1209 Citations
Crimean Congo hemorrhagic fever virus CCHFV is a tick-borne virus that can cause severe disease in humans with case fatality rates of Although structures of CCHFV glycoproteins GP and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies the structure of glycoprotein Gn and its interactions with GP and Gc have remained elusive Here we used structure-guided protein engineering to produce a stabilized GP -Gn-Gc heterotrimeric glycoprotein complex GP -GnH-DS-Gc A cryo-EM structure of this complex provides the molecular basis for GP s association on the viral surface reveals the structure of Gn and ... More
Crimean–Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus that can cause severe disease in humans with case fatality rates of 10–40%. Although structures of CCHFV glycoproteins GP38 and Gc have provided insights into viral entry and defined epitopes of neutralizing and protective antibodies, the structure of glycoprotein Gn and its interactions with GP38 and Gc have remained elusive. Here, we used structure-guided protein engineering to produce a stabilized GP38-Gn-Gc heterotrimeric glycoprotein complex (GP38-GnH-DS-Gc). A cryo-EM structure of this complex provides the molecular basis for GP38’s association on the viral surface, reveals the structure of Gn, and demonstrates that GP38-Gn restrains the Gc fusion loops in the prefusion conformation, facilitated by an N-linked glycan attached to Gn. Immunization with GP38-GnH-DS-Gc conferred 40% protection against lethal IbAr10200 challenge in mice. These data define the architecture of a GP38-Gn-Gc protomer and provide a template for structure-guided vaccine antigen development. Less
Rhomboid intramembrane serine proteases have been implicated in several pathologies and emerge as attractive pharmacological target candidates The most potent and selective rhomboid inhibitors available to date are peptidyl -ketoamides but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood This gap together with the lack of suitable in vitro models hinders ketoamide development for relevant eukaryotic rhomboid enzymes Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG human mitochondrial rhomboid PARL and human RHBDL ... More
Rhomboid intramembrane serine proteases have been implicated in several pathologies, and emerge as attractive pharmacological target candidates. The most potent and selective rhomboid inhibitors available to date are peptidyl α-ketoamides, but their selectivity for diverse rhomboid proteases and strategies to modulate it in relevant contexts are poorly understood. This gap, together with the lack of suitable in vitro models, hinders ketoamide development for relevant eukaryotic rhomboid enzymes. Here we explore the structure-activity relationship principles of rhomboid inhibiting ketoamides by medicinal chemistry and enzymatic in vitro and in-cell assays with recombinant rhomboid proteases GlpG, human mitochondrial rhomboid PARL and human RHBDL2. We use X-ray crystallography in lipid cubic phase to understand the binding mode of one of the best ketoamide inhibitors synthesised here containing branched terminal substituent bound to GlpG. In addition, to extend the interpretation of the co-crystal structure, we use quantum mechanical calculations and quantify the relative importance of interactions along the inhibitor molecule. These combined experimental analyses implicates that more extensive exploration of chemical space at the prime side is unexpectedly powerful for the selectivity of rhomboid inhibiting ketoamides. Together with variations in the peptide sequence at the non-prime side, or its non-peptidic alternatives, this strategy enables targeted tailoring of potent and selective ketoamides towards diverse rhomboid proteases including disease-relevant ones such as PARL and RHBDL2. Less
Methods of studying eukaryotic cell responses to a perturbation or of stratifying eukaryotic cells or cell lines into one or more subgroups are described The methods involve perturbing a library of cells or cell lines in the same manner and observing how the cells respond to the same perturbation The observation may be via a high throughput screening method for example cell painting and the perturbation may be for example exposure to a therapeutic agent The methods may be used for grouping cells or cell lines that respond similarly to a given therapeutic agent which may be useful for identifying ... More
Methods of studying eukaryotic cell responses to a perturbation, or of stratifying eukaryotic cells or cell lines into one or more subgroups are described. The methods involve perturbing a library of cells or cell lines in the same manner, and observing how the cells respond to the same perturbation. The observation may be via a high throughput screening method, for example, cell painting; and the perturbation may be, for example, exposure to a therapeutic agent.The methods may be used for grouping cells or cell lines that respond similarly to a given therapeutic agent, which may be useful for identifying patient groups and selecting appropriate treatments. Less
Vitamin B cobalamin or Cbl functions as a cofactor in two important enzymatic processes in human cells and life is not sustainable without it B is obtained from food and travels from the stomach through the intestine and into the bloodstream by three B -transporting proteins salivary haptocorrin HC gastric intrinsic factor IF and transcobalamin TC which all bind B with high affinity and require proteolytic degradation to liberate Cbl After intracellular delivery of dietary B Cbl in the aquo hydroxo-Cbl HOCbl form can coordinate various nucleophiles e g glutathione GSH giving rise to glutathionylcobalamin GSCbl a naturally-occurring form of ... More
Vitamin B12 (cobalamin or Cbl) functions as a cofactor in two important enzymatic processes in human cells, and life is not sustainable without it. B12 is obtained from food and travels from the stomach, through the intestine and into the bloodstream by three B12-transporting proteins: salivary haptocorrin (HC), gastric intrinsic factor (IF) and transcobalamin (TC), which all bind B12 with high affinity and require proteolytic degradation to liberate Cbl. After intracellular delivery of dietary B12, Cbl in the aquo/hydroxo-Cbl (HOCbl) form can coordinate various nucleophiles, e.g., glutathione (GSH), giving rise to glutathionylcobalamin (GSCbl), a naturally-occurring form of vitamin B12. Currently there is no data showing whether GSCbl is recognized and transported in the human body. Our crystallographic data shows for the first time the complex between a vitamin B12-transporter and GSCbl, which compared to HOCbl, binds TC equally well. Furthermore, sequence analysis and structural comparisons show that TC recognizes and transports GSCbl and that the residues involved are conserved among TCs from different organisms. Interestingly, HC and IF are not structurally tailored to bind GSCbl. This study provides new insights into the interactions between TC and Cbl. Less
The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening FFCS software suite This framework is further enriched by the option to utilize the Smart Digital User SDU software for automated data collection across the PXI PXII and PXIII beamlines In this work the newly developed HEIDI webpage https heidi psi ch is introduced a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state ... More
The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening (FFCS) software suite. This framework is further enriched by the option to utilize the Smart Digital User (SDU) software for automated data collection across the PXI, PXII and PXIII beamlines. In this work, the newly developed HEIDI webpage (https://heidi.psi.ch) is introduced: a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments. The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state transfer application programming interface (REST API). The migration of the local FFCS MongoDB instance to the cloud is highlighted and detailed. This transition ensures secure, encrypted and consistently accessible data through a robust and reliable REST API tailored for the FFCS software suite. Collectively, these advancements not only significantly elevate the user experience, but also pave the way for future expansions and improvements in the capabilities of the system. Less
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy This includes valorization of hemicellulosic fraction of plant biomass the second most abundant biopolymer from plant cell walls aiming to produce prebiotic oligosaccharides widely explored in food and feed industries In this work we conducted biochemical and biophysical characterization of a prokaryotic two-domain R champanellensis xylanase from glycoside hydrolase GH family RcXyn A and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH and GH families and a GH xylobiohydrolase RcXyn A liberates ... More
Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R. champanellensis xylanase from glycoside hydrolase (GH) family 30 (RcXyn30A), and evaluated its applicability for XOS production from glucuronoxylan in combination with two endo-xylanases from GH10 and GH11 families and a GH11 xylobiohydrolase. RcXyn30A liberates mainly long monoglucuronylated xylooligosaccharides and is inefficient in cleaving unbranched oligosaccharides. Crystallographic structure of RcXyn30A catalytic domain was solved and refined to 1.37 Å resolution. Structural analysis of the catalytic domain releveled that its high affinity for glucuronic acid substituted xylan is due to the coordination of the substrate decoration by several hydrogen bonds and ionic interactions in the subsite −2. Furthermore, the protein has a larger β5-α5 loop as compared to other GH30 xylanases, which might be crucial for creating an additional aglycone subsite (+3) of the catalytic site. Finally, RcXyn30A activity is synergic to that of GH11 xylobiohydrolase. Less
Despite their lack of a defined D structure intrinsically disordered regions IDRs of proteins play important biological roles Many IDRs contain short linear motifs SLiMs that mediate protein-protein interactions PPIs which can be regulated by post-translational modifications like phosphorylation of pathogenic missense mutations are found in IDRs and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms Here we employ peptide-based interaction proteomics to investigate disease-associated mutations affecting phosphorylation sites Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides often due to disrupted phosphorylation-dependent SLiMs We focused on a mutation of a serine phosphorylation ... More
Despite their lack of a defined 3D structure, intrinsically disordered regions (IDRs) of proteins play important biological roles. Many IDRs contain short linear motifs (SLiMs) that mediate protein-protein interactions (PPIs), which can be regulated by post-translational modifications like phosphorylation. 20% of pathogenic missense mutations are found in IDRs, and understanding how such mutations affect PPIs is essential for unraveling disease mechanisms. Here, we employ peptide-based interaction proteomics to investigate 36 disease-associated mutations affecting phosphorylation sites. Our results unveil significant differences in interactomes between phosphorylated and non-phosphorylated peptides, often due to disrupted phosphorylation-dependent SLiMs. We focused on a mutation of a serine phosphorylation site in the transcription factor GATAD1, which causes dilated cardiomyopathy. We find that this phosphorylation site mediates interaction with 14-3-3 family proteins. Follow-up experiments reveal the structural basis of this interaction and suggest that 14-3-3 binding affects GATAD1 nucleocytoplasmic transport by masking a nuclear localisation signal. Our results demonstrate that pathogenic mutations of human phosphorylation sites can significantly impact protein-protein interactions, offering insights into potential molecular mechanisms underlying pathogenesis. Less
With the rapid advancements in sequencing technologies the identification of single nucleotide mutations has surged surpassing our capacity for functional characterization Remarkably approximately of these disease-linked point mutations are situated within protein regions devoid of a well-defined D structure known as intrinsically disordered regions IDRs These IDRs are recognized for their pivotal roles in the regulation signaling and control of biological processes They can harbor short linear motifs SLiMs that act as mediators in protein-protein interactions PPIs often subject to regulation through post-translational modifications such as phosphorylation Investigating the impact of these IDR mutations on protein-protein interactions is essential for ... More
With the rapid advancements in sequencing technologies, the identification of
single nucleotide mutations has surged, surpassing our capacity for functional
characterization. Remarkably, approximately 20% of these disease-linked point
mutations are situated within protein regions devoid of a well-defined 3D structure,
known as intrinsically disordered regions (IDRs). These IDRs are recognized for
their pivotal roles in the regulation, signaling, and control of biological processes.
They can harbor short linear motifs (SLiMs) that act as mediators in protein-protein
interactions (PPIs), often subject to regulation through post-translational
modifications such as phosphorylation. Investigating the impact of these IDR
mutations on protein-protein interactions is essential for comprehending the
molecular mechanisms underlying human diseases.
In this doctoral thesis, I present a comprehensive exploration of a peptide-based
proteomics screen, employed to scrutinize 36 disease-associated mutations that
impair phosphorylation sites within IDRs. This approach entailed the immobilization
of synthetic peptides, corresponding to the mutated regions, onto a cellulose
membrane. These peptides were then utilized to capture interacting proteins from
cellular extracts. This method facilitated the simultaneous comparison of
interaction partners among wild-type, phosphorylated, and mutated peptide forms,
enabling the functional assessment of individual mutations. Our analysis
uncovered significant disparities
between the interactomes of phosphorylated and non-phosphorylated peptides,
changes frequently attributed to the disruption of phosphorylation-dependent
SLiMs.
Building on our findings, we placed particular emphasis on the S102P mutation
within the transcription factor GATAD1, a mutation associated with dilated
cardiomyopathy. Our screening demonstrated that this mutation disrupts a crucial
phosphorylation site responsible for 14-3-3 protein binding. To delve deeper into
this interaction, we conducted a thorough investigation, employing techniques such
as isothermal titration calorimetry, X-ray crystallography, and alanine scanning
coupled with mass spectrometry. Our meticulous analyses hinted at the regulatory
role of 14-3-3 binding in GATAD1's nucleocytoplasmic transport, achieved by
masking its nuclear localization signal.
In conclusion, this doctoral thesis focuses on the profound impact of pathogenic
mutations within human phosphorylation sites on protein-protein interactions. The
insights from our research shed fresh light on potential molecular mechanisms
underpinning the development of various human diseases, offering a promising
avenue for further investigation and therapeutic exploration. Less
single nucleotide mutations has surged, surpassing our capacity for functional
characterization. Remarkably, approximately 20% of these disease-linked point
mutations are situated within protein regions devoid of a well-defined 3D structure,
known as intrinsically disordered regions (IDRs). These IDRs are recognized for
their pivotal roles in the regulation, signaling, and control of biological processes.
They can harbor short linear motifs (SLiMs) that act as mediators in protein-protein
interactions (PPIs), often subject to regulation through post-translational
modifications such as phosphorylation. Investigating the impact of these IDR
mutations on protein-protein interactions is essential for comprehending the
molecular mechanisms underlying human diseases.
In this doctoral thesis, I present a comprehensive exploration of a peptide-based
proteomics screen, employed to scrutinize 36 disease-associated mutations that
impair phosphorylation sites within IDRs. This approach entailed the immobilization
of synthetic peptides, corresponding to the mutated regions, onto a cellulose
membrane. These peptides were then utilized to capture interacting proteins from
cellular extracts. This method facilitated the simultaneous comparison of
interaction partners among wild-type, phosphorylated, and mutated peptide forms,
enabling the functional assessment of individual mutations. Our analysis
uncovered significant disparities
between the interactomes of phosphorylated and non-phosphorylated peptides,
changes frequently attributed to the disruption of phosphorylation-dependent
SLiMs.
Building on our findings, we placed particular emphasis on the S102P mutation
within the transcription factor GATAD1, a mutation associated with dilated
cardiomyopathy. Our screening demonstrated that this mutation disrupts a crucial
phosphorylation site responsible for 14-3-3 protein binding. To delve deeper into
this interaction, we conducted a thorough investigation, employing techniques such
as isothermal titration calorimetry, X-ray crystallography, and alanine scanning
coupled with mass spectrometry. Our meticulous analyses hinted at the regulatory
role of 14-3-3 binding in GATAD1's nucleocytoplasmic transport, achieved by
masking its nuclear localization signal.
In conclusion, this doctoral thesis focuses on the profound impact of pathogenic
mutations within human phosphorylation sites on protein-protein interactions. The
insights from our research shed fresh light on potential molecular mechanisms
underpinning the development of various human diseases, offering a promising
avenue for further investigation and therapeutic exploration. Less
Light-driven sodium pumps NaRs are unique ion-transporting microbial rhodopsins The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base We thoroughly characterize a member of this subgroup namely the protein ErNaR from Erythrobacter sp HL- and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity which is in contrast to previously studied NaRs ... More
Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identify a subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterize a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and show that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics. Less
Spatial heterogeneity is ubiquitous across life and the universe the same is true for phase separating pharmaceutical formulations cells and tissues To interrogate these spatially-varying complicated samples simple analysis techniques such as fluorescence recovery after photobleaching FRAP can provide information on molecular transport Conventional FRAP approaches localize analysis to small spots which may not be representative of trends across the full field of view Taking advantage of strategies used for structures illumination an approach has been developed to use patterned illumination in combination with FRAP for probing large fields of view while representatively sampling Patterned illumination is used to establish ... More
Spatial heterogeneity is ubiquitous across life and the universe; the same is true for phase separating pharmaceutical formulations, cells, and tissues. To interrogate these spatially-varying complicated samples, simple analysis techniques such as fluorescence recovery after
photobleaching (FRAP) can provide information on molecular transport. Conventional FRAP
approaches localize analysis to small spots, which may not be representative of trends across the full field of view. Taking advantage of strategies used for structures illumination, an approach has been developed to use patterned illumination in combination with FRAP for probing large fields of view while representatively sampling. Patterned illumination is used to establish a concentration
gradient across a sample by irreversibly photobleaching fluorophores, such as with the simple comb pattern photobleach presented in Chapters 1 and 4. Patterned photobleaching allows spatial Fourier-domain analysis of multiple spatial harmonics simultaneously. In the spatial FT-domain the real-space photobleach signal is integrated into puncta, greatly increasing the signal to noise ratio compared to conventional point-bleach FRAP. The order of the spatial harmonic is directly related to the length scale of translational diffusion measured, with a series of harmonics accessing
diffusion over many length scales in a single experiment. Measurements of diffusion at multiple length scales informs on the diffusion mechanism by sensitively reporting on deviations away from normal diffusion. Complementing the physical hardware for inducing patterned illumination, this dissertation
introduces novel algorithms for reconstructing spatially-resolved diffusion maps in heterogeneous materials by combining Fourier domain analysis with patterned photobleaching. FT-FRAP is introduced in Chapter 1 for interrogating phase-separating samples using beam-scanning instrumentation for comb-bleach illumination. This analysis allowed disentangling separate contributions to diffusion from normal bulk diffusion and an interfacial exchange mechanism only available due to multi-harmonic analysis. The introduction of a dot-array bleach pattern using widefield microscopy is presented in Chapter 2 for high-throughput detection of mobility in simple binary systems as well as for segmentation in phase-separating pharmaceutical formulations. The analysis becomes more complicated as more components are added to the system such as a surfactant. Introduced in chapter 3, FT-FRAP with dot-array photobleaching was shown to be
useful for characterizing diffusion of phase-separating micro-domain smaller than a single pixel of the camera. Supported by simulations, a biexponential fitting model was developed for
quantification of diffusion by multiple species simultaneously. Chapter 4 introduces imaging
inside of 3D particles comprised of an active pharmaceutical ingredient (API) in
microencapsulated agglomerates which exhibited strong interfacial exchange. Multi-photon excited fluorescence enabled imaging a small focal volume within the particles. Less
photobleaching (FRAP) can provide information on molecular transport. Conventional FRAP
approaches localize analysis to small spots, which may not be representative of trends across the full field of view. Taking advantage of strategies used for structures illumination, an approach has been developed to use patterned illumination in combination with FRAP for probing large fields of view while representatively sampling. Patterned illumination is used to establish a concentration
gradient across a sample by irreversibly photobleaching fluorophores, such as with the simple comb pattern photobleach presented in Chapters 1 and 4. Patterned photobleaching allows spatial Fourier-domain analysis of multiple spatial harmonics simultaneously. In the spatial FT-domain the real-space photobleach signal is integrated into puncta, greatly increasing the signal to noise ratio compared to conventional point-bleach FRAP. The order of the spatial harmonic is directly related to the length scale of translational diffusion measured, with a series of harmonics accessing
diffusion over many length scales in a single experiment. Measurements of diffusion at multiple length scales informs on the diffusion mechanism by sensitively reporting on deviations away from normal diffusion. Complementing the physical hardware for inducing patterned illumination, this dissertation
introduces novel algorithms for reconstructing spatially-resolved diffusion maps in heterogeneous materials by combining Fourier domain analysis with patterned photobleaching. FT-FRAP is introduced in Chapter 1 for interrogating phase-separating samples using beam-scanning instrumentation for comb-bleach illumination. This analysis allowed disentangling separate contributions to diffusion from normal bulk diffusion and an interfacial exchange mechanism only available due to multi-harmonic analysis. The introduction of a dot-array bleach pattern using widefield microscopy is presented in Chapter 2 for high-throughput detection of mobility in simple binary systems as well as for segmentation in phase-separating pharmaceutical formulations. The analysis becomes more complicated as more components are added to the system such as a surfactant. Introduced in chapter 3, FT-FRAP with dot-array photobleaching was shown to be
useful for characterizing diffusion of phase-separating micro-domain smaller than a single pixel of the camera. Supported by simulations, a biexponential fitting model was developed for
quantification of diffusion by multiple species simultaneously. Chapter 4 introduces imaging
inside of 3D particles comprised of an active pharmaceutical ingredient (API) in
microencapsulated agglomerates which exhibited strong interfacial exchange. Multi-photon excited fluorescence enabled imaging a small focal volume within the particles. Less
Nowadays an efficient and robust virtual screening procedure is crucial in the drug discovery process especially when performed on large and chemically diverse databases Virtual screening methods like molecular docking and classic QSAR models are limited in their ability to handle vast numbers of compounds and to learn from scarce data respectively In this study we introduce a universal methodology that uses a machine learning-based approach to predict docking scores without the need for time-consuming molecular docking procedures The developed protocol yielded times faster binding energy predictions than classical docking-based screening The proposed predictive model learns from docking results allowing ... More
Nowadays, an efficient and robust virtual screening procedure is crucial in the drug discovery process, especially when performed on large and chemically diverse databases. Virtual screening methods, like molecular docking and classic QSAR models, are limited in their ability to handle vast numbers of compounds and to learn from scarce data, respectively. In this study, we introduce a universal methodology that uses a machine learning-based approach to predict docking scores without the need for time-consuming molecular docking procedures. The developed protocol yielded 1000 times faster binding energy predictions than classical docking-based screening. The proposed predictive model learns from docking results, allowing users to choose their preferred docking software without relying on insufficient and incoherent experimental activity data. The methodology described employs multiple types of molecular fingerprints and descriptors to construct an ensemble model that further reduces prediction errors and is capable of delivering highly precise docking score values for monoamine oxidase ligands, enabling faster identification of promising compounds. An extensive pharmacophore-constrained screening of the ZINC database resulted in a selection of 24 compounds that were synthesized and evaluated for their biological activity. A preliminary screen discovered weak inhibitors of MAO-A with a percentage efficiency index close to a known drug at the lowest tested concentration. The approach presented here can be successfully applied to other biological targets as target-specific knowledge is not incorporated at the screening phase. Less
Sphingolipid activator protein B saposin B SapB is an essential activator of globotriaosylceramide Gb catabolism by -galactosidase A However the manner by which SapB stimulates -galactosidase A activity remains unknown To uncover the molecular mechanism of SapB presenting Gb to -galactosidase A we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole Gb -NBD to a series of biochemical and structural assays involving SapB First we showed that SapB stably binds Gb -NBD using a fluorescence equilibrium binding assay isolates Gb -NBD from micelles and facilitates -galactosidase A cleavage of Gb -NBD in vitro Second we crystallized SapB in the presence of Gb -NBD ... More
Sphingolipid activator protein B (saposin B; SapB) is an essential activator of globotriaosylceramide (Gb3) catabolism by α-galactosidase A. However, the manner by which SapB stimulates α-galactosidase A activity remains unknown. To uncover the molecular mechanism of SapB presenting Gb3 to α-galactosidase A, we subjected the fluorescent substrate globotriaosylceramide-nitrobenzoxidazole (Gb3-NBD) to a series of biochemical and structural assays involving SapB. First, we showed that SapB stably binds Gb3-NBD using a fluorescence equilibrium binding assay, isolates Gb3-NBD from micelles, and facilitates α-galactosidase A cleavage of Gb3-NBD in vitro. Second, we crystallized SapB in the presence of Gb3-NBD and validated the ligand-bound assembly. Third, we captured transient interactions between SapB and α-galactosidase A by chemical cross-linking. Finally, we determined the crystal structure of SapB bound to α-galactosidase A. These findings establish general principles for molecular recognition in saposin:hydrolase complexes and highlight the utility of NBD reporter lipids in saposin biochemistry and structural biology. Less
Dysregulation of phosphorylation-dependent signaling is a hallmark of tumorigenesis Protein phosphatase PP A is an essential regulator of cell growth One scaffold subunit A binds to a catalytic subunit C to form a core AC heterodimer which together with one of many regulatory B subunits forms the active trimeric enzyme The combinatorial number of distinct PP A complexes is large which results in diverse substrate specificity and subcellular localization The detailed mechanism of PP A assembly and regulation remains elusive and reports about an important role of methylation of the carboxy terminus of PP A C are conflicting A better ... More
Dysregulation of phosphorylation-dependent signaling is a hallmark of tumorigenesis. Protein phosphatase 2 (PP2A) is an essential regulator of cell growth. One scaffold subunit (A) binds to a catalytic subunit (C) to form a core AC heterodimer, which together with one of many regulatory (B) subunits forms the active trimeric enzyme. The combinatorial number of distinct PP2A complexes is large, which results in diverse substrate specificity and subcellular localization. The detailed mechanism of PP2A assembly and regulation remains elusive and reports about an important role of methylation of the carboxy terminus of PP2A C are conflicting. A better understanding of the molecular underpinnings of PP2A assembly and regulation is critical to dissect PP2A function in physiology and disease. Here, we combined biochemical reconstitution, mass spectrometry, X-ray crystallography and functional assays to characterize the assembly of trimeric PP2A. In vitro studies demonstrated that methylation of the carboxy-terminus of PP2A C was dispensable for PP2A assembly in vitro. To corroborate these findings, we determined the X-ray crystal structure of the unmethylated PP2A Aα-B56ε-Cα trimer complex to 3.1 Å resolution. The experimental structure superimposed well with an Alphafold2Multimer prediction of the PP2A trimer. We then predicted models of all canonical PP2A complexes providing a framework for structural analysis of PP2A. In conclusion, methylation was dispensable for trimeric PP2A assembly and integrative structural biology studies of PP2A offered predictive models for all canonical PP2A complexes. Less
Carbohydrate-active enzymes from the glycoside hydrolase family GH play a key role in processing lignocellulosic biomass Although the structural features of some GH enzymes are known the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel A enzyme utilizes to depolymerize cellulosic substrates we used a combination of biochemical assays X-ray crystallography small-angle X-ray scattering and molecular dynamics simulations The results reveal that BlCel A breaks down cellulosic substrates releasing cellobiose and glucose as the major products but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose ... More
Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs. Less
The activities of the phospholipase C gamma PLC and enzymes are essential for numerous cellular processes Unsurprisingly dysregulation of PLC or PLC activity is associated with multiple maladies including immune disorders cancers and neurodegenerative diseases Therefore the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases To aid in the discovery of PLC family enzyme modulators that could be developed into therapeutic agents we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C CF -coumarin Herein the ability of PLC and PLC to enzymatically process C CF -coumarin was confirmed ... More
The activities of the phospholipase C gamma (PLCγ) 1 and 2 enzymes are essential for numerous cellular processes. Unsurprisingly, dysregulation of PLCγ1 or PLCγ2 activity is associated with multiple maladies including immune disorders, cancers, and neurodegenerative diseases. Therefore, the modulation of either of these two enzymes has been suggested as a therapeutic strategy to combat these diseases. To aid in the discovery of PLCγ family enzyme modulators that could be developed into therapeutic agents, we have synthesized a high-throughput screening-amenable micellular fluorogenic substrate called C16CF3-coumarin. Herein, the ability of PLCγ1 and PLCγ2 to enzymatically process C16CF3-coumarin was confirmed, the micellular assay conditions were optimized, and the kinetics of the reaction were determined. A proof-of-principle pilot screen of the Library of Pharmacologically Active Compounds 1280 (LOPAC1280) was performed. This new substrate allows for an additional screening methodology to identify modulators of the PLCγ family of enzymes. Less
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
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
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
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
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
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
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
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
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