1194 Citations
Corticospinal neurons CSNs synapse directly on spinal neurons a diverse assortment of cells with unique structural and functional properties necessary for body movements CSNs modulating forelimb behavior fractionate into caudal forelimb area CFA and rostral forelimb area RFA motor cortical populations Despite their prominence the full diversity of spinal neurons targeted by CFA and RFA CSNs is uncharted Here we use anatomical and RNA sequencing methods to show that CSNs synapse onto a remarkably selective group of spinal cell types favoring inhibitory populations that regulate motoneuron activity and gate sensory feedback CFA and RFA CSNs target similar spinal neuron types ... More
Corticospinal neurons (CSNs) synapse directly on spinal neurons, a diverse assortment of cells with unique structural and functional properties necessary for body movements. CSNs modulating forelimb behavior fractionate into caudal forelimb area (CFA) and rostral forelimb area (RFA) motor cortical populations. Despite their prominence, the full diversity of spinal neurons targeted by CFA and RFA CSNs is uncharted. Here, we use anatomical and RNA sequencing methods to show that CSNs synapse onto a remarkably selective group of spinal cell types, favoring inhibitory populations that regulate motoneuron activity and gate sensory feedback. CFA and RFA CSNs target similar spinal neuron types, with notable exceptions that suggest that these populations differ in how they influence behavior. Finally, axon collaterals of CFA and RFA CSNs target similar brain regions yet receive highly divergent inputs. These results detail the rules of CSN connectivity throughout the brain and spinal cord for two regions critical for forelimb behavior. Less
Evolution leads to conservation of amino acid residues in protein families Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure Surprisingly proline residues that are highly conserved in class A -lactamases were found to tolerate various substitutions without large losses in enzyme activity We investigated the roles of three conserved prolines at positions and in the -lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time For the variant Pro Thr the crystal ... More
Evolution leads to conservation of amino acid residues in protein families. Conserved proline residues are usually considered to ensure the correct folding and to stabilize the three-dimensional structure. Surprisingly, proline residues that are highly conserved in class A β-lactamases were found to tolerate various substitutions without large losses in enzyme activity. We investigated the roles of three conserved prolines at positions 107, 226, and 258 in the β-lactamase BlaC from Mycobacterium tuberculosis and found that mutations can lead to dimerization of the enzyme and an overall less stable protein that is prone to aggregate over time. For the variant Pro107Thr, the crystal structure shows dimer formation resembling domain swapping. It is concluded that the proline substitutions loosen the structure, enhancing multimerization. Even though the enzyme does not lose its properties without the conserved proline residues, the prolines ensure the long-term structural integrity of the enzyme. Less
A novel monoacylglycerol MAG has been produced for use in the in meso lipid cubic phase crystallization of membrane proteins and complexes MAG differs from monoolein the most extensively used lipid for in meso crystallization in that it is shorter in chain length by one methylene and its cis olefinic bond is two carbons closer to the glycerol headgroup These changes in structure alter the phase behavior of the hydrated lipid and the microstructure of the corresponding mesophases formed Temperature composition phase diagrams for MAG have been constructed using small- and wide-angle X-ray scattering over a range of temperatures and ... More
A novel monoacylglycerol, 7.10 MAG, has been produced for use in the in meso (lipid cubic phase) crystallization of membrane proteins and complexes. 7.10 MAG differs from monoolein, the most extensively used lipid for in meso crystallization, in that it is shorter in chain length by one methylene and its cis olefinic bond is two carbons closer to the glycerol headgroup. These changes in structure alter the phase behavior of the hydrated lipid and the microstructure of the corresponding mesophases formed. Temperature–composition phase diagrams for 7.10 MAG have been constructed using small- and wide-angle X-ray scattering over a range of temperatures and hydration levels that span those used for crystallization. The phase diagrams include lamellar crystalline, fluid isotropic, lamellar liquid-crystalline, cubic-Ia3d, and cubic-Pn3m phases, as observed with monoolein. Conspicuous by its absence is the inverted hexagonal phase which is rationalized on the basis of 7.10 MAG’s chemical constitution. The cubic phase prepared with the new lipid facilitates the growth of crystals that were used to generate high-resolution structures of intramembrane β-barrel and α-helical proteins. Compatibility of fully hydrated 7.10 MAG with cholesterol and phosphatidylcholine means that these two lipids can be used as additives to optimize crystallogenesis in screening trials with 7.10 MAG as the host lipid. Less
Proteins usually execute their biological functions through interactions with other proteins and by forming macromolecular complexes but global profiling of protein complexes directly from human tissue samples has been limited In this study we utilized co-fractionation mass spectrometry CF-MS to map protein complexes within the post-mortem human brain with experimental replicates First we used concatenated anion and cation Ion Exchange Chromatography IEX to separate native protein complexes in fractions then proceeded with Data-Independent Acquisition DIA mass spectrometry to analyze the proteins in each fraction quantifying a total of proteins with overlapping in both replicates We improved DIA quantitative accuracy by ... More
Proteins usually execute their biological functions through interactions with other proteins and by forming macromolecular complexes, but global profiling of protein complexes directly from human tissue samples has been limited. In this study, we utilized co-fractionation mass spectrometry (CF-MS) to map protein complexes within the post-mortem human brain with experimental replicates. First, we used concatenated anion and cation Ion Exchange Chromatography (IEX) to separate native protein complexes in 192 fractions, then proceeded with Data-Independent Acquisition (DIA) mass spectrometry to analyze the proteins in each fraction, quantifying a total of 4,804 proteins with 3,260 overlapping in both replicates. We improved DIA quantitative accuracy by implementing a constant amount of bovine serum albumin (BSA) in each fraction as an internal standard. Next, advanced computational pipelines, which integrate both a database-based complex analysis and an unbiased protein-protein interaction (PPI) search, were applied to identify protein complexes and construct protein-protein interaction networks in the human brain. Our study led to the identification of 486 protein complexes and 10,054 binary protein-protein interactions, which represents the first global profiling of human brain PPIs using CF-MS. Overall, this study offers a resource and tool for a wide range of human brain research, including the identification of disease-specific protein complexes in the future. Less
The mammalian orthoreovirus reovirus NS protein is required for formation of replication compartments that support viral genome replication and capsid assembly Despite its functional importance a mechanistic understanding of NS is lacking We conducted structural and biochemical analyses of a NS mutant that forms dimers instead of the higher-order oligomers formed by wildtype WT NS The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT NS filaments in complex with RNA observed using cryo-EM The interior of the helical assembly is of appropriate diameter to bind RNA The helical assembly ... More
The mammalian orthoreovirus (reovirus) σNS protein is required for formation of replication compartments that support viral genome replication and capsid assembly. Despite its functional importance, a mechanistic understanding of σNS is lacking. We conducted structural and biochemical analyses of a σNS mutant that forms dimers instead of the higher-order oligomers formed by wildtype (WT) σNS. The crystal structure shows that dimers interact with each other using N-terminal arms to form a helical assembly resembling WT σNS filaments in complex with RNA observed using cryo-EM. The interior of the helical assembly is of appropriate diameter to bind RNA. The helical assembly is disrupted by bile acids, which bind to the same site as the N-terminal arm. This finding suggests that the N-terminal arm functions in conferring context-dependent oligomeric states of σNS, which is supported by the structure of σNS lacking an N-terminal arm. We further observed that σNS has RNA chaperone activity likely essential for presenting mRNA to the viral polymerase for genome replication. This activity is reduced by bile acids and abolished by N-terminal arm deletion, suggesting that the activity requires formation of σNS oligomers. Our studies provide structural and mechanistic insights into the function of σNS in reovirus replication. Less
Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria which are valuable for bioenergy development and biotechnology Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins scaffoldins and dockerin modules in enzymes Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes tandem dockerins also exist albeit very rare in some cellulosomal components whose assembly and functional roles are currently unclear In this study we characterized the structure and mode of assembly of a tandem bimodular double-dockerin which is connected to a putative S protease in ... More
Cellulosomes are intricate cellulose-degrading multi-enzymatic complexes produced by anaerobic bacteria, which are valuable for bioenergy development and biotechnology. Cellulosome assembly relies on the selective interaction between cohesin modules in structural scaffolding proteins (scaffoldins) and dockerin modules in enzymes. Although the number of tandem cohesins in the scaffoldins is believed to determine the complexity of the cellulosomes, tandem dockerins also exist, albeit very rare, in some cellulosomal components whose assembly and functional roles are currently unclear. In this study, we characterized the structure and mode of assembly of a tandem bimodular double-dockerin, which is connected to a putative S8 protease in the cellulosome-producing bacterium, Clostridium thermocellum. Crystal and NMR structures of the double-dockerin revealed two typical type I dockerin folds with significant interactions between them. Interaction analysis by isothermal titration calorimetry and NMR titration experiments revealed that the double-dockerin displays a preference for binding to the cell-wall anchoring scaffoldin ScaD through the first dockerin with a canonical dual-binding mode, while the second dockerin module was unable to bind to any of the tested cohesins. Surprisingly, the double-dockerin showed a much higher affinity to a cohesin from the CipC scaffoldin of Clostridium cellulolyticum than to the resident cohesins from C. thermocellum. These results contribute valuable insights into the structure and assembly of the double-dockerin module, and provide the basis for further functional studies on multiple-dockerin modules and cellulosomal proteases, thus highlighting the complexity and diversity of cellulosomal components. Less
Mycobacterium tuberculosis Mtb adapt to various host environments and utilize a variety of sugars and lipids as carbon sources Among these sugars maltose and trehalose also play crucial role in bacterial physiology and virulence However some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known Here we structurally and functionally characterized a conserved hypothetical gene Rv We determined the crystal structure of Rv at resolution The crystal structure revealed that Rv adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins Our comparative structural analysis suggested that Rv could perform either ... More
Mycobacterium tuberculosis (Mtb) adapt to various host environments and utilize a variety of sugars and lipids as carbon sources. Among these sugars, maltose and trehalose, also play crucial role in bacterial physiology and virulence. However, some key enzymes involved in trehalose and maltose metabolism in Mtb are not yet known. Here we structurally and functionally characterized a conserved hypothetical gene Rv3400. We determined the crystal structure of Rv3400 at 1.7 Å resolution. The crystal structure revealed that Rv3400 adopts Rossmann fold and shares high structural similarity with haloacid dehalogenase family of proteins. Our comparative structural analysis suggested that Rv3400 could perform either phosphatase or pyrophosphatase or β-phosphoglucomutase (β-PGM) activity. Using biochemical studies, we further confirmed that Rv3400 performs β-PGM activity and hence, Rv3400 encodes for β-PGM in Mtb. Our data also confirm that Mtb β-PGM is a metal dependent enzyme having broad specificity for divalent metal ions. β-PGM converts β-D-glucose-1-phosphate to β-D-glucose-6-phosphate which is required for the generation of ATP and NADPH through glycolysis and pentose phosphate pathway, respectively. Using site directed mutagenesis followed by biochemical studies, we show that two Asp residues in the highly conserved DxD motif, D29 and D31, are crucial for enzyme activity. While D29A, D31A, D29E, D31E and D29N mutants lost complete activity, D31N mutant retained about 30% activity. This study further helps in understanding the role of β-PGM in the physiology of Mtb. Less
Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability selectivity metabolic stability and rapid clearance of peptides Here we describe the rational design synthesis and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead We have identified potent and selective inhibitors of TMPRSS matriptase hepsin and HGFA and demonstrated their improved protease selectivity metabolic stability and pharmacokinetic PK properties We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD b bound to matriptase revealing an unexpected binding conformation Cyclic biphenyl ether VD ... More
Protease inhibitor drug discovery is challenged by the lack of cellular and oral permeability, selectivity, metabolic stability, and rapid clearance of peptides. Here, we describe the rational design, synthesis, and evaluation of peptidomimetic side-chain-cyclized macrocycles which we converted into covalent serine protease inhibitors with the addition of an electrophilic ketone warhead. We have identified potent and selective inhibitors of TMPRSS2, matriptase, hepsin, and HGFA and demonstrated their improved protease selectivity, metabolic stability, and pharmacokinetic (PK) properties. We obtained an X-ray crystal structure of phenyl ether-cyclized tripeptide VD4162 (8b) bound to matriptase, revealing an unexpected binding conformation. Cyclic biphenyl ether VD5123 (11) displayed the best PK properties in mice with a half-life of 4.5 h and compound exposure beyond 24 h. These new cyclic tripeptide scaffolds can be used as easily modifiable templates providing a new strategy to overcoming the obstacles presented by linear acyclic peptides in protease inhibitor drug discovery. Less
X-ray crystallography is the most commonly employed technique to discern macromolecular structures but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging The crystallization of biomolecules is largely experimentally defined and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions At the National High-Throughput Crystallization HTX Center highly reproducible methods have been implemented to facilitate crystal growth including an automated high-throughput -well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters Plates are monitored using state-of-the-art imaging modalities over the course of weeks to provide insight into ... More
X-ray crystallography is the most commonly employed technique to discern macromolecular structures, but the crucial step of crystallizing a protein into an ordered lattice amenable to diffraction remains challenging. The crystallization of biomolecules is largely experimentally defined, and this process can be labor-intensive and prohibitive to researchers at resource-limited institutions. At the National High-Throughput Crystallization (HTX) Center, highly reproducible methods have been implemented to facilitate crystal growth, including an automated high-throughput 1,536-well microbatch-under-oil plate setup designed to sample a wide breadth of crystallization parameters. Plates are monitored using state-of-the-art imaging modalities over the course of 6 weeks to provide insight into crystal growth, as well as to accurately distinguish valuable crystal hits. Furthermore, the implementation of a trained artificial intelligence scoring algorithm for identifying crystal hits, coupled with an open-source, user-friendly interface for viewing experimental images, streamlines the process of analyzing crystal growth images. Here, the key procedures and instrumentation are described for the preparation of the cocktails and crystallization plates, imaging the plates, and identifying hits in a way that ensures reproducibility and increases the likelihood of successful crystallization. Less
Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from crystallization plates at Diamond Light Source beamline VMXi are described This approach enables high-quality room-temperature crystal structures to be determined from multiple crystals in a straightforward manner and provides very rapid feedback on the results of crystallization trials as well as enabling serial crystallography The value of room temperature structures in understanding protein structure ligand binding and dynamics is becoming increasingly recognized in the structural biology community This pipeline is accessible to users from all over the world with several available ... More
Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from crystallization plates at Diamond Light Source beamline VMXi are described. This approach enables high-quality room-temperature crystal structures to be determined from multiple crystals in a straightforward manner and provides very rapid feedback on the results of crystallization trials as well as enabling serial crystallography. The value of room temperature structures in understanding protein structure, ligand binding, and dynamics is becoming increasingly recognized in the structural biology community. This pipeline is accessible to users from all over the world with several available modes of access. Crystallization experiments that are set up can be imaged and viewed remotely with crystals identified automatically using a machine learning tool. Data are measured in a queue-based system with up to 60° rotation datasets from user-selected crystals in a plate. Data from all the crystals within a particular well or sample group are automatically merged using xia2.multiplex with the outputs straightforwardly accessed via a web browser interface. Less
The CD IGFBP axis proteins are key factors expressed in endothelial cells EC that mediate EC angiogenesis and migration Their upregulation contributes to tumor vascular abnormality and a blockade of this interaction promotes a favorable tumor microenvironment for therapeutic interventions However the interactions of these proteins with each other remain unclear In this study we determined a partial structure of the human CD IGFBP complex comprising the EGF domain of CD and the IB domain of IGFBP Mutagenesis studies confirmed interactions and specificities Cellular and mouse tumor studies demonstrated the physiological relevance of the CD IGFBP interaction in EC angiogenesis ... More
The CD93/IGFBP7 axis proteins are key factors expressed in endothelial cells (EC) that mediate EC angiogenesis and migration. Their upregulation contributes to tumor vascular abnormality and a blockade of this interaction promotes a favorable tumor microenvironment for therapeutic interventions. However, the interactions of these proteins with each other remain unclear. In this study, we determined a partial structure of the human CD93–IGFBP7 complex comprising the EGF1 domain of CD93 and the IB domain of IGFBP7. Mutagenesis studies confirmed interactions and specificities. Cellular and mouse tumor studies demonstrated the physiological relevance of the CD93–IGFBP7 interaction in EC angiogenesis. Our study provides leads for the development of therapeutic agents to precisely disrupt unwanted CD93–IGFBP7 signaling in the tumor microenvironment. Additionally, analysis of the CD93 full-length architecture provides insights into how CD93 protrudes on the cell surface and forms a flexible platform for binding to IGFBP7 and other ligands. Less
Cancer cells often exhibit DNA copy number aberrations and can vary widely in their ploidy Correct estimation of the ploidy of single-cell genomes is paramount for downstream analysis Based only on single-cell DNA sequencing information scAbsolute achieves accurate and unbiased measurement of single-cell ploidy and replication status including whole-genome duplications We demonstrate scAbsolute s capabilities using experimental cell multiplets a FUCCI cell cycle expression system and a benchmark against state-of-the-art methods scAbsolute provides a robust foundation for single-cell DNA sequencing analysis across different technologies and has the potential to enable improvements in a number of downstream analyses
Introduction Type diabetes mellitus T DM is a prevalent form of diabetes that affects - of all diabetic patients Insulin sensitizers and insulin exogenous supply could temporarily ameliorate hyperglycaemia however they are accompanied by side effects As a result new approaches are required to address insulin resistance and regenerate beta cells simultaneously The secretome of hypoxic mesenchymal stem cells SH-MSCs contains various growth factors and anti-inflammatory cytokines that could potentially enhance insulin resistance and improve pancreatic function Objectives In this study we performed SH-MSCs infusion to ameliorate HFD-induced hyperglycaemia in T DM rats Methods We created a T DM rat ... More
Introduction: Type 2 diabetes mellitus (T2DM) is a prevalent form of diabetes that affects 90 - 95 % of all diabetic patients. Insulin sensitizers and insulin exogenous supply could temporarily ameliorate hyperglycaemia; however, they are accompanied by side effects. As a result, new approaches are required to address insulin resistance and regenerate beta cells simultaneously. The secretome of hypoxic mesenchymal stem cells (SH-MSCs) contains various growth factors and anti-inflammatory cytokines that could potentially enhance insulin resistance and improve pancreatic function. Objectives: In this study, we performed SH-MSCs infusion to ameliorate HFD-induced hyperglycaemia in T2DM rats. Methods: We created a T2DM rat model using a combination of a high-fat diet (HFD) and streptozotocin (STZ) administration. Then, we administered SH-MSCs injection at doses of 250 and 500 µL and assessed the therapeutic effects of SH-MSCs. We also investigated the potential underlying mechanisms involved. Results: The administration of SH-MSCs improved hyperglycemia in rats with T2DM. Infusion of SH-MSCs at 500 µL dose decreased homeostatic model assessment for insulin resistance (HOMA-IR). Histological analysis revealed that injection of SH-MSCs alleviated morphological damage of pancreas. SH-MSCs administration also inhibit the level of IL-6 and promote the expression of CD163 type 2 macrophage. Conclusion: The results of our study indicate that SH-MSCs have the potential to improve hyperglycemia and exert a protective effect on T2DM rats. Less
Pacak-Zhuang syndrome is caused by mutations in the EPAS gene which encodes for one of the three hypoxia-inducible factor alpha HIF paralogs HIF and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia However the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood Here we devised a quantitative method for determining the dissociation constant Kd of the HIF peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase PHD using microscale thermophoresis MST and showed that neuroendocrine-associated Class HIF mutants have distinctly higher Kd than the exclusively polycythemia-associated Class HIF mutants Based on the co-crystal ... More
Pacak-Zhuang syndrome is caused by mutations in the EPAS1 gene, which encodes for one of the three hypoxia-inducible factor alpha (HIFα) paralogs HIF2α and is associated with defined but varied phenotypic presentations including neuroendocrine tumors and polycythemia. However, the mechanisms underlying the complex genotype-phenotype correlations remain incompletely understood. Here, we devised a quantitative method for determining the dissociation constant (Kd) of the HIF2α peptides containing disease-associated mutations and the catalytic domain of prolyl-hydroxylase (PHD2) using microscale thermophoresis (MST) and showed that neuroendocrine-associated Class 1 HIF2α mutants have distinctly higher Kd than the exclusively polycythemia-associated Class 2 HIF2α mutants. Based on the co-crystal structure of PHD2/HIF2α peptide complex at 1.8 Å resolution, we showed that the Class 1 mutated residues are localized to the critical interface between HIF2α and PHD2, adjacent to the PHD2 active catalytic site, while Class 2 mutated residues are localized to the more flexible region of HIF2α that makes less contact with PHD2. Concordantly, Class 1 mutations were found to significantly increase HIF2α-mediated transcriptional activation in cellulo compared to Class 2 counterparts. These results reveal a structural mechanism in which the strength of the interaction between HIF2α and PHD2 is at the root of the general genotype-phenotype correlations observed in Pacak-Zhuang syndrome. Less
Plant pathogens secrete proteins known as effectors that function in the apoplast or inside plant cells to promote virulence Effector recognition by cell-surface or cytosolic receptors results in the activation of defence pathways and plant immunity Despite their importance our general understanding of fungal effector function and recognition by immunity receptors remains poor One complication often associated with effectors is their high sequence diversity and lack of identifiable sequence motifs precluding prediction of structure or function In recent years several studies have demonstrated that fungal effectors can be grouped into structural classes despite significant sequence variation and existence across taxonomic ... More
Plant pathogens secrete proteins, known as effectors, that function in the apoplast or inside plant cells to promote virulence. Effector recognition by cell-surface or cytosolic receptors results in the activation of defence pathways and plant immunity. Despite their importance, our general understanding of fungal effector function and recognition by immunity receptors remains poor. One complication often associated with effectors is their high sequence diversity and lack of identifiable sequence motifs precluding prediction of structure or function. In recent years, several studies have demonstrated that fungal effectors can be grouped into structural classes, despite significant sequence variation and existence across taxonomic groups. Using protein X-ray crystallography, we identify a new structural class of effectors hidden within the secreted in xylem (SIX) effectors from Fusarium oxysporum f. sp. lycopersici (Fol). The recognised effectors Avr1 (SIX4) and Avr3 (SIX1) represent the founding members of the Fol dual-domain (FOLD) effector class, with members containing two distinct domains. Using AlphaFold2, we predicted the full SIX effector repertoire of Fol and show that SIX6 and SIX13 are also FOLD effectors, which we validated experimentally for SIX6. Based on structural prediction and comparisons, we show that FOLD effectors are present within three divisions of fungi and are expanded in pathogens and symbionts. Further structural comparisons demonstrate that Fol secretes effectors that adopt a limited number of structural folds during infection of tomato. This analysis also revealed a structural relationship between transcriptionally co-regulated effector pairs. We make use of the Avr1 structure to understand its recognition by the I receptor, which leads to disease resistance in tomato. This study represents an important advance in our understanding of Fol-tomato, and by extension plant–fungal interactions, which will assist in the development of novel control and engineering strategies to combat plant pathogens. Less
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy especially considering the anthropogenic effects in the global warming and natural resources depletion This includes the valorization of the hemicellulosic fraction of plant biomass aiming to produce prebiotic oligosaccharides widely explored in food and feed industries In the present work we conducted biochemical and biophysical characterization of two prokaryotic xylanases of family from Bacillus pumilus and Ruminococcus champanellensis and assessed their applicability for xylooligosaccharides production using alkaline pretreated corn cob and eucalyptus sawdust collected from a local market and ... More
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy, especially considering the anthropogenic effects in the global warming and natural resources depletion. This includes the valorization of the hemicellulosic fraction of plant biomass, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In the present work, we conducted biochemical and biophysical characterization of two prokaryotic xylanases of family 30_8 from Bacillus pumilus and Ruminococcus champanellensis, and assessed their applicability for xylooligosaccharides production using alkaline pretreated corn cob and eucalyptus sawdust collected from a local market and a sawmill shop in Araraquara, respectively. Mass spectrometry and high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis revealed that RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and proved to be highly inefficient in the cleavage of X4, X5 and X6, whereas BpXyn30A produces both linear and branched oligosaccharides. Crystallographic structure of BpXyn30A and RcXyn30A catalytic domain were solved and refined to 2.16 Å and 1.37Å resolution, respectively. Structural analysis of the enzymes binding cleft showed a conserved set of amino acids interacting with glucuronic acid substitution in the subsite -2b by several hydrogen bonds and ionic interactions, a characteristic shared between true glucunoxylanases. Furthermore, RcXyn30A has a larger β5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3). Finally, B. pumilus xylanase obtained higher conversion yields from pretreated biomasses than RcXyn30A, although the latter presents a specific activity against glucuronoxylan 9 times higher than the former. Less
Periplasmic solute-binding proteins SBPs are key ligand recognition components of bacterial ATP-binding cassette ABC transporters that allow bacteria to import nutrients and metabolic precursors from the environment Periplasmic SBPs comprise a large and diverse family of proteins of which only a small number have been empirically characterized In this work we identify a set of unique uncharacterized proteins within the SBP bac family that are found in conserved operons comprising genes encoding i ABC transport systems and ii putative amidases from the FmdA AmdA family From these uncharacterized SBP bac proteins we characterize a representative periplasmic SBP from Mesorhizobium sp ... More
Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from Mesorhizobium sp. A09 (MeAmi_SBP) and show that MeAmi_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of MeAmi_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca2+ binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca2+ binding are conserved amongst the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that MeAmi_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization. Less
Hematopoietic stem cells HSCs develop from the hemogenic endothelium HE in the aorta- gonads-and mesonephros AGM region and reside within Intra-aortic hematopoietic clusters IAHC along with hematopoietic progenitors HPC The signalling mechanisms that distinguish HSCs from HPCs are unknown Notch signaling is essential for arterial specification IAHC formation and HSC activity but current studies on how Notch segregates these different fates are inconsistent We now demonstrate that Notch activity is highest in a subset of GFI HSC-primed HE cells and is gradually lost with HSC maturation We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH ... More
Hematopoietic stem cells (HSCs) develop from the hemogenic endothelium (HE) in the aorta- gonads-and mesonephros (AGM) region and reside within Intra-aortic hematopoietic clusters (IAHC) along with hematopoietic progenitors (HPC). The signalling mechanisms that distinguish HSCs from HPCs are unknown. Notch signaling is essential for arterial specification, IAHC formation and HSC activity, but current studies on how Notch segregates these different fates are inconsistent. We now demonstrate that Notch activity is highest in a subset of, GFI1 + , HSC-primed HE cells, and is gradually lost with HSC maturation. We uncover that the HSC phenotype is maintained due to increasing levels of NOTCH1 and JAG1 interactions on the surface of the same cell (cis) that renders the NOTCH1 receptor from being activated. Forced activation of the NOTCH1 receptor in IAHC activates a hematopoietic differentiation program. Our results indicate that NOTCH1-JAG1 cis-inhibition preserves the HSC phenotype in the hematopoietic clusters of the embryonic aorta. Less
Infected cell protein ICP is an immediate-early regulatory protein of herpes simplex virus HSV- that possesses E ubiquitin ligase activity ICP transactivates viral genes in part through its C-terminal dimer domain residues Deletion of this dimer domain results in reduced viral gene expression lytic infection and reactivation from latency Since ICP 's dimer domain is associated with its transactivation activity and efficient viral replication we wanted to determine the structure of this specific domain The C-terminus of ICP was purified from bacteria and analyzed by X-ray crystallography to solve its structure Each subunit or monomer in the ICP dimer is ... More
Infected cell protein 0 (ICP0) is an immediate-early regulatory protein of herpes simplex virus 1 (HSV-1) that possesses E3 ubiquitin ligase activity. ICP0 transactivates viral genes, in part, through its C-terminal dimer domain (residues 555–767). Deletion of this dimer domain results in reduced viral gene expression, lytic infection, and reactivation from latency. Since ICP0's dimer domain is associated with its transactivation activity and efficient viral replication, we wanted to determine the structure of this specific domain. The C-terminus of ICP0 was purified from bacteria and analyzed by X-ray crystallography to solve its structure. Each subunit or monomer in the ICP0 dimer is composed of nine β-strands and two α-helices. Interestingly, two adjacent β-strands from one monomer “reach” into the adjacent subunit during dimer formation, generating two β-barrel-like structures. Additionally, crystallographic analyses indicate a tetramer structure is formed from two β-strands of each dimer, creating a “stacking” of the β-barrels. The structural protein database searches indicate the fold or structure adopted by the ICP0 dimer is novel. The dimer is held together by an extensive network of hydrogen bonds. Computational analyses reveal that ICP0 can either form a dimer or bind to SUMO1 via its C-terminal SUMO-interacting motifs but not both. Understanding the structure of the dimer domain will provide insights into the activities of ICP0 and, ultimately, the HSV-1 life cycle. Less
ADP-ribosyltransferases PARP and PARP play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations Recently a study showed that Histone PARylation Factor HPF forms a joint active site with PARP The interaction of HPF with PARP alters the modification site from Aspartate Glutamate to Serine which has been shown to be a key ADP-ribosylation event in the context of DNA damage Therefore disruption of PARP -HPF interaction could ... More
ADP-ribosyltransferases PARP1 and PARP2 play a major role in DNA repair mechanism by detecting the DNA damage and inducing poly-ADP-ribosylation dependent chromatin relaxation and recruitment of repair proteins. Catalytic PARP inhibitors are used as anticancer drugs especially in the case of tumors arising from sensitizing mutations. Recently, a study showed that Histone PARylation Factor (HPF1) forms a joint active site with PARP1/2. The interaction of HPF1 with PARP1/2 alters the modification site from Aspartate/Glutamate to Serine, which has been shown to be a key ADP-ribosylation event in the context of DNA damage. Therefore, disruption of PARP1/2-HPF1 interaction could be an alternative strategy for drug development to block the PARP1/2 activity. In this study, we describe a FRET based high-throughput screening assay to screen inhibitor libraries against PARP-HPF1 interaction. We optimized the conditions for FRET signal and verified the interaction by competing the FRET pair in multiple ways. The assay is robust and easy to automate. Validatory screening showed the robust performance of the assay, and we discovered two compounds Dimethylacrylshikonin and Alkannin, with µM inhibition potency against PARP1/2-HPF1 interaction. The assay will facilitate the discovery of inhibitors against HPF1-PARP1/2 complex and to develop potentially new effective anticancer agents. Less
Osteoclasts are over-activated as we age which results in bone loss Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts indicating that Src function is essential in osteoclasts G-protein-coupled receptors GPCRs are the targets for of approved drugs but it is still unclear how GPCRs regulate Src kinase activity Here we reveal that GPR activation by its natural ligand Kisspeptin- Kp- causes Dusp to dephosphorylate Src at Tyr Mechanistically Gpr recruits both active Src and the Dusp phosphatase at its proline arginine-rich motif in its C terminus We show that Kp- binding to Gpr ... More
Osteoclasts are over-activated as we age, which results in bone loss. Src deficiency in mice leads to severe osteopetrosis due to a functional defect in osteoclasts, indicating that Src function is essential in osteoclasts. G-protein-coupled receptors (GPCRs) are the targets for ∼35% of approved drugs but it is still unclear how GPCRs regulate Src kinase activity. Here, we reveal that GPR54 activation by its natural ligand Kisspeptin-10 (Kp-10) causes Dusp18 to dephosphorylate Src at Tyr 416. Mechanistically, Gpr54 recruits both active Src and the Dusp18 phosphatase at its proline/arginine-rich motif in its C terminus. We show that Kp-10 binding to Gpr54 leads to the up-regulation of Dusp18. Kiss1, Gpr54 and Dusp18 knockout mice all exhibit osteoclast hyperactivation and bone loss, and Kp-10 abrogated bone loss by suppressing osteoclast activity in vivo. Therefore, Kp-10/Gpr54 is a promising therapeutic target to abrogate bone resorption by Dusp18-mediated Src dephosphorylation. Less
Melanocortin receptor MC -R antagonists are actively sought for treating cancer cachexia We determined the structures of complexes with PG- and SBL-MC- These peptides differ from SHU by substituting His with Pro and inserting Gly or Arg The structures revealed two subpockets at the TM -TM -TM domains separated by N Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study Most ligands retained the SHU potency but several SBL-MC- -derived peptides significantly enhanced MC -R selectivity over MC -R by - to -fold We also investigated MC -R coupling to the K ... More
Melanocortin 4 receptor (MC4-R) antagonists are actively sought for treating cancer cachexia. We determined the structures of complexes with PG-934 and SBL-MC-31. These peptides differ from SHU9119 by substituting His6 with Pro6 and inserting Gly10 or Arg10. The structures revealed two subpockets at the TM7-TM1-TM2 domains, separated by N2857.36. Two peptide series based on the complexed peptides led to an antagonist activity and selectivity SAR study. Most ligands retained the SHU9119 potency, but several SBL-MC-31-derived peptides significantly enhanced MC4-R selectivity over MC1-R by 60- to 132-fold. We also investigated MC4-R coupling to the K+ channel, Kir7.1. Some peptides activated the channel, whereas others induced channel closure independently of G protein coupling. In cell culture studies, channel activation correlated with increased feeding, while a peptide with Kir7.1 inhibitory activity reduced eating. These results highlight the potential for targeting the MC4-R:Kir7.1 complex for treating positive and restrictive eating disorders. Less
Memory encodes past experiences thereby enabling future plans The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks These transcriptional signatures implicate neuropeptide and BDNF signalling MAPK and CREB activation ubiquitination pathways and synaptic connectivity as key components of long-term memory Notably upon long-term memory ... More
Memory encodes past experiences, thereby enabling future plans. The basolateral amygdala is a centre of salience networks that underlie emotional experiences and thus has a key role in long-term fear memory formation1. Here we used spatial and single-cell transcriptomics to illuminate the cellular and molecular architecture of the role of the basolateral amygdala in long-term memory. We identified transcriptional signatures in subpopulations of neurons and astrocytes that were memory-specific and persisted for weeks. These transcriptional signatures implicate neuropeptide and BDNF signalling, MAPK and CREB activation, ubiquitination pathways, and synaptic connectivity as key components of long-term memory. Notably, upon long-term memory formation, a neuronal subpopulation defined by increased Penk and decreased Tac expression constituted the most prominent component of the memory engram of the basolateral amygdala. These transcriptional changes were observed both with single-cell RNA sequencing and with single-molecule spatial transcriptomics in intact slices, thereby providing a rich spatial map of a memory engram. The spatial data enabled us to determine that this neuronal subpopulation interacts with adjacent astrocytes, and functional experiments show that neurons require interactions with astrocytes to encode long-term memory. Less
Dihydroorotate dehydrogenase DHODH is a mitochondrial enzyme that affects many aspects essential to cell proliferation and survival Recently DHODH has been identified as a potential target for acute myeloid leukemia therapy Herein we describe the identification of potent DHODH inhibitors through a scaffold hopping approach emanating from a fragment screen followed by structure-based drug design to further improve the overall profile and reveal an unexpected novel binding mode Additionally these compounds had low P-gp efflux ratios allowing for applications where exposure to the brain would be required
The adenosine subfamily G protein-coupled receptors A AR and A BR have been identified as promising cancer immunotherapy candidates One of the A AR A BR dual antagonists AB has progressed to a phase II clinical trial to treat rectal cancer However the precise mechanism underlying its dual-antagonistic properties remains elusive Herein we report crystal structures of the A AR complexed with AB and a selective A AR antagonist The structures revealed a common binding mode on A AR wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets In contrast the cAMP assay and A ... More
The adenosine subfamily G protein-coupled receptors A2AR and A2BR have been identified as promising cancer immunotherapy candidates. One of the A2AR/A2BR dual antagonists, AB928, has progressed to a phase II clinical trial to treat rectal cancer. However, the precise mechanism underlying its dual-antagonistic properties remains elusive. Herein, we report crystal structures of the A2AR complexed with AB928 and a selective A2AR antagonist 2–118. The structures revealed a common binding mode on A2AR, wherein the ligands established extensive interactions with residues from the orthosteric and secondary pockets. In contrast, the cAMP assay and A2AR and A2BR molecular dynamics simulations indicated that the ligands adopted distinct binding modes on A2BR. Detailed analysis of their chemical structures suggested that AB928 readily adapted to the A2BR pocket, while 2–118 did not due to intrinsic differences. This disparity potentially accounted for the difference in inhibitory efficacy between A2BR and A2AR. This study serves as a valuable structural template for the future development of selective or dual inhibitors targeting A2AR/A2BR for cancer therapy. Less
Exposure to environmental pollutants is linked to numerous toxic outcomes warranting concern about the effect of pollutants on human health To assess the threat of pollutant exposure it is essential to understand their biological activity Unfortunately gaps remain for many pollutants specific biological activity and molecular targets A superfamily of signaling proteins G-protein-coupled receptors GPCRs has been shown as potential targets for pollutant activity However research investigating the pollutant activity at the GPCRome is scarce This work explores pollutant activity across a library of human GPCRs by leveraging modern high-throughput screening techniques devised for drug discovery and pharmacology We designed ... More
Exposure to environmental pollutants is linked to numerous toxic outcomes, warranting concern about the effect of pollutants on human health. To assess the threat of pollutant exposure, it is essential to understand their biological activity. Unfortunately, gaps remain for many pollutants’ specific biological activity and molecular targets. A superfamily of signaling proteins, G-protein-coupled receptors (GPCRs), has been shown as potential targets for pollutant activity. However, research investigating the pollutant activity at the GPCRome is scarce. This work explores pollutant activity across a library of human GPCRs by leveraging modern high-throughput screening techniques devised for drug discovery and pharmacology. We designed and implemented a pilot screen of eight pollutants at 314 human GPCRs and discovered specific polychlorinated biphenyl (PCB) activity at sphingosine-1-phosphate and melatonin receptors. The method utilizes open-source resources available to academic and governmental institutions to enable future campaigns that screen large numbers of pollutants. Thus, we present a novel high-throughput approach to assess the biological activity and specific targets of pollutants. Less
In the fields of human health and agricultural research low coverage whole-genome sequencing followed by imputation to a large haplotype reference panel has emerged as a cost-effective alternative to genotyping arrays for assaying large numbers of samples However a systematic comparison of library preparation methods tailored for low coverage sequencing remains absent in the existing literature In this study we evaluated one full sized kit from IDT and miniaturized and evaluated three Illumina-compatible library preparation kits the KAPA HyperPlus kit Roche the DNA Prep kit Illumina and an IDT kit using human DNA samples Metrics evaluated included imputation concordance with ... More
In the fields of human health and agricultural research, low coverage whole-genome sequencing followed by imputation to a large haplotype reference panel has emerged as a cost-effective alternative to genotyping arrays for assaying large numbers of samples. However, a systematic comparison of library preparation methods tailored for low coverage sequencing remains absent in the existing literature. In this study, we evaluated one full sized kit from IDT and miniaturized and evaluated three Illumina-compatible library preparation kits—the KAPA HyperPlus kit (Roche), the DNA Prep kit (Illumina), and an IDT kit—using 96 human DNA samples. Metrics evaluated included imputation concordance with high-depth genotypes, coverage, duplication rates, time for library preparation, and additional optimization requirements. Despite slightly elevated duplication rates in IDT kits, we find that all four kits perform well in terms of imputation accuracy, with IDT kits being only marginally less performant than Illumina and Roche kits. Laboratory handling of the kits was similar: thus, the choice of a kit will largely depend on (1) existing or planned infrastructure, such as liquid handling capabilities, (2) whether a specific characteristic is desired, such as the use of full-length adapters, shorter processing times, or (3) use case, for instance, long vs short read sequencing. Our findings offer a comprehensive resource for both commercial and research workflows of low-cost library preparation methods suitable for high-throughput low coverage whole genome sequencing. Less
We herein describe the development and application of a modular technology platform which incorporates recent advances in plate-based microscale chemistry automated purification in situ quantification and robotic liquid handling to enable rapid access to high-quality chemical matter already formatted for assays In using microscale chemistry and thus consuming minimal chemical matter the platform is not only efficient but also follows green chemistry principles By reorienting existing high-throughput assay technology the platform can generate a full package of relevant data on each set of compounds in every learning cycle The multiparameter exploration of chemical and property space is hereby driven by ... More
We herein describe the development and application of a modular technology platform which incorporates recent advances in plate-based microscale chemistry, automated purification, in situ quantification, and robotic liquid handling to enable rapid access to high-quality chemical matter already formatted for assays. In using microscale chemistry and thus consuming minimal chemical matter, the platform is not only efficient but also follows green chemistry principles. By reorienting existing high-throughput assay technology, the platform can generate a full package of relevant data on each set of compounds in every learning cycle. The multiparameter exploration of chemical and property space is hereby driven by active learning models. The enhanced compound optimization process is generating knowledge for drug discovery projects in a time frame never before possible. Less
Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels This process is mediated by the parasite s polymorphic erythrocyte membrane protein PfEMP adhesion proteins A subset of PfEMP variants that bind human endothelial protein C receptor EPCR through their CIDR domains is responsible for severe malaria pathogenesis A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP variants Here we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDR The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of CIDR domains representing five of ... More
Plasmodium falciparum pathology is driven by the accumulation of parasite-infected erythrocytes in microvessels. This process is mediated by the parasite’s polymorphic erythrocyte membrane protein 1 (PfEMP1) adhesion proteins. A subset of PfEMP1 variants that bind human endothelial protein C receptor (EPCR) through their CIDRα1 domains is responsible for severe malaria pathogenesis. A longstanding question is whether individual antibodies can recognize the large repertoire of circulating PfEMP1 variants. Here, we describe two broadly reactive and binding-inhibitory human monoclonal antibodies against CIDRα1. The antibodies isolated from two different individuals exhibited a similar and consistent EPCR-binding inhibition of 34 CIDRα1 domains, representing five of the six subclasses of CIDRα1. Both antibodies inhibited EPCR binding of both recombinant full-length and native PfEMP1 proteins as well as parasite sequestration in bioengineered 3D brain microvessels under physiologically relevant flow conditions. Structural analyses of the two antibodies in complex with two different CIDRα1 antigen variants reveal similar binding mechanisms that depend on interactions with three highly conserved amino acid residues of the EPCR-binding site in CIDRα1. These broadly reactive antibodies likely represent a common mechanism of acquired immunity to severe malaria and offer novel insights for the design of a vaccine or treatment targeting severe malaria. Less
To identify starting points for therapeutics targeting SARS-CoV- the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket Of soaked fragments a total of hits bound either in the active site hits a remote binding pocket three hits or at crystal-packing interfaces two hits Notably two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue The structures ... More
To identify starting points for therapeutics targeting SARS-CoV-2, the Paul Scherrer Institute and Idorsia decided to collaboratively perform an X-ray crystallographic fragment screen against its main protease. Fragment-based screening was carried out using crystals with a pronounced open conformation of the substrate-binding pocket. Of 631 soaked fragments, a total of 29 hits bound either in the active site (24 hits), a remote binding pocket (three hits) or at crystal-packing interfaces (two hits). Notably, two fragments with a pose that was sterically incompatible with a more occluded crystal form were identified. Two isatin-based electrophilic fragments bound covalently to the catalytic cysteine residue. The structures also revealed a surprisingly strong influence of the crystal form on the binding pose of three published fragments used as positive controls, with implications for fragment screening by crystallography. Less
The tripartite ATP-independent periplasmic TRAP transporters use an extra cytoplasmic substrate binding protein SBP to transport a wide variety of substrates in bacteria and archaea The SBP can adopt an open- or closed state depending on the presence of substrate The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient Insights from experimental structures structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein Here we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM ... More
The tripartite ATP-independent periplasmic (TRAP) transporters use an extra cytoplasmic substrate binding protein (SBP) to transport a wide variety of substrates in bacteria and archaea. The SBP can adopt an open- or closed state depending on the presence of substrate. The two transmembrane domains of TRAP transporters form a monomeric elevator whose function is strictly dependent on the presence of a sodium ion gradient. Insights from experimental structures, structural predictions and molecular modeling have suggested a conformational coupling between the membrane elevator and the substrate binding protein. Here, we use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from Haemophilus influenzae in different conformational states. The SBP, HiSiaP, is locked in its substrate-bound form and the transmembrane elevator, HiSiaQM, is locked in either its assumed inward- or outward-facing states. We characterize the disulfide-locked constructs and use single-molecule total internal reflection fluorescence (TIRF) microscopy to study their interactions. Our experiments demonstrate that the SBP and the transmembrane elevator are indeed conformationally coupled, meaning that the open and closed state of the SBP recognize specific conformational states of the transporter and vice versa. Less
Monoolein-based liquid crystal phases are established media that are researched for various biological applications including drug delivery While water is the most common solvent for self-assembly some ionic liquids ILs can support lipidic self-assembly However currently there is limited knowledge of IL-lipid phase behavior in ILs In this study the lyotropic liquid crystal phase behavior of monoolein was investigated in six protic ILs known to support amphiphile self-assembly namely ethylammonium nitrate ethanolammonium nitrate ethylammonium formate ethanolammonium formate ethylammonium acetate and ethanolammonium acetate These ILs were selected to identify specific ion effects on monoolein self-assembly specifically increasing the alkyl chain length ... More
Monoolein-based liquid crystal phases are established media that are researched for various biological applications, including drug delivery. While water is the most common solvent for self-assembly, some ionic liquids (ILs) can support lipidic self-assembly. However, currently, there is limited knowledge of IL-lipid phase behavior in ILs. In this study, the lyotropic liquid crystal phase behavior of monoolein was investigated in six protic ILs known to support amphiphile self-assembly, namely ethylammonium nitrate, ethanolammonium nitrate, ethylammonium formate, ethanolammonium formate, ethylammonium acetate, and ethanolammonium acetate. These ILs were selected to identify specific ion effects on monoolein self-assembly, specifically increasing the alkyl chain length of the cation or anion, the presence of a hydroxyl group in the cation, and varying the anion. The lyotropic liquid crystal phases with 20–80 wt. % of monoolein were characterized over a temperature range from 25 to 65 °C using synchrotron small angle x-ray scattering and cross-polarized optical microscopy. These results were used to construct partial phase diagrams of monoolein in each of the six protic ILs, with inverse hexagonal, bicontinuous cubic, and lamellar phases observed. Protic ILs containing the ethylammonium cation led to monoolein forming lamellar and bicontinuous cubic phases, while those containing the ethanolammonium cation formed inverse hexagonal and bicontinuous cubic phases. Protic ILs containing formate and acetate anions favored bicontinuous cubic phases across a broader range of protic IL concentrations than those containing the nitrate anion. Less
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold They are generally described as electron carriers that rely on a T copper centre coordinated by four ligands provided by the folded polypeptide The discovery of novel cupredoxins demonstrates the high diversity of this family with variations in terms of copper-binding ligands copper centre geometry redox potential as well as biological function AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans AcoP presents original features including high resistance to acidic pH and a constrained green-type copper centre of high ... More
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper centre coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in terms of copper-binding ligands, copper centre geometry, redox potential, as well as biological function. AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features, including high resistance to acidic pH and a constrained green-type copper centre of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structures, including native reduced AcoP at 1.65 Å resolution, unveil a typical cupredoxin fold. The presence of extended loops, never observed in previously characterized cupredoxins, might account for the interaction of AcoP with physiological partners. The Cu-ligand distances, determined by both X-ray diffraction and EXAFS, show that the AcoP metal centre seems to present both T1 and T1.5 features, in turn suggesting that AcoP might not fit well to the coupled distortion model. The crystal structures of two AcoP mutants confirm that the active centre of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active centre rigidity due to the presence of an extensive hydrogen bond network. Finally, we show that other cupredoxins do not perfectly follow the coupled distortion model as well, raising the suspicion that further alternative models to describe copper centre geometries need to be developed, while the importance of rack-induced contributions should not be underestimated. Less
TP is the most frequently mutated gene in human cancer While it is well understood that the ability of p to act as a transcription factor is required for tumor suppression the key target genes downstream of p required for tumor suppression are still incompletely understood We first set out to characterize a rare African-specific germline variant of TP in the DNA binding domain Tyr His Y H Although we find that Y H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p target genes Y H mice develop spontaneous cancers ... More
TP53 is the most frequently mutated gene in human cancer. While it is well understood that the ability of p53 to act as a transcription factor is required for tumor suppression, the key target genes downstream of p53 required for tumor suppression are still incompletely understood. We first set out to characterize a rare, African-specific, germline variant of TP53 in the DNA binding domain, Tyr107His (Y107H). Although we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes, Y107H mice develop spontaneous cancers and metastases. We identified the p53 target gene, PADI4, to be exquisitely sensitive to p53 mutation, and loss of PADI4 is seen in Y107H and other transcriptionally competent p53 hypomorphs. PADI4 is a regulator of histone modification and gene transcription via citrullination, which is the process of deiminating arginine to the non-natural amino acid citrulline. Our TCGA analysis reveals PADI4 is downregulated or mutated in multiple human cancers. Surprisingly, we show that PADI4 is sufficient to suppress tumor growth and sensitize wild-type p53 cells to chemotherapeutics. We further show that PADI4 is potently tumor suppressive in vivo, and complete tumor suppression by PADI4 requires an intact immune system. We find PADI4 enhances the transactivation of p53 targets and genes involved in immune activation. In addition, we identify a p53–PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. We have further found that PADI4 interacts and modifies p53 via citrullination at key residues within the oligomerization and C-terminal domain of p53. PADI4 colocalizes with p53 on chromatin at non-canonical p53 target genes and genes devoid of a p53 response element. Citrullination of p53 may alter p53 function through enhanced oligomerization or binding of p53 to DNA. The findings from this study reveal PADI4 as not only a key target gene of p53, but a core regulator of p53 activity and target specificity through a novel protein modification. This work highlights the need to reassess the role of PADI4 in cancer, and provides insight into critical downstream target genes important for tumor suppression by p53. Less
Predictive drug testing of patient-derived tumor organoids PDTOs holds promise for personalizing treatment of metastatic colorectal cancer mCRC but prospective data are limited to chemotherapy regimens with conflicting results We describe a unified framework for PDTO-based predictive testing across standard-of-care chemotherapy and biologic and targeted therapy options In an Australian community cohort PDTO predictions based on treatment-naive patients n and response rates from first-line mCRC clinical trials achieve accuracy for forecasting responses in patients receiving palliative treatments patients treatments Similar assay accuracy is achieved in a prospective study of third-line or later mCRC treatment AGITG FORECAST- n patients Resistant predictions ... More
Predictive drug testing of patient-derived tumor organoids (PDTOs) holds promise for personalizing treatment of metastatic colorectal cancer (mCRC), but prospective data are limited to chemotherapy regimens with conflicting results. We describe a unified framework for PDTO-based predictive testing across standard-of-care chemotherapy and biologic and targeted therapy options. In an Australian community cohort, PDTO predictions based on treatment-naive patients (n = 56) and response rates from first-line mCRC clinical trials achieve 83% accuracy for forecasting responses in patients receiving palliative treatments (18 patients, 29 treatments). Similar assay accuracy is achieved in a prospective study of third-line or later mCRC treatment, AGITG FORECAST-1 (n = 30 patients). “Resistant” predictions are associated with inferior progression-free survival; misclassification rates are similar by regimen. Liver metastases are the optimal site for sampling, with testing achievable within 7 weeks for 68.8% cases. Our findings indicate that PDTO drug panel testing can provide predictive information for multifarious standard-of-care therapies for mCRC. Less
Membrane proteins are indispensable for every living organism yet their structural organization remains underexplored Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography which have significantly increased the structural coverage of membrane proteins across various kingdoms certain scientific methods such as time-resolved crystallography still mostly rely on crystallization techniques such as lipidic cubic phase LCP or in meso crystallization In this study we present an open-access blueprint for a humidity control chamber designed for LCP in meso crystallization experiments using a Gryphon crystallization robot Using this chamber we have obtained crystals of a transmembrane aspartate transporter ... More
Membrane proteins are indispensable for every living organism, yet their structural organization remains underexplored. Despite the recent advancements in single-particle cryogenic electron microscopy and cryogenic electron tomography, which have significantly increased the structural coverage of membrane proteins across various kingdoms, certain scientific methods, such as time-resolved crystallography, still mostly rely on crystallization techniques, such as lipidic cubic phase (LCP) or in meso crystallization. In this study, we present an open-access blueprint for a humidity control chamber designed for LCP/in meso crystallization experiments using a Gryphon crystallization robot. Using this chamber, we have obtained crystals of a transmembrane aspartate transporter GltTk from Thermococcus kodakarensis in a lipidic environment using in meso crystallization. The data collected from these crystals allowed us to perform an analysis of lipids bound to the transporter. With this publication of our open-access design of a humidity chamber, we aim to improve the accessibility of in meso protein crystallization for the scientific community. Less
StayGold is an exceptionally bright and stable fluorescent protein that is highly resistant to photobleaching Despite favorable fluorescence properties use of StayGold as a fluorescent tag is limited because it forms a natural dimer Here we report the structure of StayGold and generate a derivative mStayGold that retains the brightness and photostability of the original protein while being fully monomeric
Microbial natural products are specialized metabolites that are sources of many bioactive compounds including antibiotics antifungals antiparasitics anticancer agents and probes of biology The assembly of libraries of producers of natural products has traditionally been the province of the pharmaceutical industry This sector has gathered significant historical collections of bacteria and fungi to identify new drug leads with outstanding outcomes - upwards of of drug scaffolds originate from such libraries Despite this success the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds ... More
Microbial natural products are specialized metabolites that are sources of many bioactive
compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of
biology. The assembly of libraries of producers of natural products has traditionally been the
province of the pharmaceutical industry. This sector has gathered significant historical
collections of bacteria and fungi to identify new drug leads with outstanding outcomes - upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we
report a perspective on our efforts to assemble a library of natural product-producing microbes
and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds. Less
compounds including antibiotics, antifungals, antiparasitics, anticancer agents, and probes of
biology. The assembly of libraries of producers of natural products has traditionally been the
province of the pharmaceutical industry. This sector has gathered significant historical
collections of bacteria and fungi to identify new drug leads with outstanding outcomes - upwards of 60% of drug scaffolds originate from such libraries. Despite this success, the repeated rediscovery of known compounds and the resultant diminishing chemical novelty contributed to a pivot from this source of bioactive compounds toward more tractable synthetic compounds in the drug industry. The advent of advanced mass spectrometry tools, along with rapid whole genome sequencing and in silico identification of biosynthetic gene clusters that encode the machinery necessary for the synthesis of specialized metabolites, offers the opportunity to revisit microbial natural product libraries with renewed vigor. Assembling a suitable library of microbes and extracts for screening requires the investment of resources and the development of methods that have customarily been the proprietary purview of large pharmaceutical companies. Here, we
report a perspective on our efforts to assemble a library of natural product-producing microbes
and the establishment of methods to extract and fractionate bioactive compounds using resources available to most academic labs. We validate the library and approach through a series of screens for antimicrobial and cytotoxic agents. This work serves as a blueprint for establishing libraries of microbial natural product producers and bioactive extract fractions suitable for screens of bioactive compounds. Less
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins yet have very similar spectra when in the oxidized state Recently we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue Q V or red I V A Q shifts of fluorescence emission maxima Here we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q V an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I V A Q variant Consequently in Q V excitation emission and phosphorescence spectra are shifted whereas in I V A ... More
Flavins such as flavin mononucleotide or flavin adenine dinucleotide are bound by diverse proteins, yet have very similar spectra when in the oxidized state. Recently, we developed new variants of flavin-binding protein CagFbFP exhibiting notable blue (Q148V) or red (I52V A85Q) shifts of fluorescence emission maxima. Here, we use time-resolved and low-temperature spectroscopy to show that whereas the chromophore environment is static in Q148V, an additional protein-flavin hydrogen bond is formed upon photoexcitation in the I52V A85Q variant. Consequently, in Q148V, excitation, emission, and phosphorescence spectra are shifted, whereas in I52V A85Q, excitation and low-temperature phosphorescence spectra are relatively unchanged, while emission spectrum is altered. We also determine the x-ray structures of the two variants to reveal the flavin environment and complement the spectroscopy data. Our findings illustrate two distinct color-tuning mechanisms of flavin-binding proteins and could be helpful for the engineering of new variants with improved optical properties. Less
NADH cytochrome b oxidoreductase Ncb or is a cytosolic ferric reductase implicated in diabetes and neurological conditions Ncb or comprises cytochrome b b and cytochrome b reductase b R domains separated by a CHORD-Sgt CS linker domain Ncb or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme While full-length human Ncb or has proven resistant to crystallization we have succeeded in obtaining high-resolution atomic structures of the b domain and a construct containing the CS and b R domains CS b R Ncb or also contains an N-terminal intrinsically ... More
NADH cytochrome b5 oxidoreductase (Ncb5or) is a cytosolic ferric reductase implicated in diabetes and neurological conditions. Ncb5or comprises cytochrome b5 (b5) and cytochrome b5 reductase (b5R) domains separated by a CHORD-Sgt1 (CS) linker domain. Ncb5or redox activity depends on proper inter-domain interactions to mediate electron transfer from NADH or NADPH via FAD to heme. While full-length human Ncb5or has proven resistant to crystallization, we have succeeded in obtaining high-resolution atomic structures of the b5 domain and a construct containing the CS and b5R domains (CS/b5R). Ncb5or also contains an N-terminal intrinsically disordered region of 50 residues that has no homologs in other protein families in animals but features a distinctive, conserved L34MDWIRL40 motif also present in reduced lateral root formation (RLF) protein in rice and increased recombination center 21 in baker's yeast, all attaching to a b5 domain. After unsuccessful attempts at crystallizing a human Ncb5or construct comprising the N-terminal region naturally fused to the b5 domain, we were able to obtain a high-resolution atomic structure of a recombinant rice RLF construct corresponding to residues 25–129 of human Ncb5or (52% sequence identity; 74% similarity). The structure reveals Trp120 (corresponding to invariant Trp37 in Ncb5or) to be part of an 11-residue α-helix (S116QMDWLKLTRT126) packing against two of the four helices in the b5 domain that surround heme (α2 and α5). The Trp120 side chain forms a network of interactions with the side chains of four highly conserved residues corresponding to Tyr85 and Tyr88 (α2), Cys124 (α5), and Leu47 in Ncb5or. Circular dichroism measurements of human Ncb5or fragments further support a key role of Trp37 in nucleating the formation of the N-terminal helix, whose location in the N/b5 module suggests a role in regulating the function of this multi-domain redox enzyme. This study revealed for the first time an ancient origin of a helical motif in the N/b5 module as reflected by its existence in a class of cytochrome b5 proteins from three kingdoms among eukaryotes. Less
For optimal bioperformance the drug in an amorphous solid dispersion ASD should ideally not undergo crystallization in the solid dosage form during storage or from the supersaturated solution generated upon dissolution Incomplete processing during hot melt extrusion HME can lead to residual crystallinity Commonly residual crystallinity is evaluated using techniques such as powder X-ray diffraction pXRD However residual crystallinity at levels below the detection limit of pXRD can be detrimental to the ASD performance The goal of this study was to evaluate the impact of different levels of residual crystallinity in an ASD containing the fast-crystallizing drug posaconazole PCZ and ... More
For optimal bioperformance, the drug in an amorphous solid dispersion (ASD) should ideally not undergo crystallization in the solid dosage form during storage or from the supersaturated solution generated upon dissolution. Incomplete processing during hot melt extrusion (HME) can lead to residual crystallinity. Commonly, residual crystallinity is evaluated using techniques such as powder X-ray diffraction (pXRD). However, residual crystallinity at levels below the detection limit of pXRD can be detrimental to the ASD performance. The goal of this study was to evaluate the impact of different levels of residual crystallinity in an ASD containing the fast-crystallizing drug posaconazole (PCZ) and hydroxypropyl methylcellulose acetate succinate (HPMCAS) on dissolution and additional crystallization. ASDs with and without residual crystallinity at 10, 25, and 50 wt % drug loadings were prepared using HME, processing at temperatures below and above the critical temperature, which was calculated using the Flory–Huggins theory. Some of the ASDs contained levels of residual crystallinity that were below the quantification limit of pXRD, requiring the use of second harmonic generation (SHG) imaging. The impact of residual crystallinity on dissolution was studied by using two-stage dissolution. Additional characterization in support of dissolution measurements included SHG imaging and particle size evolution with focused beam reflectance measurement (FBRM) using pH-shift experiments. The 10 wt % ASD processed below the critical solution temperature contained residual crystallinity of 0.3%, which promoted rapid crystallization when the ASD was in a solution environment. Real-time monitoring of both the solid and solution phases revealed that PCZ in ASDs containing residual crystals underwent crystallization both in the matrix and from solution. The study supports the need to select a sufficiently sensitive crystallinity estimation technique, a suitable discriminatory dissolution technique, and appropriate HME processing conditions in order to optimize and achieve successful performance of ASDs of fast-crystallizing drugs. Less
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection It is responsible for US deaths annually Neutrophils are an integral part of the innate immune response and rapidly clear pathogens from circulation using neutrophil extracellular traps NETs which are released through a process called NETosis NETs prevent dissemination of pathogens by entrapment in externalized chromatin containing deactivating enzymes While we have learned much about the mechanisms underlying NETosis we are yet to translate it to improved therapies or patient outcomes This gap may be attributable to the models used to study NETosis Current models used ... More
Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection (1). It is responsible for ~370,000 US deaths annually (2). Neutrophils are an integral part of the innate immune response and rapidly clear pathogens from circulation using neutrophil extracellular traps (NETs), which are released through a process called NETosis (3). NETs prevent dissemination of pathogens by entrapment in externalized chromatin containing deactivating enzymes. While we have learned much about the mechanisms underlying NETosis, we are yet to translate it to improved therapies or patient outcomes. This gap may be attributable to the models used to study NETosis. Current models used to investigate NETosis are limited and routinely employ unnatural triggers such as phorbol 12-myristate 13-acetate (PMA). PMA is not a physiological trigger present in the immune system and may bypass the natural pathways that regulate NETs production. Mouse models that use isolated neutrophils and neutrophil-like cells induced from immortalized cell lines do not completely reflect the complex cellular and molecular biology underlying neutrophil activation and NETosis, especially in a whole-blood environment. Therefore, it is crucial to study how specific factors, known to be upregulated in disease, interact and potentially induce NETosis. Here we use high-throughput screening and natural NETosis triggers to develop a more biologically relevant ex vivo NETosis (Synthetic-Sepsis™) model.
Whole blood was collected from healthy donors and aliquoted into a 384 well plate using a Formulatrix Mantis liquid handler. This plate contained small molecules associated with neutrophils or NETosis activation, such as interleukins: Il-1b, IL-5, IL-6, IL-8, IL-15, IL-17, IL-18 and other molecules TNF- α, LT-α, IFN-γ, G-CSF, GM-CSF, E-selectin, PAF-16, CXCL1, CXCL2, LTB4, CXCL5, CCL2, CCL3, fMLP, Ferritin, HMGB1, C5a and LPS. We used a combinatorial pooling strategy designed using JMP software to identify which combinations of small molecules could stimulate NET formation. NETosis was assessed using Sytox green intercalation at 5 minute intervals for up to 24 hours using a Molecular Devices plate reader. PMA was utilized as a positive control for NETosis induction at varying concentrations.
Using our combinatorial pooling approach of the various factors, we successfully induced NETosis in an ex vivo whole blood system using naturally occurring cytokines and chemokines at physiologically relevant concentrations. We found that different combinations of factors evoke distinct neutrophil responses both in the time of NET generation and/or magnitude of NET-associated intercalation signal. We observed inter-donor variability in response time and amplitude however, similar small molecule pools induced consistent responses across donors. Furthermore, our findings suggest that at least four naturally occurring factors are necessary to induce NETosis in our system. Although some factors activate similar pathways, they are unable to induce a signal alone and as the number of factors increased beyond four, there was an enhanced NET response. Interestingly, we found either TNF-α or LT-α was required to cause a NETosis response, underlining the potentially significant roles these factors play in inflammatory disease. These results suggest an underlying master regulatory mechanism, such that certain factors are essential but not individually sufficient to trigger NETosis.
To our knowledge, we report the first ex-vivo model using naturally occurring cytokines and chemokines to induce NETosis in whole blood. These findings emphasize the importance of expanding our understanding of neutrophil physiology in a biologically relevant context with physiological triggers to induce NETosis. This approach could reveal new dimensions in our understanding of disease pathology and risk factors and might unearth potential therapeutic targets providing novel strategies for disease intervention and treatment. Further investigation of these factors is underway to further understand the release of NETs in natural and pathological states. Less
Whole blood was collected from healthy donors and aliquoted into a 384 well plate using a Formulatrix Mantis liquid handler. This plate contained small molecules associated with neutrophils or NETosis activation, such as interleukins: Il-1b, IL-5, IL-6, IL-8, IL-15, IL-17, IL-18 and other molecules TNF- α, LT-α, IFN-γ, G-CSF, GM-CSF, E-selectin, PAF-16, CXCL1, CXCL2, LTB4, CXCL5, CCL2, CCL3, fMLP, Ferritin, HMGB1, C5a and LPS. We used a combinatorial pooling strategy designed using JMP software to identify which combinations of small molecules could stimulate NET formation. NETosis was assessed using Sytox green intercalation at 5 minute intervals for up to 24 hours using a Molecular Devices plate reader. PMA was utilized as a positive control for NETosis induction at varying concentrations.
Using our combinatorial pooling approach of the various factors, we successfully induced NETosis in an ex vivo whole blood system using naturally occurring cytokines and chemokines at physiologically relevant concentrations. We found that different combinations of factors evoke distinct neutrophil responses both in the time of NET generation and/or magnitude of NET-associated intercalation signal. We observed inter-donor variability in response time and amplitude however, similar small molecule pools induced consistent responses across donors. Furthermore, our findings suggest that at least four naturally occurring factors are necessary to induce NETosis in our system. Although some factors activate similar pathways, they are unable to induce a signal alone and as the number of factors increased beyond four, there was an enhanced NET response. Interestingly, we found either TNF-α or LT-α was required to cause a NETosis response, underlining the potentially significant roles these factors play in inflammatory disease. These results suggest an underlying master regulatory mechanism, such that certain factors are essential but not individually sufficient to trigger NETosis.
To our knowledge, we report the first ex-vivo model using naturally occurring cytokines and chemokines to induce NETosis in whole blood. These findings emphasize the importance of expanding our understanding of neutrophil physiology in a biologically relevant context with physiological triggers to induce NETosis. This approach could reveal new dimensions in our understanding of disease pathology and risk factors and might unearth potential therapeutic targets providing novel strategies for disease intervention and treatment. Further investigation of these factors is underway to further understand the release of NETs in natural and pathological states. Less
The phospholipase D PLD family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids PLD is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes yielding a soluble active form The deficiency of PLD leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors While the mechanism of PLD phospholipase activity has been extensively characterized not much is known about how PLDs bind and hydrolyze nucleic acids Here we determined the high-resolution crystal structure of the luminal N-glycosylated domain of ... More
The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3. Less
The pathogen Paracoccidioides lutzii Pb is found in South America countries Colombia Ecuador Venezuela and Brazil especially in the central west and north regions of the latter It belongs to the Ajellomycetaceae family Onygenales order and is typically thermodimorphic presenting yeast cells when it grows in animal tissues but mycelia when in the environment where it produces the infectious propagule This fungus is one of the etiologic agents of Paracoccidioidomycosis PCM the most important endemic fungal infection in Latin America Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic ... More
The pathogen Paracoccidioides lutzii (Pb01) is found in South America countries Colombia, Ecuador, Venezuela and Brazil, especially in the central, west, and north regions of the latter. It belongs to the Ajellomycetaceae family, Onygenales order, and is typically thermodimorphic, presenting yeast cells when it grows in animal tissues, but mycelia when in the environment, where it produces the infectious propagule. This fungus is one of the etiologic agents of Paracoccidioidomycosis (PCM), the most important endemic fungal infection in Latin America. Investigations on its genome have contributed to a better understanding about its metabolism and revealed the complexity of several metabolic glycolytic pathways. Glyceraldehyde-3-Phosphate Dehydrogenase from Paracoccidioides lutzii (PlGAPDH) is considered a moonlighting protein and participates in several biological processes of this pathogen. The enzyme was expressed and purified, as seen in SDS-PAGE gel, crystallized and had its three dimensional structure (3D) determined in complex with NAD+, a sulphate ion and d-galactonic acid, therefore, a type of ‘GAA site’. It is the first GAPDH structure to show this chemical type in this site and how this protein can bind an acid derived from oxidation of a linear hexose. Less
The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria Among the most prevalent are aminoglycoside acetyltransferases AACs that inactivate the antibiotics through acetyl coenzyme A-mediated modification Most AACs are members of the GCN superfamily of acyltransferases which lack conserved active site residues that participate in catalysis ApmA is the first reported AAC belonging to the left-handed -helix superfamily These enzymes are characterized by an essential active site histidine that acts as an active site base Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other ... More
The efficacy of aminoglycoside antibiotics is waning due to the acquisition of diverse resistance mechanisms by bacteria. Among the most prevalent are aminoglycoside acetyltransferases (AACs) that inactivate the antibiotics through acetyl coenzyme A-mediated modification. Most AACs are members of the GCN5 superfamily of acyltransferases which lack conserved active site residues that participate in catalysis. ApmA is the first reported AAC belonging to the left-handed β-helix superfamily. These enzymes are characterized by an essential active site histidine that acts as an active site base. Here we show that ApmA confers broad-spectrum aminoglycoside resistance with a molecular mechanism that diverges from other detoxifying left-handed β-helix superfamily enzymes and canonical GCN5 AACs. We find that the active site histidine plays different functions depending on the acetyl-accepting aminoglycoside substrate. This flexibility in the mechanism of a single enzyme underscores the plasticity of antibiotic resistance elements to co-opt protein catalysts in the evolution of drug detoxification. Less
This study aimed to identify inhibitors of the translocated intimin receptor Tir of enteropathogenic Escherichia coli EPEC EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide Because Tir is a key virulence factor involved in EPEC pathogenesis inhibiting its function is a potential strategy for controlling EPEC infections Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells Three sites were targeted using the cocrystal structure published earlier A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay The ... More
This study aimed to identify inhibitors of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC). EPEC is an intestinal pathogen that causes diarrhea and is a major health concern worldwide. Because Tir is a key virulence factor involved in EPEC pathogenesis, inhibiting its function is a potential strategy for controlling EPEC infections. Virtual screening was applied to chemical libraries to search for compounds that inhibit Tir-mediated bacterial adherence to host cells. Three sites were targeted using the cocrystal structure published earlier. A selection of compounds was then assessed in a cell-based infection model and fluorescence microscopy assay. The results of this study provide a basis for further optimization and testing of Tir inhibitors as potential therapeutic agents for EPEC infections. Less
Spatial tissue proteomics combining microscopy-based cell phenotyping with ultra-sensitive mass spectrometry MS -based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease However optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue are currently lacking Here we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed paraffin-embedded FFPE material Benchmarking in the murine liver resulted in up to quantified proteins from single hepatocyte contours and nearly proteins from -cell ... More
Spatial tissue proteomics combining microscopy-based cell phenotyping with ultra-sensitive mass spectrometry (MS)-based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease. However, optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue, are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed, paraffin-embedded (FFPE) material. Benchmarking in the murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions with high quantitative reproducibility. Applied to human tonsil, we profiled 146 microregions including spatially defined T and B lymphocyte niches and quantified cell type specific markers, cytokines, immune cell regulators and transcription factors. These rich data also highlighted proteome dynamics in spatially defined zones of activated germinal centers, illuminating sites undergoing active B-cell proliferation and somatic hypermutation. Our results demonstrate the power of spatially-resolved proteomics for tissue phenotyping by integrating high-content imaging, laser microdissection, and ultra-sensitive mass spectrometry. This approach has broad implications for a wide range of biomedical applications, including early disease profiling, drug target discovery and biomarker research. Less
Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption from photosystems However it is unclear how the antenna transfers energy efficiently to these photosystems We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein each composed of two protomers but with different quaternary structures arising from a diverse subunit family We report crystal structures of the major phycobiliprotein from each spectrotype Two-thirds of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors These are supplemented by a newly discovered open-braced form ... More
Cryptophyte algae have a unique phycobiliprotein light-harvesting antenna that fills a spectral gap in chlorophyll absorption from photosystems. However, it is unclear how the antenna transfers energy efficiently to these photosystems. We show that the cryptophyte Hemiselmis andersenii expresses an energetically complex antenna comprising three distinct spectrotypes of phycobiliprotein, each composed of two αβ protomers but with different quaternary structures arising from a diverse α subunit family. We report crystal structures of the major phycobiliprotein from each spectrotype. Two-thirds of the antenna consists of open quaternary form phycobiliproteins acting as primary photon acceptors. These are supplemented by a newly discovered open-braced form (~15%), where an insertion in the α subunit produces ~10 nm absorbance red-shift. The final components (~15%) are closed forms with a long wavelength spectral feature due to substitution of a single chromophore. This chromophore is present on only one β subunit where asymmetry is dictated by the corresponding α subunit. This chromophore creates spectral overlap with chlorophyll, thus bridging the energetic gap between the phycobiliprotein antenna and the photosystems. We propose that the macromolecular organization of the cryptophyte antenna consists of bulk open and open-braced forms that transfer excitations to photosystems via this bridging closed form phycobiliprotein. Less
Gonorrhea a prevalent sexually transmitted infection affecting millions annually worldwide is caused by Neisseria gonorrhoeae Ngo a superbug resistant to all antibiotic classes Compounding the challenge the absence of protective immunity upon infection allows for reinfection and a viable vaccine against gonococcal infection remains elusive In response to infection the human host deploys nutritional immunity sequestering essential metals like iron from invading bacteria using metal binding proteins To overcome this Ngo employs outer-membrane TonB-dependent transporters TdTs like HpuAB to acquire iron from host metal binding proteins such as hemoglobin Hb Part of our study focused on HpuA the lipoprotein component ... More
Gonorrhea, a prevalent sexually transmitted infection affecting millions annually worldwide, is caused by Neisseria gonorrhoeae (Ngo), a "superbug" resistant to all antibiotic classes. Compounding the challenge, the absence of protective immunity upon infection allows for reinfection, and a viable vaccine against gonococcal infection remains elusive.
In response to infection, the human host deploys nutritional immunity, sequestering essential metals like iron from invading bacteria, using metal binding proteins. To overcome this, Ngo employs outer-membrane TonB-dependent transporters (TdTs), like HpuAB, to acquire iron from host metal binding proteins, such as hemoglobin (Hb).
Part of our study focused on HpuA, the lipoprotein component of the HpuAB system. Mutations targeting hydrophobic residues crucial for Hb interaction were studied. Results demonstrated that without HpuB, strains failed to grow, emphasizing HpuB's role in iron internalization. Notably, when HpuB is produced, deletion and insertion mutations in loop 2 of HpuA affected growth and binding to Hb.
Further investigations into HpuB, the transmembrane protein of the HpuAB system, uncovered essential loop regions for binding and growth on Hb as a sole iron source. Deletion mutations in loops 2, 3, and 4 facilitated binding and growth independently of HpuA production. Intriguingly, mutations in loop 7 abrogated binding and impaired growth in the absence of HpuA, but partial growth and full binding recovery occurred when HpuA was present. This highlighted the importance of loop 7 in iron acquisition and suggested a potential role for both HpuA and HpuB in the binding Hb.
As a combination of non-binding TdT mutants is hypothesized to have the potential to improve vaccine efficacy and provide protection, identifying non-binding HpuB mutants could be important. In summary, this research sheds light on the intricacies of the HpuAB system, contributing valuable insights that could inform the development of an effective gonorrhea vaccine. Less
In response to infection, the human host deploys nutritional immunity, sequestering essential metals like iron from invading bacteria, using metal binding proteins. To overcome this, Ngo employs outer-membrane TonB-dependent transporters (TdTs), like HpuAB, to acquire iron from host metal binding proteins, such as hemoglobin (Hb).
Part of our study focused on HpuA, the lipoprotein component of the HpuAB system. Mutations targeting hydrophobic residues crucial for Hb interaction were studied. Results demonstrated that without HpuB, strains failed to grow, emphasizing HpuB's role in iron internalization. Notably, when HpuB is produced, deletion and insertion mutations in loop 2 of HpuA affected growth and binding to Hb.
Further investigations into HpuB, the transmembrane protein of the HpuAB system, uncovered essential loop regions for binding and growth on Hb as a sole iron source. Deletion mutations in loops 2, 3, and 4 facilitated binding and growth independently of HpuA production. Intriguingly, mutations in loop 7 abrogated binding and impaired growth in the absence of HpuA, but partial growth and full binding recovery occurred when HpuA was present. This highlighted the importance of loop 7 in iron acquisition and suggested a potential role for both HpuA and HpuB in the binding Hb.
As a combination of non-binding TdT mutants is hypothesized to have the potential to improve vaccine efficacy and provide protection, identifying non-binding HpuB mutants could be important. In summary, this research sheds light on the intricacies of the HpuAB system, contributing valuable insights that could inform the development of an effective gonorrhea vaccine. Less
MbtI from Mycobacterium tuberculosis Mtb is a Mg -dependent salicylate synthase belonging to the chorismate-utilizing enzyme CUE family As a fundamental player in iron acquisition MbtI promotes the survival and pathogenicity of Mtb in the infected host Hence it has emerged in the last decade as an innovative potential target for the anti-virulence therapy of tuberculosis In this context -phenylfuran- -carboxylic acids have been identified as potent MbtI inhibitors The first co-crystal structure of MbtI in complex with a member of this class was described in showing the enzyme adopting an open configuration Due to the high mobility of the ... More
MbtI from Mycobacterium tuberculosis (Mtb) is a Mg2+-dependent salicylate synthase, belonging to the chorismate-utilizing enzyme (CUE) family. As a fundamental player in iron acquisition, MbtI promotes the survival and pathogenicity of Mtb in the infected host. Hence, it has emerged in the last decade as an innovative, potential target for the anti-virulence therapy of tuberculosis. In this context, 5-phenylfuran-2-carboxylic acids have been identified as potent MbtI inhibitors. The first co-crystal structure of MbtI in complex with a member of this class was described in 2020, showing the enzyme adopting an open configuration. Due to the high mobility of the loop adjacent to the binding pocket, large portions of the amino acid chain were not defined in the electron density map, hindering computational efforts aimed at structure-driven ligand optimization. Herein, we report a new, high-resolution co-crystal structure of MbtI with a furan-based derivative, in which the closed configuration of the enzyme allowed tracing the entirety of the active site pocket in the presence of the bound inhibitor. Moreover, we describe a new crystal structure of MbtI in open conformation and in complex with the known inhibitor methyl-AMT, suggesting that in vitro potency is not related to the observed enzyme conformation. These findings will prove fundamental to enhance the potency of this series via rational structure-based drug-design approaches. Less