eukaryotes To explore this added complexity we conducted a protein protein interaction screen in human cells This led to the identification of the eumetazoan-specific SPATA SPATA L CINP C ORF LCC complex as a key regulator of ribosome biogenesis Structural analyses using cryo-EM and X-ray crystallography defined the architecture of LCC Functional studies following acute depletion revealed that each component is essential for pre- S maturation Swapping endogenous LCC components with mutant versions pinpointed critical functional interactions and showed ... More
The ribosome is a universally conserved and essential protein complex, but its biogenesis in mammals is more complex than in single-celled eukaryotes. To explore this added complexity, we conducted a protein–protein interaction screen in human cells. This led to the identification of the eumetazoan-specific SPATA5–SPATA5L1–CINP–C1ORF109 (55LCC) complex as a key regulator of ribosome biogenesis. Structural analyses using cryo-EM and X-ray crystallography defined the architecture of 55LCC. Functional studies following acute depletion revealed that each component is essential for pre-60S maturation. Swapping endogenous 55LCC components with mutant versions pinpointed critical functional interactions and showed that SPATA5’s ATPase activity is more important than SPATA5L1’s. Our findings support that SPATA5 evolved from the solitary yeast ATPase Drg1 into the multiprotein 55LCC complex in metazoans. This work provides insights into the complexity of ribosome biogenesis and lays the foundation for deeper exploration of 55LCC’s role in pre-60S maturation. Less
Abstract Background Recombinant human NELL- rhNELL- is a potent osteogenic protein with therapeutic potential in regenerative medicine A stable formulation is essential to prevent aggregation during production filling storage and clinical use Methodology A four-stage rational formulation strategy was used identify intrinsic aggregation risks of rhNELL- screen polysorbate- and cyclodextrin-based formulations to enhance colloidal and conformational stability test lead candidates under agitation freeze thaw pH shifts and elevated temperature Analytical techniques included PEG challenge differential scanning fluorimetry DSF isothermal chemical denaturation ICD and dynamic light scattering DLS Aggregation was assessed via visible particles VP opalescence subvisible particles SVP Micro Flow ... More
Abstract
Background
Recombinant human NELL-1 (rhNELL-1) is a potent osteogenic protein with therapeutic potential in regenerative medicine. A stable formulation is essential to prevent aggregation during production, filling, storage, and clinical use.
Methodology
A four-stage rational formulation strategy was used: (1) identify intrinsic aggregation risks of rhNELL-1; (2) screen polysorbate- and cyclodextrin-based formulations to enhance colloidal and conformational stability; (3–4) test lead candidates under agitation, freeze/thaw, pH shifts, and elevated temperature. Analytical techniques included PEG challenge, differential scanning fluorimetry (DSF), isothermal chemical denaturation (ICD), and dynamic light scattering (DLS). Aggregation was assessed via visible particles (VP), opalescence, subvisible particles (SVP, Micro Flow Imaging), SDS-PAGE, and ultra-high performance size exclusion chromatography (UP-SEC).
Results
rhNELL-1 was prone to self-association via hydrophobic and electrostatic interactions. Polysorbate 20 (PS20) and hydroxypropyl beta cyclodextrin (HPB-LB-BCD) improved protein stability. PS20 markedly reduced VP and SVP formation. While HPB-LB-BCD alone did not further reduce SVP beyond PS20, it enhanced thermal stress resistance. PS20 was more effective under agitation.
Conclusions
Two lead formulations containing potassium phosphate/Tris buffer, sorbitol, PS20, and HPB-LB-BCD demonstrated strong resistance to aggregation under multiple stresses. PS20 mitigated interfacial stress, while HPB-LB-BCD suppressed solution-phase aggregation, especially at high temperatures. This systematic approach offers a framework for stabilizing other aggregation-prone proteins. Less
Background
Recombinant human NELL-1 (rhNELL-1) is a potent osteogenic protein with therapeutic potential in regenerative medicine. A stable formulation is essential to prevent aggregation during production, filling, storage, and clinical use.
Methodology
A four-stage rational formulation strategy was used: (1) identify intrinsic aggregation risks of rhNELL-1; (2) screen polysorbate- and cyclodextrin-based formulations to enhance colloidal and conformational stability; (3–4) test lead candidates under agitation, freeze/thaw, pH shifts, and elevated temperature. Analytical techniques included PEG challenge, differential scanning fluorimetry (DSF), isothermal chemical denaturation (ICD), and dynamic light scattering (DLS). Aggregation was assessed via visible particles (VP), opalescence, subvisible particles (SVP, Micro Flow Imaging), SDS-PAGE, and ultra-high performance size exclusion chromatography (UP-SEC).
Results
rhNELL-1 was prone to self-association via hydrophobic and electrostatic interactions. Polysorbate 20 (PS20) and hydroxypropyl beta cyclodextrin (HPB-LB-BCD) improved protein stability. PS20 markedly reduced VP and SVP formation. While HPB-LB-BCD alone did not further reduce SVP beyond PS20, it enhanced thermal stress resistance. PS20 was more effective under agitation.
Conclusions
Two lead formulations containing potassium phosphate/Tris buffer, sorbitol, PS20, and HPB-LB-BCD demonstrated strong resistance to aggregation under multiple stresses. PS20 mitigated interfacial stress, while HPB-LB-BCD suppressed solution-phase aggregation, especially at high temperatures. This systematic approach offers a framework for stabilizing other aggregation-prone proteins. Less
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 ... 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 both with visible and UV light images and integrates a Lab-In-The-Loop approach combining ML and expert inputs for continuous 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 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 both in fundamental and translational research. Less
Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase composed of factor Xa fXa and fVa The affinity of fXa for fVa is low with assembly and function dependent on phospholipid PL membranes Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL We created a similar high-affinity PL-independent human prothrombinase with mutations to human fXa M The increase in affinity enabled cryogenic electron microscopy cryo-EM structure determination of M -prothrombinase to a resolution of All protein domains were well resolved ... More
Thrombin is generated from prothrombin through cleavage at two sites by the enzyme prothrombinase, composed of factor Xa (fXa) and fVa. The affinity of fXa for fVa is low, with assembly and function dependent on phospholipid (PL) membranes. Some snakes have evolved venom versions of fXa that bind to fVa with high affinity and efficiently activate prothrombin in the absence of PL. We created a similar high-affinity, PL-independent human prothrombinase with 17 mutations to human fXa (M17). The increase in affinity enabled cryogenic electron microscopy (cryo-EM) structure determination of M17-prothrombinase to a resolution of 3.3 Å. All protein domains were well resolved in the map, except for the Gla domain of fXa. The main contacts involve the serine protease and EGF2 domains of fXa and the A2 and A3 domains of fVa, resulting in the burying of a total surface area of 4,900 Å2. The map is of sufficient quality to resolve side chain interactions, including several key M17 mutations. To aid in the placement of the loop Cterminal to the A2 domain (a2-loop), we solved a high-resolution crystal structure of fXa in complex with a synthetic a2 peptide. The acidic a2-loop interacts with the basic heparin binding site of fXa, involving a conserved antiparallel -strand interaction. The M17-prothrombinase structure is compatible with data from biochemical and mutagenesis research and provides important new insights into the assembly and function of the prothrombinase complex. Less
Severe acute respiratory syndrome coronavirus SARS-CoV- continues to threaten global health This underpins the need for novel therapeutics against this virus Nonstructural protein Nsp of SARS-CoV- is a multifunctional protein with an essential role in viral replication As such it presents itself as an attractive target for drug discovery Here we describe two crystallographic fragment-screening campaigns against Nsp one using the established F X-Entry Screen and one using a new chemically and structurally diverse fragment library which we call the KIT library Together hits could be identified from screened fragments which constitutes the highest hit rate reported for Nsp to ... More
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten global health. This underpins the need for novel therapeutics against this virus. Nonstructural protein 1 (Nsp1) of SARS-CoV-2 is a multifunctional protein with an essential role in viral replication. As such, it presents itself as an attractive target for drug discovery. Here, we describe two crystallographic fragment-screening campaigns against Nsp1, one using the established F2X-Entry Screen and one using a new, chemically and structurally diverse fragment library, which we call the KIT library. Together, 21 hits could be identified from 192 screened fragments, which constitutes the highest hit rate reported for Nsp1 to date. Many hits bind to a key functional region and interact with residues involved in cellular mRNA cleavage, ribosome binding and viral RNA recognition. Furthermore, most of the identified fragments share a common binding mode, providing promising starting points for further optimization into drug-like compounds that can disrupt the role of Nsp1 in viral replication. Less
Discovery and artificial intelligence-guided mechanistic elucidation of a narrow-spectrum antibiotic
Current clinical antibiotics are largely broad-spectrum agents that can alter the gut microbiome and promote colonization by Enterobacteriaceae which are often drug resistant This includes adherent-invasive Escherichia coli AIEC particularly in patients with inflammatory bowel disease in which dysbiosis creates a niche for this pathogen to colonize There is an urgent and unmet need for novel narrow-spectrum and microbiome-sparing antibiotics Here we screened bioactive small molecules for antibacterial activity against AIEC and discovered enterololin an antibacterial compound with targeted activity against Enterobacteriaceae species Enterololin could overcome intrinsic and acquired resistance mechanisms in clinical isolates when combined with a subinhibitory concentration ... More
Current clinical antibiotics are largely broad-spectrum agents that can alter the gut microbiome and promote colonization by Enterobacteriaceae, which are often drug resistant. This includes adherent-invasive Escherichia coli (AIEC), particularly in patients with inflammatory bowel disease, in which dysbiosis creates a niche for this pathogen to colonize. There is an urgent and unmet need for novel narrow-spectrum and microbiome-sparing antibiotics. Here we screened 10,747 bioactive small molecules for antibacterial activity against AIEC and discovered enterololin, an antibacterial compound with targeted activity against Enterobacteriaceae species. Enterololin could overcome intrinsic and acquired resistance mechanisms in clinical isolates when combined with a subinhibitory concentration of SPR741, a polymyxin B analogue used here to increase outer membrane permeability in Gram-negative bacteria. Molecular substructure- and deep learning-guided mechanism-of-action investigations revealed that enterololin perturbs lipoprotein trafficking through a mechanism involving the LolCDE complex, laboratory-evolved resistant mutants predominantly mapped to lolC and lolE, with an in vitro frequency of resistance of ~10-8 to 10-7. Enterololin showed low mammalian cytotoxicity (HEK293 half-maximal inhibitory concentration ~100 µg ml-1) and suppressed AIEC infection in mouse models when administered in combination with SPR741, while largely preserving the overall microbiome composition. This study highlights the utility of deep learning methods for predicting molecular interactions and identifies a promising Enterobacteriaceae-specific antibacterial candidate for further development. Less
Antibody-drug conjugates ADCs are a promising therapeutic modality that enables the delivery of cytotoxic drugs to the target cells that express the corresponding antigen However the purification of ADCs while ensuring product safety homogeneity and stability is a challenging task due to their complex and fragile structure Size exclusion chromatography SEC the conventional method for ADC purification is time-consuming as it requires multiple column washes and equilibration steps Moreover subsequent formulation of ADCs typically using dead-end filtration DEF further complicates the production workflow We compared SEC DEF with the Pulse a miniaturized and automated tangential flow filtration system for purification ... More
Antibody-drug conjugates (ADCs) are a promising therapeutic modality that enables the delivery of cytotoxic drugs to the target cells that express the corresponding antigen. However, the purification of ADCs while ensuring product safety, homogeneity, and stability is a challenging task due to their complex and fragile structure. Size exclusion chromatography (SEC), the conventional method for ADC purification, is time-consuming as it requires multiple column washes and equilibration steps. Moreover, subsequent formulation of ADCs, typically using dead-end filtration (DEF), further complicates the production workflow. We compared SEC+DEF with the µPulse®, a miniaturized and automated tangential flow filtration system, for purification and formulation of ADCs. Quality analysis revealed that both approaches were equally gentle as comparable drug-to-antibody ratios (DARs) and monomer purities were observed in the purified samples. Most importantly, both methods exhibited equivalent cleanup efficiency with a 99.8% reduction in free linker-drug concentration. The endotoxin loads comprised 0.11 EU mg-1 for the µPulse and 0.07 EU mg-1 for SEC+DEF, ensuring validation of the safe application of purified ADCs in living systems. However, the µPulse performed purification and formulation of ADCs simultaneously as compared to SEC+DEF, which required multiple manual interventions. Our results indicate that the µPulse is a gentle, single-step, and walk-away approach for the purification of ADCs. Less
Ferritins are a widespread family of proteins involved in iron homeostasis While classic ferritins consist of four -helices and form -meric nanocages related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity Here by analyzing the available genomic data we identify a family of double ferritin-like proteins DFLPs composed of two four-helical domains which arose by duplication of a ferritin fold protein We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens TmDFLP and CaDFLP and show that they form homodimers and bind heme We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase ... More
Ferritins are a widespread family of proteins involved in iron homeostasis. While classic ferritins consist of four α-helices and form 24-meric nanocages, related ferritin-like proteins display other types of assemblies and sometimes lack any iron storage capacity. Here, by analyzing the available genomic data, we identify a family of double ferritin-like proteins (DFLPs) composed of two four-helical domains, which arose by duplication of a ferritin fold protein. We characterize representative DFLPs from Thermocrinis minervae and Caldanaerovirga acetigignens, TmDFLP and CaDFLP, and show that they form homodimers and bind heme. We determine the X-ray structure of TmDFLP and demonstrate its ferroxidase activity. Furthermore, we show that some DFLPs, including TmDFLP and CaDFLP, are highly likely to be targeted into encapsulin shells. Our work expands the range of known iron metabolism systems and highlights the power of genome mining for discovery of new proteins. Less
Flap endonuclease FEN is a long-standing target of interest in the DNA damage response DDR field due to its therapeutic potential in BRCA mutant cancers To-date there have only been a handful of FEN inhibitors reported in the literature most of which display modest selectivity and or weak cellular activity As such there is a need for more advanced pharmacological tools to probe the biology of FEN Here we report the discovery of MSC the first potent selective and orally bioavailable FEN inhibitor We describe our metal-chelating fragment screening approach and structure-based optimization to identify MSC using structural insights to ... More
Flap endonuclease 1 (FEN1) is a long-standing target of interest in the DNA damage response (DDR) field due to its therapeutic potential in BRCA mutant cancers. To-date there have only been a handful of FEN1 inhibitors reported in the literature, most of which display modest selectivity and/or weak cellular activity. As such, there is a need for more advanced pharmacological tools to probe the biology of FEN1. Here, we report the discovery of MSC778, the first potent, selective, and orally bioavailable FEN1 inhibitor. We describe our metal-chelating fragment screening approach and structure-based optimization to identify MSC778, using structural insights to drive design. Consistent with FEN1 inhibition, MSC778 selectively kills BRCA2-deficient cells and potentiates the activity of PARPi niraparib in vivo to induce tumor stasis in a BRCA2 KO DLD-1 mouse xenograft. Furthermore, we illustrate how development of this approach has the potential for addressing nucleases as a target class. Less
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space Here we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor NCS- protein-protein interactions PPIs This study represents a complete implementation of a single high-value design-make-test-analyze cycle that directly yields compounds with micromolar affinity with the potential to modulate NCS- interactions with key targets including the G-protein chaperone Ric- A and the dopamine D and cannabinoid CB receptors X-ray crystallographic fragment screening CFS revealed diverse interaction patterns within the NCS- hydrophobic crevice Algorithmically guided fragment evolution and automated synthesis ... More
Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space. Here, we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor 1 (NCS-1) protein-protein interactions (PPIs). This study represents a complete implementation of a single high-value design-make-test-analyze cycle that directly yields compounds with micromolar affinity with the potential to modulate NCS-1 interactions with key targets, including the G-protein chaperone Ric-8A and the dopamine D2 and cannabinoid CB1 receptors. X-ray crystallographic fragment screening (CFS) revealed diverse interaction patterns within the NCS-1 hydrophobic crevice. Algorithmically guided fragment evolution and automated synthesis enabled the rapid generation of over 250 derivatives, with biophysical validation using LC-MS and Grating-coupled interferometry. Structural analyses highlighted key pharmacophores, with selected compounds exhibiting favorable drug-like properties and potential blood-brain barrier penetration, making them promising candidates for neurodegenerative and neurodevelopmental disorders. Our results demonstrate the feasibility of accelerated hit-to-lead development at synchrotrons, demonstrating a robust, scalable platform for PPI-targeting drug discovery. The generated chemically diverse scaffolds provide a strong foundation for future therapeutic optimization. Less
The enzymatic degradation of polyethylene terephthalate PET offers a sustainable solution for PET recycling Over the past two decades more than PETases have been characterized primarily exhibiting similar sequences and structures Here we report new PET-degrading hydrolases including HaloPETase from the marine Halopseudomonas lineage thereby extending the narrow sequence space by novel features at the active site The crystal structure of HaloPETase was determined to a resolution of revealing a unique active site architecture and a lack of the canonical -stacking clamp found in PETases so far Further variations in active site composition and loop structures were observed Additionally we ... More
The enzymatic degradation of polyethylene terephthalate (PET) offers a sustainable solution for PET recycling. Over the past two decades, more than 100 PETases have been characterized, primarily exhibiting similar sequences and structures. Here, we report new PET-degrading α/β hydrolases, including HaloPETase1 from the marine Halopseudomonas lineage, thereby extending the narrow sequence space by novel features at the active site. The crystal structure of HaloPETase1 was determined to a resolution of 1.16 Å, revealing a unique active site architecture and a lack of the canonical π-stacking clamp found in PETases so far. Further, variations in active site composition and loop structures were observed. Additionally, we found five more enzymes from the same lineage, two of which have a high similarity to type IIa bacterial PETases, while the other three resemble HaloPETase1. All these enzymes exhibited high salt tolerance ranging from 2.5 to 5 M NaCl leading to higher total product releases upon PET degradation at 40 or 50 °C. Based on these findings, we propose an extension of the existing PETase classification system to include type III PETases. Less
Poly-ADP-ribosylation at sites of DNA damage catalyzed by PARP enzymes activates the DNA damage response chromatin remodeling and DNA repair The modification is reversed by two enzymes in humans PARG which efficiently hydrolyzes the poly-ADP-ribose chains and ARH which is the key enzyme for removing the last proximal mono-ADP-ribose from serine residues While inhibitor development has largely focused on PARPs and PARG no potent and selective inhibitors for ARH are currently available We optimized a FRET-based competition assay for ARH and carried out high-throughput screening of small-molecule inhibitors One hit compound with a potency of M was discovered and through ... More
Poly-ADP-ribosylation at sites of DNA damage, catalyzed by PARP enzymes, activates the DNA damage response, chromatin remodeling, and DNA repair. The modification is reversed by two enzymes in humans: PARG, which efficiently hydrolyzes the poly-ADP-ribose chains, and ARH3, which is the key enzyme for removing the last proximal mono-ADP-ribose from serine residues. While inhibitor development has largely focused on PARPs and PARG, no potent and selective inhibitors for ARH3 are currently available. We optimized a FRET-based competition assay for ARH3 and carried out high-throughput screening of small-molecule inhibitors. One hit compound, 1, with a potency of 22 μM was discovered, and through structure–activity relationship studies and synthesis, we improved its potency 10-fold to 2 μM (compound 27, MDOLL-0286). We demonstrate that the compound inhibits ARH3’s poly-ADP-ribose hydrolytic activity on cellular substrates. Intriguingly, it does not effectively inhibit the hydrolysis of mono-ADP-ribosylation from natural protein substrates. This is despite the fact that the cocrystal structure of compound 1 bound to ARH3 reveals its overlap with the enzyme’s ADP-ribose binding site, agreeing with the competition in the FRET assay. The first experimental ARH3 inhibitor complex provides a valuable starting point for developing more potent chemical probes to study DNA damage response mechanisms in the future. Less
Liquid dispensing technologies that enhance experimental efficiency and deliver high accuracy across multiple volume ranges are critically important in high-throughput experimentation HTE workflows for reaction exploration and optimization The Mantis liquid handler stands out as a compact highly modular system featuring a wide array of input options and near plug-and-play functionality with carousels all while employing positive displacement dispensing technology for precise liquid delivery that is largely independent of liquid properties However the system was originally designed to handle aqueous-based biological media at low volumes L dispense limiting its applications in nonaqueous chemistry Herein we report a hardware development effort ... More
Liquid dispensing technologies that enhance experimental efficiency and deliver high accuracy across multiple volume
ranges are critically important in high-throughput experimentation (HTE) workflows for reaction exploration and optimization. The
Mantis liquid handler stands out as a compact, highly modular system featuring a wide array of input options and near plug-and-play
functionality with carousels, all while employing positive displacement dispensing technology for precise liquid delivery that is largely
independent of liquid properties. However, the system was originally designed to handle aqueous-based biological media at low
volumes (1−5 μL/dispense), limiting its applications in nonaqueous chemistry. Herein, we report a hardware development effort
from a cross-industry collaboration aimed at enhancing Mantis’ capabilities to handle organic solvents and chemical reagents, even at
larger deliverable volumes (up to 50 μL/dispense). Various chemistry examples are provided to demonstrate the implementation of
newly developed chip designs, which allow the acquisition of accurate, reproducible, and robust data, enabling more efficient
workflows for new reaction discovery, reaction optimization, and data set production. Most importantly, by implementing the Mantis
liquid handler from the outset, an increase of 77% in HTE execution efficiency relative to a manual workflow incorporating
traditional liquid handling operations was achieved in an amide coupling demonstration. Less
ranges are critically important in high-throughput experimentation (HTE) workflows for reaction exploration and optimization. The
Mantis liquid handler stands out as a compact, highly modular system featuring a wide array of input options and near plug-and-play
functionality with carousels, all while employing positive displacement dispensing technology for precise liquid delivery that is largely
independent of liquid properties. However, the system was originally designed to handle aqueous-based biological media at low
volumes (1−5 μL/dispense), limiting its applications in nonaqueous chemistry. Herein, we report a hardware development effort
from a cross-industry collaboration aimed at enhancing Mantis’ capabilities to handle organic solvents and chemical reagents, even at
larger deliverable volumes (up to 50 μL/dispense). Various chemistry examples are provided to demonstrate the implementation of
newly developed chip designs, which allow the acquisition of accurate, reproducible, and robust data, enabling more efficient
workflows for new reaction discovery, reaction optimization, and data set production. Most importantly, by implementing the Mantis
liquid handler from the outset, an increase of 77% in HTE execution efficiency relative to a manual workflow incorporating
traditional liquid handling operations was achieved in an amide coupling demonstration. Less
An enzyme activity-based workflow for the identification and characterization of covalent inhibitors
The field of covalent drug development has advanced rapidly offering promising therapeutic potential due to the ability of these drugs to form slowly reversible or irreversible bonds with target proteins resulting in prolonged pharmacodynamic effects This distinctive mechanism of action has sparked resurging interest in covalent inhibitors across various disease areas including oncology neurological disorders and infectious diseases However characterization of covalent inhibitors poses unique challenges highlighting the need for simplified and robust assay methods This protocol describes an enzyme activity-based workflow designed to identify and characterize covalent inhibitors efficiently By streamlining the evaluation process this approach enhances the reliability ... More
The field of covalent drug development has advanced rapidly, offering promising therapeutic potential due to the ability of these drugs to form slowly reversible or irreversible bonds with target proteins, resulting in prolonged pharmacodynamic effects. This distinctive mechanism of action has sparked resurging interest in covalent inhibitors across various disease areas, including oncology, neurological disorders, and infectious diseases. However, characterization of covalent inhibitors poses unique challenges, highlighting the need for simplified and robust assay methods. This protocol describes an enzyme activity-based workflow designed to identify and characterize covalent inhibitors efficiently. By streamlining the evaluation process, this approach enhances the reliability and reproducibility of covalent inhibitor assessment, ultimately accelerating the discovery and optimization of novel covalent therapeutics. Less
In more and more drug discovery projects crystallographic fragment screening CFS is employed as an early screening method Here we demonstrate that choosing the right crystal form has a profound influence on the hit rates and hence success and speed of downstream lead generation Two CFS campaigns with the same fragment library and an almost identical experimental setup were carried out against the two crystal forms of the SARS-CoV- main protease While both crystal forms exhibit similar diffraction properties the observed hit rates in the two campaigns were vastly different For the monoclinic crystals a hit rate of was determined ... More
In more and more drug discovery projects, crystallographic fragment screening (CFS) is employed as an early screening method. Here, we demonstrate that choosing the right crystal form has a profound influence on the hit rates and hence success and speed of downstream lead generation. Two CFS campaigns with the same fragment library and an almost identical experimental setup were carried out against the two crystal forms of the SARS-CoV-2 main protease.While both crystal forms exhibit similar diffraction properties, the observed hit rates in the two campaigns were vastly different. For the monoclinic crystals a hit rate of 3% was determined, while a hit rate of 16% was observed for the orthorhombic crystals. These findings align with the more open molecular packing in the orthorhombic crystals where the solvent channels leading to the active sites are about twice larger than in the monoclinic crystal form. Our results highlight the critical importance of the crystal system in a crystallographic fragment-screening campaign and identify this parameter as one of the most important ones to be optimized during preparation of a campaign. Less
Glutarimide analogs such as thalidomide redirect the E ubiquitin ligase CRL CRBN to induce degradation of certain zinc finger ZF proteins Although the core structural motif recognized by CRBN has been characterized it does not fully explain substrate specificity To explore the role of residues adjacent to this core motif we constructed a comprehensive ZF reporter library of reporters derived from human ZF proteins and conducted a library-on-library screen with glutarimide analogs to identify compounds that collectively degrade ZF reporters Cryo-electron microscopy and crystal structures of ZFs in complex with CRBN revealed the importance of interactions beyond the core ZF ... More
Glutarimide analogs, such as thalidomide, redirect the E3 ubiquitin ligase CRL4CRBN to induce degradation of certain zinc finger (ZF) proteins. Although the core structural motif recognized by CRBN has been characterized, it does not fully explain substrate specificity. To explore the role of residues adjacent to this core motif, we constructed a comprehensive ZF reporter library of 9,097 reporters derived from 1,655 human ZF proteins and conducted a library-on-library screen with 29 glutarimide analogs to identify compounds that collectively degrade 38 ZF reporters. Cryo-electron microscopy and crystal structures of ZFs in complex with CRBN revealed the importance of interactions beyond the core ZF degron. We used systematic mutagenesis of ZFs and CRBN to identify modes of neosubstrate recruitment requiring distinct amino acids. Finally, we found subtle chemical variations in glutarimide analogs that alter target scope and selectivity, thus providing a roadmap for their rational design. Less
Deep learning has revolutionized soluble protein design yet de novo transmembrane TM protein engineering remains hindered by scarce structural data complex membrane-specific interactions and conformational dynamics We developed TMDiffusion TMDF a joint all-heavy-atom sequence structure diffusion model trained to capture the full interaction diversity of natural TM proteins including weak and polar contact networks TMDF designs diverse TM architectures associating domains inhibitors and conformational switches in a single step achieving experimental success A crystal structure of designed proteins matches predictions with atomic accuracy Leveraging TMDF we built synthetic single-pass receptors whose de novo TM domains toggle between conformations enabling precise ... More
Deep learning has revolutionized soluble protein design, yet de novo transmembrane (TM) protein engineering remains hindered by scarce structural data, complex membrane-specific interactions and conformational dynamics. We developed TMDiffusion (TMDF), a joint all-heavy-atom sequence–structure diffusion model trained to capture the full interaction diversity of natural TM proteins, including weak and polar contact networks. TMDF designs diverse TM architectures—associating domains, inhibitors, and conformational switches—in a single step, achieving >70% experimental success. A crystal structure of designed proteins matches predictions with atomic accuracy. Leveraging TMDF, we built synthetic single-pass receptors whose de novo TM domains toggle between conformations, enabling precise control of signalling outputs consistent with predicted equilibria. These results show that membrane-adapted DL models can accurately encode and program TM association energetics and conformations. TMDF establishes a general framework for bottom-up design of TM proteins with programmable functions, advancing both mechanistic studies of membrane proteins and development of next-generation therapeutics. Less
Transcription factors TFs regulate gene expression by engaging chromatin remodeling complexes yet the structural principles governing these critical interactions remain poorly defined Here we uncover the molecular mechanism by which lineage-specific pioneer transcription factor PU encoded by SPI directly engages the BAF mSWI SNF chromatin remodeling complex First using a variety of genomic approaches we establish that BAF collaborates with PU to regulate transcription in AML cells Then using a combination of biochemistry and biophysics mass spectrometry-based protein footprinting and crystallography we map the PU -BAF A interface to a disordered region of PU that adopts a helical conformation upon ... More
Transcription factors (TFs) regulate gene expression by engaging chromatin remodeling complexes, yet the structural principles governing these critical interactions remain poorly defined. Here, we uncover the molecular mechanism by which lineage-specific pioneer transcription factor PU.1 (encoded by SPI1) directly engages the BAF (mSWI/SNF) chromatin remodeling complex. First, using a variety of genomic approaches, we establish that BAF collaborates with PU.1 to regulate transcription in AML cells. Then, using a combination of biochemistry and biophysics, mass spectrometry-based protein footprinting, and crystallography, we map the PU.1-BAF60A interface to a disordered region of PU.1 that adopts a helical conformation upon binding to the YEATS-like domain of BAF60A. Disruption of this functionally critical interface via knockdown abrogates the ability of PU.1 to rescue cell viability. Finally, we conducted a high-throughput screen that yielded small molecules which selectively bind BAF60A and disrupt PU.1 binding. Co-crystal structures reveal distinct compound binding modes that converge on a critical PU.1-BAF60A interaction hotspot. These findings define, for the first time, the structural interface between a pioneer transcription factor and the BAF complex and establish a platform that enables targeting transcription factor-chromatin remodeling complex interactions in cancer. Less
Dual-specificity mitogen-activated protein kinase MAPK phosphatases MKPs directly dephosphorylate and inactivate the MAPKs Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored Here we sought to define the molecular mechanism of MKP regulation through an allosteric site within its catalytic domain We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y is required to maintain the structural integrity of the allosteric pocket Along with molecular dynamics simulations these data provide insight into how changes in ... More
Dual-specificity mitogen-activated protein kinase (MAPK) phosphatases (MKPs) directly dephosphorylate and inactivate the MAPKs. Although the catalytic mechanism of dephosphorylation of the MAPKs by the MKPs is established, a complete molecular picture of the regulatory interplay between the MAPKs and MKPs still remains to be fully explored. Here, we sought to define the molecular mechanism of MKP5 regulation through an allosteric site within its catalytic domain. We demonstrate using crystallographic and NMR spectroscopy approaches that residue Y435 is required to maintain the structural integrity of the allosteric pocket. Along with molecular dynamics simulations, these data provide insight into how changes in the allosteric pocket propagate conformational flexibility in the surrounding loops to reorganize catalytically crucial residues in the active site. Furthermore, Y435 contributes to the interaction with p38 MAPK and JNK, thereby promoting dephosphorylation. Collectively, these results highlight the role of Y435 in the allosteric site as a novel mode of MKP5 regulation by p38 MAPK and JNK Less
Transport and Golgi Organization Homolog TANGO protein deficiency disorder TDD is a rare autosomal recessive disorder characterized by multi-systemic abnormalities and significant phenotypic variability including neurodevelopmental delay seizures intermittent ataxia hypothyroidism rhabdomyolysis life-threatening metabolic derangements and cardiac arrhythmias Mutations in TANGO result in mitochondrial dysfunction abnormal lipid homeostasis with cardiolipin deficiency and impaired Golgi-ER trafficking in TANGO patient-derived cells Despite the wide recognition of the clinical manifestations of TDD and numerous molecular studies the precise function of TANGO and the pathophysiology of TDD remain poorly understood A computationally derived three-dimensional structure model suggested that TANGO adopts an -fold similar to ... More
Transport and Golgi Organization 2 Homolog (TANGO2) protein deficiency disorder (TDD) is a rare autosomal recessive disorder characterized by multi-systemic abnormalities and significant phenotypic variability including neurodevelopmental delay, seizures, intermittent ataxia, hypothyroidism, rhabdomyolysis, life-threatening metabolic derangements, and cardiac arrhythmias. Mutations in TANGO2 result in mitochondrial dysfunction, abnormal lipid homeostasis with cardiolipin deficiency, and impaired Golgi-ER trafficking in TANGO2 patient-derived cells. Despite the wide recognition of the clinical manifestations of TDD and numerous molecular studies, the precise function of TANGO2 and the pathophysiology of TDD remain poorly understood. A computationally derived three-dimensional structure model suggested that TANGO2 adopts an αββα-fold, similar to the N-terminal nucleophile aminohydrolase (Ntn) superfamily of proteins, but the experimentally verified structure has not been available thus far. Here, we present the first crystal structure of the recombinant human TANGO2, determined at 1.70 Å resolution. The X-ray structure data confirmed its predicted tertiary fold with similarity to the Ntn-hydrolase family of proteins, and the comparative analysis of the active site architecture, including residues involved in catalysis and putative ligand binding site, suggests a potential hydrolase function. Additional examination of the common mutation sites found in TDD patients provides insight regarding their potential effect on protein structure integrity. Less
This protocol describes the crystallization of Enterovirus EV- A protease mutant C A containing the VP - A junction in the active site The crystals form within - hours using a crystallization screen composed of M NaCl and ethanol The crystal structure was determined using X-ray diffraction resulting in hexagonal prism crystals in space group P with unit cell dimensions of and an average resolution of The protein was expressed using the plasmid Enterovirus Coxsackievirus A A protease
This protocol describes the crystallization of Enterovirus EV- A protease mutant C A containing the VP - A junction in the active site The crystals form within - hours using a crystallization screen composed of M NaCl and ethanol The crystal structure was determined using X-ray diffraction resulting in hexagonal prism crystals in space group P with unit cell dimensions of and an average resolution of The protein was expressed using the plasmid Enterovirus Coxsackievirus A A protease
Genetic studies have identified thousands of individual disease-associated non-coding alleles but identification of the causal alleles and their functions remain critical bottlenecks Even though CRISPR-Cas editing has enabled targeted modification of DNA inefficient editing leads to heterogeneous outcomes across individual cells limiting the ability to detect functional consequences of disease alleles To overcome these challenges we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits assays the transcriptome and measures cell surface protein expression We apply this approach to investigate the effects of gene disruption deletions in regulatory regions and non-coding single nucleotide polymorphisms We identify ... More
Genetic studies have identified thousands of individual disease-associated non-coding alleles, but identification of the causal alleles and their functions remain critical bottlenecks. Even though CRISPR-Cas editing has enabled targeted modification of DNA, inefficient editing leads to heterogeneous outcomes across individual cells, limiting the ability to detect functional consequences of disease alleles. To overcome these challenges, we present a multi-omic single cell sequencing approach that directly identifies genomic DNA edits, assays the transcriptome, and measures cell surface protein expression. We apply this approach to investigate the effects of gene disruption, deletions in regulatory regions, and non-coding single nucleotide polymorphisms. We identify the specific effects of individual SNPs, including the state-specific effects of an IL2RA autoimmune variant in primary human T cells. Multimodal functional genomic single cell assays including DNA sequencing bridge a crucial gap in our understanding of complex human diseases by directly identifying causal variation in primary human cells. Less
Electroporation-mediated gene delivery is a cornerstone of synthetic biology offering several advantages over other methods higher efficiencies broader applicability and simpler sample preparation Yet electroporation protocols are often challenging to integrate into highly multiplexed workflows owing to limitations in their scalability and tunability These challenges ultimately increase the time and cost per transformation As a result rapidly screening genetic libraries exploring combinatorial designs or optimizing electroporation parameters requires extensive iterations consuming large quantities of expensive custom-made DNA and cell lines or primary cells To address these limitations we have developed a High-Throughput Microfluidic Electroporation HTME platform that includes a -well ... More
Electroporation-mediated gene delivery is a cornerstone of synthetic biology, offering several advantages over other methods: higher efficiencies, broader applicability, and simpler sample preparation. Yet, electroporation protocols are often challenging to integrate into highly multiplexed workflows, owing to limitations in their scalability and tunability. These challenges ultimately increase the time and cost per transformation. As a result, rapidly screening genetic libraries, exploring combinatorial designs, or optimizing electroporation parameters requires extensive iterations, consuming large quantities of expensive custom-made DNA and cell lines or primary cells. To address these limitations, we have developed a High-Throughput Microfluidic Electroporation (HTME) platform that includes a 384-well electroporation plate (E-Plate) and control electronics capable of rapidly electroporating all wells in under a minute with individual control of each well. Fabricated using scalable and cost-effective printed-circuit-board (PCB) technology, the E-Plate significantly reduces consumable costs and reagent consumption by operating on nano to microliter volumes. Furthermore, individually addressable wells facilitate rapid exploration of large sets of experimental conditions to optimize electroporation for different cell types and plasmid concentrations/types. Use of the standard 384-well footprint makes the platform easily integrable into automated workflows, thereby enabling end-to-end automation. We demonstrate transformation of E. coli with pUC19 to validate the HTME’s core functionality, achieving at least a single colony forming unit in more than 99% of wells and confirming the platform’s ability to rapidly perform hundreds of electroporations with customizable conditions. This work highlights the HTME’s potential to significantly accelerate synthetic biology Design-Build-Test-Learn (DBTL) cycles by mitigating the transformation/transfection bottleneck. Less
This review highlights the development and evolution of three macromolecular crystallography MX beamlines at the Swiss Light Source SLS over the past two decades We discuss key advancements in X-ray optics detectors goniometers sample changers and MX methodology emphasizing their impact on high-throughput and high-resolution structural biology Our contributions are presented within the broader context of global efforts in synchrotron-based MX Looking ahead we explore the future experiments enabled by SLS and new opportunities at SwissFEL to enhance experimental capabilities and drive scientific discoveries
Fungal cell walls composed of polysaccharides and proteins play critical roles in adaptation cell division and protection against environmental stress Their polyglucan components are continuously remodeled by various types of glycosyl hydrolases GHs and transferases GTs In Saccharomyces cerevisiae and other ascomycetes enzymes of the Dfg subfamily which belong as GTs to the GH family cleave an linkage between glucosamine and mannose to facilitate covalent linkage of GPI-anchored proteins to the cell wall s polyglucans In contrast the functions of other fungal GH subfamilies are not understood We characterized CtGH from the sordariomycete Chaetomium thermophilum a member of the Fungi ... More
Fungal cell walls, composed of polysaccharides and proteins, play critical roles in adaptation, cell division, and protection against environmental stress. Their polyglucan components are continuously remodeled by various types of glycosyl hydrolases (GHs) and transferases (GTs). In Saccharomyces cerevisiae and other ascomycetes, enzymes of the Dfg5 subfamily, which belong as GTs to the GH76 family, cleave an α1,4 linkage between glucosamine and mannose to facilitate covalent linkage of GPI-anchored proteins to the cell wall’s polyglucans. In contrast, the functions of other fungal GH76 subfamilies are not understood. We characterized CtGH76 from the sordariomycete Chaetomium thermophilum, a member of the Fungi/Bacteria-mixed GH76 subfamily, revealing conserved structural features and functional divergence within the GH76 family. Notably, our structural characterization by X-ray crystallography combined with glycan fragment screening indicated that CtGH76 can recognize GPI-anchors like members of the Dfg5 subfamily but shows a broader promiscuity toward other glycans with central α1,6-mannobiose motifs due to the presence of an elongated glycan binding canyon. These findings provide new insights into GH76 enzyme diversity and fungal cell wall maturation. Less
Malaria remains a global health threat exacerbated by emerging resistance to antimalarial therapies and insecticides climate-driven outbreaks and limited chemoprotective options Here we report the characterization of RUPB- the first orally bioavailable inhibitor of Plasmodium falciparum cGMP-dependent protein kinase PfPKG RUPB- prevents infection by P falciparum and P cynomolgi sporozoites including the formation of hypnozoites by the latter A single oral dose blocks liver infection by P berghei sporozoites in vivo demonstrating efficacy consistent with further development as a once-weekly prophylaxis based on pharmacokinetic modeling The compound retains activity against field isolates resistant to chloroquine mefloquine cycloguanil sulfadoxine and pyrimethamine ... More
Malaria remains a global health threat exacerbated by emerging resistance to antimalarial therapies and insecticides, climate-driven outbreaks, and limited chemoprotective options. Here, we report the characterization of RUPB-61, the first orally bioavailable inhibitor of Plasmodium falciparum cGMP-dependent protein kinase (PfPKG). RUPB-61 prevents infection by P. falciparum and P. cynomolgi sporozoites, including the formation of hypnozoites by the latter. A single oral dose blocks liver infection by P. berghei sporozoites in vivo, demonstrating efficacy consistent with further development as a once-weekly prophylaxis based on pharmacokinetic modeling. The compound retains activity against field isolates resistant to chloroquine, mefloquine, cycloguanil, sulfadoxine and pyrimethamine, suggesting low likelihood of cross-resistance to existing antimalarials. Structural studies and free energy-based modeling guided compound design and prospectively validated the predictive accuracy of an in silico model of PfPKG interactions with this chemotype. While selectivity profiling identified off-target activity against human kinases, structural modeling provides a clear path for optimization. These results establish PfPKG inhibitors as promising candidates for chemoprotection and support further preclinical development of the RUPB-61 chemotype. Less
Malaria poses an enormous threat to human health With ever-increasing resistance to currently deployed antimalarials new targets and starting point compounds with novel mechanisms of action need to be identified Here we explore the antimalarial activity of the Streptomyces sp natural product -O-sulfamoyl- -chloroadenosine dealanylascamycin DACM and compare it with the synthetic adenosine monophosphate AMP mimic -O-sulfamoyladenosine AMS These nucleoside sulfamates exhibit potent inhibition of P falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin Exposure of P falciparum to DACM leads to inhibition of protein translation driven by eIF phosphorylation We show that DACM ... More
Malaria poses an enormous threat to human health. With ever-increasing resistance to currently deployed antimalarials, new targets and starting point compounds with novel mechanisms of action need to be identified. Here, we explore the antimalarial activity of the Streptomyces sp natural product, 5ʹ-O-sulfamoyl-2-chloroadenosine (dealanylascamycin, DACM) and compare it with the synthetic adenosine monophosphate (AMP) mimic, 5-O-sulfamoyladenosine (AMS). These nucleoside sulfamates exhibit potent inhibition of P. falciparum growth with an efficacy comparable to that of the current front-line antimalarial dihydroartemisinin. Exposure of P. falciparum to DACM leads to inhibition of protein translation, driven by eIF2α phosphorylation. We show that DACM targets multiple amino acyl tRNA synthetase (aaRS) targets, including the cytoplasmic aspartyl tRNA synthetase (AspRS). The mechanism involves hijacking of the reaction product, leading to the formation of a tightly bound inhibitory amino acid-sulfamate conjugate. We show that recombinant P. falciparum and P. vivax AspRS are susceptible to hijacking by DACM and AMS, generating Asp-DACM and Asp-AMS adducts that stabilize these proteins. By contrast, human AspRS appears less susceptible to hijacking. X-ray crystallography reveals that apo P. vivax AspRS exhibits a stabilized flipping loop over the active site that is poised to bind substrates. By contrast, human AspRS exhibits disorder in an extended region around the flexible flipping loop as well as in a loop in motif II. These structural differences may underpin the decreased susceptibility of human AspRS to reaction-hijacking by DACM and AMS. Our work reveals Plasmodium AspRS as a promising antimalarial target and highlights structural features that underpin differences in the susceptibility of aaRSs to reaction hijacking inhibition. Less
Glaucoma is a neurodegenerative condition involving optic nerve damage and retinal ganglion cells death Animal studies suggested that the pathway linking these events can be mediated by mobile zinc secreted into the intraretinal space and exerting cytotoxic effects Whether this mechanism is relevant for human glaucoma and what are the targets of extracellular zinc is unknown We report that increased zinc content in the aqueous humor and retina is indeed a characteristic of glaucomatous neuropathy and excess extracellular zinc may be recognized by the key retinal neurotrophic factor PEDF Biophysical and X-ray crystallographic studies show that PEDF coordinates zinc ions ... More
Glaucoma is a neurodegenerative condition involving optic nerve damage and retinal ganglion cells death. Animal studies suggested that the pathway linking these events can be mediated by mobile zinc secreted into the intraretinal space and exerting cytotoxic effects. Whether this mechanism is relevant for human glaucoma and what are the targets of extracellular zinc is unknown. We report that increased zinc content in the aqueous humor and retina is indeed a characteristic of glaucomatous neuropathy, and excess extracellular zinc may be recognized by the key retinal neurotrophic factor PEDF. Biophysical and X-ray crystallographic studies show that PEDF coordinates zinc ions in five types of intermolecular high-affinity sites, leading to a decrease in negative surface charge and reversible oligomerization of the protein, thereby masking the target recognition sites responsible for its neurotrophic and antiangiogenic activities and collagen binding. Notably, PEDF secretion is enhanced in both glaucoma and retinal cell models in response to zinc stress; however, zinc binding negatively affects axogenic, differentiative and prosurvival functions of PEDF by suppressing its ability to activate receptor PEDF-R/PNPLA2. We suggest that glaucomatous neurodegeneration is associated with direct inhibition of PEDF signaling by extracellular zinc, making their complex a promising target for neuroprotective therapy. Less