1194 Citations
Coproporphyrin ferrochelatases CpfCs are enzymes catalyzing the penultimate step in the coproporphyrin-dependent CPD heme biosynthesis pathway which is mainly utilized by monoderm bacteria Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades nevertheless many mechanistic questions remain unanswered to date Especially CpfCs which are found in the CPD pathway are currently in the spotlight of research This pathway was identified in and revealed that the correct substrate for these ferrochelatases is coproporphyrin III cpIII instead of protoporphyrin IX as believed prior the discovery of the CPD pathway The chemistry of cpIII which has four ... More
Coproporphyrin ferrochelatases (CpfCs) are enzymes catalyzing the penultimate step in the coproporphyrin-dependent (CPD) heme biosynthesis pathway, which is mainly utilized by monoderm bacteria. Ferrochelatases insert ferrous iron into a porphyrin macrocycle and have been studied for many decades, nevertheless many mechanistic questions remain unanswered to date. Especially CpfCs, which are found in the CPD pathway, are currently in the spotlight of research. This pathway was identified in 2015 and revealed that the correct substrate for these ferrochelatases is coproporphyrin III (cpIII) instead of protoporphyrin IX, as believed prior the discovery of the CPD pathway. The chemistry of cpIII, which has four propionates, differs significantly from protoporphyrin IX, which features two propionate and two vinyl groups. These findings let us to thoroughly describe the physiological cpIII-ferrochelatase complex in solution and in the crystal phase. Here, we present the first crystallographic structure of the CpfC from the representative monoderm pathogen Listeria monocytogenes bound to its physiological substrate, cpIII, together with the in-solution data obtained by resonance Raman and UV–vis spectroscopy, for wild-type ferrochelatase and variants, analyzing propionate interactions. The results allow us to evaluate the porphyrin distortion and provide an in-depth characterization of the catalytically-relevant binding mode of cpIII prior to iron insertion. Our findings are discussed in the light of the observed structural restraints and necessities for this porphyrin-enzyme complex to catalyze the iron insertion process. Knowledge about this initial situation is essential for understanding the preconditions for iron insertion in CpfCs and builds the basis for future studies. Less
Mitogen-activated protein kinase MAPK phosphatase MKP is responsible for regulating the activity of the stress-responsive MAPKs and has been put forth as a potential therapeutic target for a number of diseases including dystrophic muscle disease a fatal rare disease which has neither a treatment nor cure In previous work we identified Compound -dimethyl- - - methylthio - -dihydrothieno -h quinazolin- -yl thio butan- -one as the lead compound of a novel class of MKP inhibitors In this work we explore the structure-activity relationship for inhibition of MKP through modifications to the scaffold and functional groups present in A series of ... More
Mitogen-activated protein kinase (MAPK) phosphatase 5 (MKP5) is responsible for regulating the activity of the stress-responsive MAPKs and has been put forth as a potential therapeutic target for a number of diseases, including dystrophic muscle disease a fatal rare disease which has neither a treatment nor cure. In previous work, we identified Compound 1 (3,3-dimethyl-1-((9-(methylthio)-5,6-dihydrothieno[3,4-h]quinazolin-2-yl)thio)butan-2-one) as the lead compound of a novel class of MKP5 inhibitors. In this work, we explore the structure-activity relationship for inhibition of MKP5 through modifications to the scaffold and functional groups present in 1. A series of derivative compounds was designed, synthesized, and evaluated for inhibition of MKP5. In addition, the X-ray crystal structures of six enzyme-inhibitor complexes were solved, further elucidating the necessary requirements for MKP5 inhibition. We found that the parallel-displaced π-π interaction between the inhibitor three-ring core and Tyr435 is critical for modulating potency, and that modifications to the core and functionalization at the C-9 position are essential for ensuring proper positioning of the core for this interaction. These results lay the foundation from which more potent MKP5 allosteric inhibitors can be developed for potential therapeutics towards the treatment of dystrophic muscle disease. Less
Applying rational design we developed kDa cyanobacteriochrome-based near-infrared NIR-I fluorescent protein miRFP nano miRFP nano efficiently binds endogenous biliverdin chromophore and brightly fluoresces in mammalian cells and tissues miRFP nano has maximal emission at nm and an emission tail in the short-wave infrared SWIR region allowing deep-penetrating off-peak fluorescence imaging in vivo The miRFP nano structure reveals the molecular basis of its red shift We demonstrate superiority of miRFP nano-enabled SWIR imaging over NIR-I imaging of microbes in the mouse digestive tract mammalian cells injected into the mouse mammary gland and NF-kB activity in a mouse model of liver inflammation
Microfluidic paper-based analytical devices microPADs are an emerging platform for point-of-care assays with potential applications ranging from medical diagnostics to environmental monitoring A standard approach for signal generation and amplification on microPADs is the use of enzymes and chromogenic substrates which produce colored products and enable the qualitative and quantitative detection of analytes Horseradish peroxidase HRP is one of the most commonly used enzymes for this application While a wide variety of chromogenic substrates and substrate solutions are available for HRP these were all originally developed and optimized for other platforms and have not been systematically compared on microPADs In ... More
Microfluidic paper-based analytical devices (microPADs) are an emerging platform for point-of-care assays with potential applications ranging from medical diagnostics to environmental monitoring. A standard approach for signal generation and amplification on microPADs is the use of enzymes and chromogenic substrates, which produce colored products and enable the qualitative and quantitative detection of analytes. Horseradish peroxidase (HRP) is one of the most commonly used enzymes for this application. While a wide variety of chromogenic substrates and substrate solutions are available for HRP, these were all originally developed and optimized for other platforms and have not been systematically compared on microPADs. In this work, 33 chromogenic substrate solutions for HRP containing one of six different substrate molecules were evaluated in a standardized colorimetric assay and compared on the basis of limit of detection (LOD), lower limit of quantitation (LLOQ), upper limit of quantitation (ULOQ), sensitivity, dynamic range, stability, and cost. While the substrates tetramethylbenzidine (TMB) and o-phenylenediamine (OPD) were found to achieve the best overall analytical performance, each substrate had characteristics that may make them appealing for specific applications. The results of this work will facilitate the selection of substrate molecules and the formulation of substrate solutions for future colorimetric paper-based assays involving HRP. Less
Increasing rate of genetic gain for key agronomic traits through genomic selection requires the development of new molecular methods to run genome-wide single nucleotide polymorphisms SNPs The main limitation of current methods is the cost is too high to screen breeding populations Molecular inversion probes MIPs is a targeted genotyping-by-sequencing method that could be used for soybeans that is both cost effective high-throughput and provides high data quality to screen breeder s germplasm for genomic selection A K MIP SNP set was developed for soybean with uniformly distributed markers across the genome The SNPs were selected to maximize the number ... More
Increasing rate of genetic gain for key agronomic traits through genomic selection requires the development of new molecular methods to run genome-wide single nucleotide polymorphisms (SNPs). The main limitation of current methods is the cost is too high to screen breeding populations. Molecular inversion probes (MIPs) is a targeted genotyping-by-sequencing method that could be used for soybeans that is both cost effective, high-throughput, and provides high data quality to screen breeder’s germplasm for genomic selection. A 1K MIP SNP set was developed for soybean with uniformly distributed markers across the genome. The SNPs were selected to maximize the number of informative markers in germplasm being tested in soybean breeding programs located in the North Central and Mid-South regions of the United States. The 1K SNP MIP set was tested on diverse germplasm and a recombinant inbred line population. Targeted sequencing with MIPs obtained an 85% enrichment for the targeted SNPs. MIP’s genotyping accuracy was 93% overall while homozoygous call accuracy was 98% with less than 10% missing data. The accuracy of MIPs combined with its low per sample cost makes it a powerful tool to enable genomic selection within soybean breeding programs. Less
Coelenterazine-v CTZ-v a synthetic vinylene-bridged -extended derivative is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths However Ca -regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence Here we report the crystal structure of semi-synthetic Ca -discharged obelin-v bound with the reaction product determined at resolution Comparison of the crystal structure of Ca -discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure However the drastic changes in CTZ-binding ... More
Coelenterazine-v (CTZ-v), a synthetic vinylene-bridged π-extended derivative, is able to significantly alter bioluminescence spectra of different CTZ-dependent luciferases and photoproteins by shifting them towards longer wavelengths. However, Ca2+-regulated photoproteins activated with CTZ-v display very low bioluminescence activities that hampers its usage as a substrate of photoprotein bioluminescence. Here, we report the crystal structure of semi-synthetic Ca2+-discharged obelin-v bound with the reaction product determined at 2.1 Å resolution. Comparison of the crystal structure of Ca2+-discharged obelin-v with those of other obelins before and after bioluminescence reaction reveals no considerable changes in the overall structure. However, the drastic changes in CTZ-binding cavity are observed owing to the completely different reaction product, coelenteramine-v (CTM-v). Since CTM-v is certainly the main product of obelin-v bioluminescence and is considered to be a product of the “dark” pathway of dioxetanone intermediate decomposition, it explains the low bioluminescence activity of obelin and apparently of other photoproteins with CTZ-v. Less
The past fifty years have been marked by the surge of neurodegenerative diseases Unfortunately current treatments are only symptomatic Hence the search for new and innovative therapeutic targets for curative treatments becomes a major challenge Among these targets the adenosine A A receptor A AAR has been the subject of much research in recent years In this paper we report the design synthesis and pharmacological analysis of quinazoline derivatives as A AAR antagonists with high ligand efficiency This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM- More precisely we ... More
The past fifty years have been marked by the surge of neurodegenerative diseases. Unfortunately, current treatments are only symptomatic. Hence, the search for new and innovative therapeutic targets for curative treatments becomes a major challenge. Among these targets, the adenosine A2A receptor (A2AAR) has been the subject of much research in recent years. In this paper, we report the design, synthesis and pharmacological analysis of quinazoline derivatives as A2AAR antagonists with high ligand efficiency. This class of molecules has been discovered by a virtual screening and bears no structural semblance with reference antagonist ZM-241385. More precisely, we identified a series of 2-aminoquinazoline as promising A2AAR antagonists. Among them, one compound showed a high affinity towards A2AAR (21a, Ki = 20 nM). We crystallized this ligand in complex with A2AAR, confirming one of our predicted docking poses and opening up possibilities for further optimization to derive selective ligands for specific adenosine receptor subtypes. Less
Transmembrane ion transport is a key process in living cells Active transport of ions is carried out by various ion transporters including microbial rhodopsins MRs MRs perform diverse functions such as active and passive ion transport photo-sensing and others In particular MRs can pump various monovalent ions like Na K Cl I NO The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp PCC and is named Synechocystis halorhodopsin SyHR The structural study of SyHR may help to understand what makes an MR pump divalent ions Here we present the crystal structure ... More
Transmembrane ion transport is a key process in living cells. Active transport of ions is carried out by various ion transporters including microbial rhodopsins (MRs). MRs perform diverse functions such as active and passive ion transport, photo-sensing, and others. In particular, MRs can pump various monovalent ions like Na+, K+, Cl−, I−, NO3−. The only characterized MR proposed to pump sulfate in addition to halides belongs to the cyanobacterium Synechocystis sp. PCC 7509 and is named Synechocystis halorhodopsin (SyHR). The structural study of SyHR may help to understand what makes an MR pump divalent ions. Here we present the crystal structure of SyHR in the ground state, the structure of its sulfate-bound form as well as two photoreaction intermediates, the K and O states. These data reveal the molecular origin of the unique properties of the protein (exceptionally strong chloride binding and proposed pumping of divalent anions) and sheds light on the mechanism of anion release and uptake in cyanobacterial halorhodopsins. The unique properties of SyHR highlight its potential as an optogenetics tool and may help engineer different types of anion pumps with applications in optogenetics. Less
The transcription factor TEAD together with its coactivator YAP TAZ is a key transcriptional modulator of the Hippo pathway Activation of TEAD transcription by YAP has been implicated in a number of malignancies and this complex represents a promising target for drug discovery However both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules mainly due to a lack of druggable pockets TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition Here we employed ... More
The transcription factor TEAD, together with its coactivator YAP/TAZ, is a key transcriptional modulator of the Hippo pathway. Activation of TEAD transcription by YAP has been implicated in a number of malignancies, and this complex represents a promising target for drug discovery. However, both YAP and its extensive binding interfaces to TEAD have been difficult to address using small molecules, mainly due to a lack of druggable pockets. TEAD is post-translationally modified by palmitoylation that targets a conserved cysteine at a central pocket, which provides an opportunity to develop cysteine-directed covalent small molecules for TEAD inhibition. Here, we employed covalent fragment screening approach followed by structure-based design to develop an irreversible TEAD inhibitor MYF-03–69. Using a range of in vitro and cell-based assays we demonstrated that through a covalent binding with TEAD palmitate pocket, MYF-03–69 disrupts YAP-TEAD association, suppresses TEAD transcriptional activity and inhibits cell growth of Hippo signaling defective malignant pleural mesothelioma (MPM). Further, a cell viability screening with a panel of 903 cancer cell lines indicated a high correlation between TEAD-YAP dependency and the sensitivity to MYF-03–69. Transcription profiling identified the upregulation of proapoptotic BMF gene in cancer cells that are sensitive to TEAD inhibition. Further optimization of MYF-03–69 led to an in vivo compatible compound MYF-03–176, which shows strong antitumor efficacy in MPM mouse xenograft model via oral administration. Taken together, we disclosed a story of the development of covalent TEAD inhibitors and its high therapeutic potential for clinic treatment for the cancers that are driven by TEAD-YAP alteration. Less
Histone deacetylase HDAC is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain HDAC is involved in various biological processes such as cell motility or stress responses and has been implicated in pathologies ranging from cancer to neurodegeneration Due to this broad range of functions there has been considerable interest in developing HDAC -specific small molecule inhibitors several of which are already available The crystal structure of the tandem catalytic domains of zebrafish HDAC has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains Further dissection of the biochemical properties ... More
Histone deacetylase 6 (HDAC6) is an atypical lysine deacetylase with tandem catalytic domains and an ubiquitin-binding zinc finger domain. HDAC6 is involved in various biological processes, such as cell motility or stress responses, and has been implicated in pathologies ranging from cancer to neurodegeneration. Due to this broad range of functions, there has been considerable interest in developing HDAC6-specific small molecule inhibitors, several of which are already available. The crystal structure of the tandem catalytic domains of zebrafish HDAC6 has revealed an arrangement with twofold symmetry and extensive surface interaction between the catalytic domains. Further dissection of the biochemical properties of HDAC6 and the development of novel inhibitors will benefit from being able to routinely express high-quality protein. We present here our optimized protocol for expression and crystallization of the zebrafish tandem catalytic domains. Less
Colibactin a DNA cross-linking agent produced by gut bacteria is implicated in colorectal cancer Its biosynthesis uses a prodrug resistance mechanism a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm This activation is mediated by ClbP an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain Although the transmembrane domain is required for colibactin activation its role in catalysis is unclear Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP ... More
Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-d-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads. Less
The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer CRC development Understanding colibactin s biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota Here we report small molecule boronic acid inhibitors of colibactin biosynthesis Designed to mimic the biosynthetic precursor precolibactin these compounds potently inhibit the colibactin-activating peptidase ClbP Using biochemical assays and crystallography we show that they engage the ClbP binding pocket forming a covalent bond with the catalytic serine These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block ... More
The human gut bacterial genotoxin colibactin is a possible key driver of colorectal cancer (CRC) development. Understanding colibactin’s biological effects remains difficult owing to the instability of the proposed active species and the complexity of the gut microbiota. Here, we report small molecule boronic acid inhibitors of colibactin biosynthesis. Designed to mimic the biosynthetic precursor precolibactin, these compounds potently inhibit the colibactin-activating peptidase ClbP. Using biochemical assays and crystallography, we show that they engage the ClbP binding pocket, forming a covalent bond with the catalytic serine. These inhibitors reproduce the phenotypes observed in a clbP deletion mutant and block the genotoxic effects of colibactin on eukaryotic cells. The availability of ClbP inhibitors will allow precise, temporal control over colibactin production, enabling further study of its contributions to CRC. Finally, application of our inhibitors to related peptidase-encoding pathways highlights the power of chemical tools to probe natural product biosynthesis. Less
Voltage-gated sodium ion channels Nav are central to action potential initiation through regulating the entry of sodium ions Na into excitable cells including cardiomyocytes and neurones The -subunit of Nav consists of four homologous domains DI-DIV each consisting of six transmembrane helices S -S Helices S -S of each domain forms the lining of the central pore through which sodium ions Na enters the cell upon channel activation Helices S -S of each domain form the voltage sensor which becomes displaced in response to changes in intracellular potential Additionally Nav channels include an extracellular turret region whose role in channel ... More
Voltage-gated sodium ion channels (Nav) are central to action potential initiation through regulating the entry of sodium ions (Na+) into excitable cells including cardiomyocytes and neurones. The α-subunit of Nav consists of four homologous domains (DI-DIV), each consisting of six transmembrane helices (S1-S6). Helices S5-S6 of each domain forms the lining of the central pore through which sodium ions (Na+) enters the cell upon channel activation. Helices S1-S4 of each domain form the voltage sensor which becomes displaced in response to changes in intracellular potential. Additionally, Nav channels include an extracellular turret region, whose role in channel function is poorly understood. The C-terminal domain (CTD) of Nav, connected to the DIV-S6, interacts with various other proteins including calmodulin (CaM) and fibroblast growth factor (FGF13) and mediates various regulatory roles. The subtype Nav1.5 is primarily expressed in the heart where it initiates the cardiac action potential whereas Nav1.7 is found in the peripheral nervous system where it is associated with nociception.
Various Nav associated pathologies have been associated with mutations in the extracellular turret region; however, their molecular mechanism is not well understood. In the Nav1.5 structure determined by cryogenic electron microscopy (cryoEM), the wild-type residues that correspond to some of these mutants form a complex salt bridge at the interface between the DII and DIII turret loops. Furthermore, adjacent aromatic residues could potentially form cation-π interactions with the complex salt bridge. This region was examined using site-directed mutagenesis, electrophysiology and in silico modelling, confirming functional roles for the inter-domain salt-bridges and the aromatic residues. Evidence that disruption of these contacts perturbs the geometry of the DEKA selectivity ring and both the outer and inner pore vestibules that are crucial for sodium ion permeability were provided. These findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms.
Further experiments performed preliminary studies that focussed on the CTD of Nav1.5 and Nav1.7, seeking to better understand the role of its regulation by Ca2+ and CaM, using various techniques such as ELISA, isothermal titration calorimetry (ITC) and Bio-Layer interferometry (BLI). The CTDs and CaM recombinant proteins were cloned using the Gateway cloning method, expressed in BL21 (DE3) cells using auto-induction, and purified via affinity chromatography and size exclusion chromatography. Also, attempts were made to determine the yet unresolved structure of Nav1.7 using x-ray crystallography. Finally, using an in-house phage display library of single chain fragment variable (scFv) antibodies, specific binders to the CTD of Nav1.5 and Nav1.7, were found and purified. These scFvs could have gating effects on their Nav channel targets, which might prove therapeutically applicable. Less
Various Nav associated pathologies have been associated with mutations in the extracellular turret region; however, their molecular mechanism is not well understood. In the Nav1.5 structure determined by cryogenic electron microscopy (cryoEM), the wild-type residues that correspond to some of these mutants form a complex salt bridge at the interface between the DII and DIII turret loops. Furthermore, adjacent aromatic residues could potentially form cation-π interactions with the complex salt bridge. This region was examined using site-directed mutagenesis, electrophysiology and in silico modelling, confirming functional roles for the inter-domain salt-bridges and the aromatic residues. Evidence that disruption of these contacts perturbs the geometry of the DEKA selectivity ring and both the outer and inner pore vestibules that are crucial for sodium ion permeability were provided. These findings provide insights into a class of pathological mutations occurring not only in Nav1.5 but also in other sodium channel isoforms.
Further experiments performed preliminary studies that focussed on the CTD of Nav1.5 and Nav1.7, seeking to better understand the role of its regulation by Ca2+ and CaM, using various techniques such as ELISA, isothermal titration calorimetry (ITC) and Bio-Layer interferometry (BLI). The CTDs and CaM recombinant proteins were cloned using the Gateway cloning method, expressed in BL21 (DE3) cells using auto-induction, and purified via affinity chromatography and size exclusion chromatography. Also, attempts were made to determine the yet unresolved structure of Nav1.7 using x-ray crystallography. Finally, using an in-house phage display library of single chain fragment variable (scFv) antibodies, specific binders to the CTD of Nav1.5 and Nav1.7, were found and purified. These scFvs could have gating effects on their Nav channel targets, which might prove therapeutically applicable. Less
Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane s and manipulate their infected host To date secretion systems have been identified including the type IX secretion system T SS associated with human avian and farmed-fish diseases As a bacterial secretion system the T SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria PorX is a highly conserved protein that regulates the transcription of essential T SS components and additionally mediates the function of T SS via direct interaction with PorL the rotary motor protein ... More
Pathogenic bacteria utilize specialized macromolecular secretion systems to transport virulence factors across membrane(s) and manipulate their infected host. To date, 11 secretion systems have been identified, including the type IX secretion system (T9SS) associated with human, avian and farmed-fish diseases. As a bacterial secretion system, the T9SS also facilitates gliding motility and the degradation of different macromolecules by the secretion of metabolic enzymes in nonpathogenic bacteria. PorX is a highly conserved protein that regulates the transcription of essential T9SS components and additionally mediates the function of T9SS via direct interaction with PorL, the rotary motor protein of the T9SS. PorX is also a member of a two-component system regulatory cascade, where it serves as the response regulator that relays a signal transduced from a conserved sensor histidine kinase, PorY, to a designated sigma factor. Here, the recombinant expression and purification of PorX homologous proteins from the pathogenic bacterium Porphyromonas gingivalis and the nonpathogenic bacterium Flavobacterium johnsoniae are reported. A bioinformatical characterization of the different domains comprising the PorX protein is also provided, and the crystallization and X-ray analysis of PorX from F. johnsoniae are reported. Less
Intrinsically disordered proteins IDPs often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks Among these the IDP hub protein G BP can form complexes with cytoplasmic phosphoprotein Caprin and ubiquitin peptidase USP the resulting control of USP activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR However while the identities of protein interactors are known for many IDP hub proteins the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood Here we describe in vitro analysis of these G BP ... More
Intrinsically disordered proteins (IDPs) often coordinate transient interactions with multiple proteins to mediate complex signals within large protein networks. Among these, the IDP hub protein G3BP1 can form complexes with cytoplasmic phosphoprotein Caprin1 and ubiquitin peptidase USP10; the resulting control of USP10 activity contributes to a pathogenic virulence system that targets endocytic recycling of the ion channel CFTR. However, while the identities of protein interactors are known for many IDP hub proteins, the relationship between pairwise affinities and the extent of protein recruitment and activity is not well understood. Here we describe in vitro analysis of these G3BP1 affinities, and show tryptophan substitutions of specific G3BP1 residues reduce its affinity for both USP10 and Caprin1. We show that these same mutations reduce the stability of complexes between the full-length proteins, suggesting that co-purification can serve as a surrogate measure of interaction strength. The crystal structure of G3BP1 TripleW (F15W/F33W/F124W) mutant reveals a clear reorientation of the side chain of W33, creating a steric clash with USP10 and Caprin1. Furthermore, an amino-acid scan of USP10 and Caprin1 peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific substitutions with the potential to create higher affinity peptides. Taken together, these data show that small changes in component binding affinities can have significant effects on the composition of cellular interaction hubs. These specific protein mutations can be harnessed to manipulate complex protein networks, informing future investigations into roles of these networks in cellular processes. Less
The multifunctional human Parkinson s disease protein PARK DJ is an attractive therapeutic target due to its link with early-onset Parkinson s disease upregulation in various cancers and contribution to chemoresistance However only a few compounds have been identified to bind PARK due to the lack of a dedicated chemical toolbox We report the creation of such a toolbox and showcase the application of each of its components The selective PARK submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys Installment of different dyes onto the inhibitor delivered two PARK probes The Rhodamine probe provides a ... More
The multifunctional human Parkinson’s disease protein 7 (PARK7/DJ1) is an attractive therapeutic target due to its link with early-onset Parkinson’s disease, upregulation in various cancers, and contribution to chemoresistance. However, only a few compounds have been identified to bind PARK7 due to the lack of a dedicated chemical toolbox. We report the creation of such a toolbox and showcase the application of each of its components. The selective PARK7 submicromolar inhibitor with a cyanimide reactive group covalently modifies the active site Cys106. Installment of different dyes onto the inhibitor delivered two PARK7 probes. The Rhodamine110 probe provides a high-throughput screening compatible FP assay, showcased by screening a compound library (8000 molecules). The SulfoCy5-equipped probe is a valuable tool to assess the effect of PARK7 inhibitors in a cell lysate. Our work creates new possibilities to explore PARK7 function in a physiologically relevant setting and develop new and improved PARK7 inhibitors. Less
Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets Despite recent advances in the field efforts to discover such compounds have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different PARP enzymes of the ARTD family We here expanded the methods and engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold Compared to the free enzymes the scaffold-based system results in an improved activity for the tested PARPs due to improved ... More
Human mono-ADP-ribosylating PARP enzymes have been linked to several clinically relevant processes and many of these PARPs have been suggested as potential drug targets. Despite recent advances in the field, efforts to discover such compounds have been hindered by the lack of tools to rapidly screen for high potency compounds and profile them against the different PARP enzymes of the ARTD family. We here expanded the methods and engineered mono-ART catalytic fragments to be incorporated into a cellulosome-based octavalent scaffold. Compared to the free enzymes, the scaffold-based system results in an improved activity for the tested PARPs due to improved solubility, stability and the proximity of the catalytic domains, altogether boosting their activity beyond 10-fold in the case of PARP12. This allows us to measure their enhanced activity using a simple and easily accessible homogeneous NAD+ conversion assay, facilitating its automation to reduce the assay volume and lowering the assay costs. The approach will enable the discovery of more potent compounds due to increased assay sensitivity and it can be applied to compound screening campaigns as well as inhibitor profiling. Less
Single-cell nucleosome methylome and transcriptome scNMT sequencing is a recently developed method that allows multiomics profiling of single cells In this scNMT protocol we describe profiling of cells from mouse brain and pancreatic organoids using liquid handling platforms to increase throughput from -well to -well plate format Our approach miniaturizes reaction volumes and incorporates the latest Smart-seq protocol to obtain higher numbers of detected genes and genomic DNA gDNA CpGs per cell We outline normalization steps to optimally distribute per-cell sequencing depth
The main focus of this project was chicken protein MICAL which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton The prominent role of the MICAL protein is primarily associated with axon guidance as it destabilizes actin filaments through its oxidative activity We focused on elucidating the molecular mechanisms of chicken MICAL autoinhibition using molecular and structural biology methods together with new protein structure prediction methods Chicken MICAL was produced in Sf insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL protein ... More
The main focus of this project was chicken protein MICAL1, which is involved in the semaphorin-plexin signalling pathway and has a significant effect on the rearrangement of the cytoskeleton. The prominent role of the MICAL1 protein is primarily associated with axon guidance, as it destabilizes actin filaments through its oxidative activity. We focused on elucidating the molecular mechanisms of chicken MICAL1 autoinhibition using molecular and structural biology methods together with new protein structure prediction methods. Chicken MICAL1 was produced in Sf9 insect cells using a baculovirus expression system and we produced both full-length and truncated versions of chicken MICAL1 protein. We kinetically characterized the protein and determined its oligomeric state in solution. We made great efforts to solve the protein structure using crystallography, electron microscopy and protein structure prediction in Alphafold 2. Based on the results of these experiments and assays, we conclude that MICAL1 proteins are regulated through their C terminal domain, which interacts with the monooxygenase domain. The part of this interaction is the autoinhibition of chicken MICAL1. We excluded the possibility that chicken MICAL1 is regulated by changing its oligomeric state. The results of this master's thesis. Less
Autofluorescence-detected photothermal mid-infrared AF-PTIR microscopy was shown to enable parts-per-million detection of -indomethacin impurity in -indomethacin samples Subtle differences in the photothermal response of the UV-autofluorescence of two indomethacin crystal polymorphs were used for sub-micron chemical discrimination based on fingerprint region mid-IR spectroscopy The AF-PTIR assignment was independently confirmed by second harmonic generation SHG microscopy which was shown to reduce the total analysis time by rapidly identifying the suitable fields of view AF-PTIR microscopy has the potential to assist in the early identification of crystal form impurities in the solid dosage forms development pipeline
Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers Here we show the identification and development of an affinity-enhanced T cell receptor TCR that recognizes a peptide derived from the most common KRAS mutant KRASG D presented in the context of HLA-A The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG D over KRASWT While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG D ... More
Neoantigens derived from somatic mutations are specific to cancer cells and are ideal targets for cancer immunotherapy. KRAS is the most frequently mutated oncogene and drives the pathogenesis of several cancers. Here we show the identification and development of an affinity-enhanced T cell receptor (TCR) that recognizes a peptide derived from the most common KRAS mutant, KRASG12D, presented in the context of HLA-A*11:01. The affinity of the engineered TCR is increased by over one million-fold yet fully able to distinguish KRASG12D over KRASWT. While crystal structures reveal few discernible differences in TCR interactions with KRASWT versus KRASG12D, thermodynamic analysis and molecular dynamics simulations reveal that TCR specificity is driven by differences in indirect electrostatic interactions. The affinity enhanced TCR, fused to a humanized anti-CD3 scFv, enables selective killing of cancer cells expressing KRASG12D. Our work thus reveals a molecular mechanism that drives TCR selectivity and describes a soluble bispecific molecule with therapeutic potential against cancers harboring a common shared neoantigen. Less
Modulators of the G protein-coupled A A adenosine receptor A AAR have been considered promising agents to treat Parkinson s disease inflammation cancer and central nervous system disorders Herein we demonstrate that a thiophene modification at the C position in the common adenine scaffold converted an A AAR agonist into an antagonist We synthesized and characterized a novel A AAR antagonist LJ- with Ki nM X-ray crystallographic structures of in complex with two thermostabilized A AAR constructs were solved at and resolutions In contrast to A AAR agonists which simultaneously interact with both Ser and His only transiently contacts His ... More
Modulators of the G protein-coupled A2A adenosine receptor (A2AAR) have been considered promising agents to treat Parkinson’s disease, inflammation, cancer, and central nervous system disorders. Herein, we demonstrate that a thiophene modification at the C8 position in the common adenine scaffold converted an A2AAR agonist into an antagonist. We synthesized and characterized a novel A2AAR antagonist, 2 (LJ-4517), with Ki = 18.3 nM. X-ray crystallographic structures of 2 in complex with two thermostabilized A2AAR constructs were solved at 2.05 and 2.80 Å resolutions. In contrast to A2AAR agonists, which simultaneously interact with both Ser2777.42 and His2787.43, 2 only transiently contacts His2787.43, which can be direct or water-mediated. The n-hexynyl group of 2 extends into an A2AAR exosite. Structural analysis revealed that the introduced thiophene modification restricted receptor conformational rearrangements required for subsequent activation. This approach can expand the repertoire of adenosine receptor antagonists that can be designed based on available agonist scaffolds. Less
Nonribosomal peptide synthetase heterocyclization Cy domains generate biologically important oxazoline thiazoline groups found in natural products including pharmaceuticals and virulence factors such as some siderophores Cy domains catalyze consecutive condensation and cyclodehydration reactions although the mechanism is unknown To better understand Cy domain catalysis here we report the crystal structure of the second Cy domain Cy of yersiniabactin synthetase from the causative agent of the plague Yersinia pestis Our high-resolution structure of Cy adopts a conformation that enables exploration of interactions with the extended thiazoline-containing cyclodehydration intermediate and the acceptor carrier protein CP to which it is tethered We also ... More
Nonribosomal peptide synthetase heterocyclization (Cy) domains generate biologically important oxazoline/thiazoline groups found in natural products, including pharmaceuticals and virulence factors such as some siderophores. Cy domains catalyze consecutive condensation and cyclodehydration reactions, although the mechanism is unknown. To better understand Cy domain catalysis, here we report the crystal structure of the second Cy domain (Cy2) of yersiniabactin synthetase from the causative agent of the plague, Yersinia pestis. Our high-resolution structure of Cy2 adopts a conformation that enables exploration of interactions with the extended thiazoline-containing cyclodehydration intermediate and the acceptor carrier protein (CP) to which it is tethered. We also report complementary electrostatic interfaces between Cy2 and its donor CP that mediate donor binding. Finally, we explored domain flexibility through normal mode analysis and identified small-molecule fragment-binding sites that may inform future antibiotic design targeting Cy function. Our results suggest how CP binding may influence global Cy conformations, with consequences for active-site remodeling to facilitate the separate condensation and cyclodehydration steps as well as potential inhibitor development. Less
Chickpea is a crop that is known as a source of high-quality proteins CL-AI which belongs to the S globulin and cupin superfamily was initially identified in chickpea seeds CL-AI has recently been shown to inhibit various types of -amylases To determine its molecular mechanism the crystal structure of CL-AI was solved at a final resolution of Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association and each molecule is divided into an -chain and a -chain CL-AI has high structural similarity to other S globulins and canonical metal-dependent enzyme-related cupin proteins ... More
Chickpea is a crop that is known as a source of high-quality proteins. CL-AI, which belongs to the 11S globulin and cupin superfamily, was initially identified in chickpea seeds. CL-AI has recently been shown to inhibit various types of α-amylases. To determine its molecular mechanism, the crystal structure of CL-AI was solved at a final resolution of 2.2 Å. Structural analysis indicated that each asymmetric unit contains three molecules with threefold symmetry and a head-to-tail association, and each molecule is divided into an α-chain and a β-chain. CL-AI has high structural similarity to other 11S globulins and canonical metal-dependent enzyme-related cupin proteins, whereas its stimilarity to α-amylase inhibitor from Phaseolus vulgaris is quite low. The structure presented here will provide insight into the function of CL-AI. Less
The recent discovery of comammox complete ammonia oxidation Nitrospira has upended the long-held nitrification paradigm Although comammox Nitrospira have been identified in wastewater treatment systems the conditions for their dominance over canonical ammonia oxidizers remain unclear Here we report the dominance of comammox Nitrospira in a moving bed biofilm reactor MBBR fed with synthetic mainstream wastewater Integrated S rRNA gene amplicon sequencing fluorescence in situ hybridization FISH and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen DO concentration above mg O L The dominance of comammox Nitrospira over canonical ... More
The recent discovery of comammox (complete ammonia oxidation) Nitrospira has upended the long-held nitrification paradigm. Although comammox Nitrospira have been identified in wastewater treatment systems, the conditions for their dominance over canonical ammonia oxidizers remain unclear. Here, we report the dominance of comammox Nitrospira in a moving bed biofilm reactor (MBBR) fed with synthetic mainstream wastewater. Integrated 16S rRNA gene amplicon sequencing, fluorescence in situ hybridization (FISH), and metagenomic sequencing methods demonstrated the selective enrichment of comammox bacteria when the MBBR was operated at a dissolved oxygen (DO) concentration above 6 mg O2/L. The dominance of comammox Nitrospira over canonical ammonia oxidizers (i.e., Nitrosomonas) was attributed to the low residual ammonium concentration (0.02–0.52 mg N/L) formed in the high-DO MBBR. Two clade A comammox Nitrospira were identified, which are phylogenetically close to Candidatus Nitrospira nitrosa. Interestingly, cryosectioning-FISH showed these two comammox species spatially distributed on the surface of the biofilm. Moreover, the ammonia-oxidizing activity of comammox Nitrospira-dominated biofilms was susceptible to the oxygen supply, which dropped by half with the DO concentration decrease from 6 to 2 mg O2/L. These features collectively suggest a low apparent oxygen affinity for the comammox Nitrospira-dominated biofilms in the high-DO nitrifying MBBR. Less
We herein report the development of an automation platform for rapid purification and quantification of chemical libraries including reformatting of chemical matter to mM DMSO stock solutions This fully integrated workflow features tailored conditions for preparative reversed-phase RP HPLC-MS on microscale based on analytical data online fraction QC and CAD-based quantification as well as automated reformatting to enable rapid purification of chemical libraries This automated workflow is entirely solution-based eliminating the need to weigh or handle solids This increases process efficiency and creates a link between high-throughput synthesis and profiling of novel chemical matter with respect to biological and physicochemical ... More
We herein report the development of an automation platform for rapid purification and quantification of chemical libraries including reformatting of chemical matter to 10 mM DMSO stock solutions. This fully integrated workflow features tailored conditions for preparative reversed-phase (RP) HPLC-MS on microscale based on analytical data, online fraction QC and CAD-based quantification as well as automated reformatting to enable rapid purification of chemical libraries. This automated workflow is entirely solution-based, eliminating the need to weigh or handle solids. This increases process efficiency and creates a link between high-throughput synthesis and profiling of novel chemical matter with respect to biological and physicochemical properties in relevant assays. Less
Polyurethane-based hydrogels are relatively inexpensive and mechanically robust biomaterials with ideal properties for various applications including drug delivery prosthetics implant coatings soft robotics and tissue engineering In this report we present a simple method for synthesizing and casting biocompatible polyurethane-poly ethylene glycol PU-PEG hydrogels with tunable mechanical properties non-fouling characteristics and sustained tolerability as an implantable material or coating The hydrogels are synthesized via a simple one-pot method using commercially available precursors and low toxicity solvents and reagents yielding a consistent and biocompatible gel platform primed for long-term biomaterial applications The mechanical and physical properties of the gels are easily ... More
Polyurethane-based hydrogels are relatively inexpensive and mechanically robust biomaterials with ideal properties for various applications, including drug delivery, prosthetics, implant coatings, soft robotics, and tissue engineering. In this report, we present a simple method for synthesizing and casting biocompatible polyurethane-poly(ethylene glycol) (PU-PEG) hydrogels with tunable mechanical properties, non-fouling characteristics, and sustained tolerability as an implantable material or coating. The hydrogels are synthesized via a simple one-pot method using commercially available precursors and low toxicity solvents and reagents, yielding a consistent and biocompatible gel platform primed for long-term biomaterial applications. The mechanical and physical properties of the gels are easily controlled by varying the curing concentration, producing networks with complex shear moduli of 0.82 kPa – 190 kPa, similar to a range of human soft tissues. When evaluated against a mechanically-matched PDMS formulation the PU-PEG hydrogels demonstrated favorable non-fouling characteristics including comparable adsorption of plasma proteins (albumin and fibrinogen) and significantly reduced cellular adhesion. Moreover, preliminary murine implant studies reveal a mild foreign body response after 41 days. Due to the tunable mechanical properties, excellent biocompatibility, and sustained in vivo tolerability of these hydrogels, we propose that this method offers a simplified platform for fabricating soft PU-based biomaterials for a variety of applications. Less
Mice deficient for all ten-eleven translocation TET genes exhibit early gastrulation lethality However separating cause and effect in such embryonic failure is challenging To isolate cell-autonomous effects of TET loss we used temporal single-cell atlases from embryos with partial or complete mutant contributions Strikingly when developing within a wild-type embryo Tet-mutant cells retain near-complete differentiation potential whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential We map de-repressions of early epiblast factors e g Dppa and Gdf and failure to activate multiple signaling from nascent mesoderm Lefty FGF and Notch as likely ... More
Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, Tet-mutant cells retain near-complete differentiation potential, whereas embryos solely comprising mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors (e.g., Dppa4 and Gdf3) and failure to activate multiple signaling from nascent mesoderm (Lefty, FGF, and Notch) as likely cell-intrinsic drivers of TET loss phenotypes. We further suggest loss of enhancer demethylation as the underlying mechanism. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications. Less
Akt is a Ser Thr protein kinase that plays a central role in metabolism and cancer Regulation of Akt s activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology PH domain and its kinase domain that can be relieved by C-tail phosphorylation PH domain mutant E K Akt is a well-established oncogene Previously we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds Chu et al Here we discover unexpectedly that a single mutation R A Akt exhibits intensified ... More
Akt is a Ser/Thr protein kinase that plays a central role in metabolism and cancer. Regulation of Akt’s activity involves an autoinhibitory intramolecular interaction between its pleckstrin homology (PH) domain and its kinase domain that can be relieved by C-tail phosphorylation. PH domain mutant E17K Akt is a well-established oncogene. Previously, we reported that the conformation of autoinhibited Akt may be shifted by small molecule allosteric inhibitors limiting the mechanistic insights from existing X-ray structures that have relied on such compounds (Chu et al., 2020). Here, we discover unexpectedly that a single mutation R86A Akt exhibits intensified autoinhibitory features with enhanced PH domain-kinase domain affinity. Structural and biochemical analysis uncovers the importance of a key interaction network involving Arg86, Glu17, and Tyr18 that controls Akt conformation and activity. Our studies also shed light on the molecular basis for E17K Akt activation as an oncogenic driver. Less
The bioactive lysophospholipid sphingosine- -phosphate S P acts via five different subtypes of S P receptors S PRs - S P - S P is predominantly expressed in nervous and immune systems regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders as well as carcinogenesis Several S PR therapeutic drugs have been developed to treat these diseases however they lack receptor subtype selectivity which leads to side effects In this article we describe a resolution room temperature crystal structure of the human S P receptor in complex with a selective ... More
The bioactive lysophospholipid sphingosine-1-phosphate (S1P) acts via five different subtypes of S1P receptors (S1PRs) - S1P1-5. S1P5 is predominantly expressed in nervous and immune systems, regulating the egress of natural killer cells from lymph nodes and playing a role in immune and neurodegenerative disorders, as well as carcinogenesis. Several S1PR therapeutic drugs have been developed to treat these diseases; however, they lack receptor subtype selectivity, which leads to side effects. In this article, we describe a 2.2 Å resolution room temperature crystal structure of the human S1P5 receptor in complex with a selective inverse agonist determined by serial femtosecond crystallography (SFX) at the Pohang Accelerator Laboratory X-Ray Free Electron Laser (PAL-XFEL) and analyze its structure-activity relationship data. The structure demonstrates a unique ligand-binding mode, involving an allosteric sub-pocket, which clarifies the receptor subtype selectivity and provides a template for structure-based drug design. Together with previously published S1PR structures in complex with antagonists and agonists, our structure with S1P5-inverse agonist sheds light on the activation mechanism and reveals structural determinants of the inverse agonism in the S1PR family. Less
We studied the prevalent cytotoxic CD T cell response mounted against severe acute respiratory syndrome coronavirus SARS-CoV- Spike glycoprotein - epitope sequence YLQPRTFLL via the most frequent human leukocyte antigen HLA class I worldwide HLA A The Spike P L mutation that has arisen in at least different SARS-CoV- lineages to date including in lineages classified as variants of concern was not recognized by the large CD T cell response seen across cohorts of HLA A convalescent patients and individuals vaccinated against SARS-CoV- despite these responses comprising of over different individual T cell receptors Viral escape at prevalent T cell ... More
We studied the prevalent cytotoxic CD8 T cell response mounted against severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent
human leukocyte antigen (HLA) class I worldwide, HLA A*02. The Spike P272L mutation that has arisen in at
least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘‘variants of concern,’’
was not recognized by the large CD8 T cell response seen across cohorts of HLA A*02+ convalescent patients
and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different
individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may
be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike,
providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting
the need for monitoring T cell escape in new SARS-CoV-2 variants. Less
coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent
human leukocyte antigen (HLA) class I worldwide, HLA A*02. The Spike P272L mutation that has arisen in at
least 112 different SARS-CoV-2 lineages to date, including in lineages classified as ‘‘variants of concern,’’
was not recognized by the large CD8 T cell response seen across cohorts of HLA A*02+ convalescent patients
and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different
individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may
be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike,
providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting
the need for monitoring T cell escape in new SARS-CoV-2 variants. Less
We studied the prevalent cytotoxic CD T cell response mounted against severe acute respiratory syndrome coronavirus SARS-CoV- Spike glycoprotein - epitope sequence YLQPRTFLL via the most frequent human leukocyte antigen HLA class I worldwide HLA A The Spike P L mutation that has arisen in at least different SARS-CoV- lineages to date including in lineages classified as variants of concern was not recognized by the large CD T cell response seen across cohorts of HLA A convalescent patients and individuals vaccinated against SARS-CoV- despite these responses comprising of over different individual T cell receptors Viral escape at prevalent T cell ... More
We studied the prevalent cytotoxic CD8 T cell response mounted against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent human leukocyte antigen (HLA) class I worldwide, HLA A∗02. The Spike P272L mutation that has arisen in at least 112 different SARS-CoV-2 lineages to date, including in lineages classified as “variants of concern,” was not recognized by the large CD8 T cell response seen across cohorts of HLA A∗02+ convalescent patients and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike, providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting the need for monitoring T cell escape in new SARS-CoV-2 variants. Less
Acute myelogenous leukemia AML a disease of the blood and bone marrow is characterized by the inability of myeloblasts to differentiate into mature cell types Dihydroorotate dehydrogenase DHODH is an enzyme well-known in the pyrimidine biosynthesis pathway however small molecule DHODH inhibitors were recently shown to induce differentiation in multiple AML subtypes Using virtual screening and structure-based drug design approaches a new series of N-heterocyclic -pyridyl carboxamide DHODH inhibitors were discovered Two lead compounds and have potent biochemical and cellular DHODH activity favorable physicochemical properties and efficacy in a preclinical model of AML
Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of D protein structures which has recently been further augmented by revolutionary advances in cryoEM However a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts limiting the overview of inherent protein physiological dynamics which play a critical role in the biological functions of proteins Here a room-temperature X-ray crystallo graphic method using temperature as a trigger to record movie-like structural snapshots has been developed The method has been used to show how TL a Da fragment undergoes ... More
Continuous developments in cryogenic X-ray crystallography have provided most of our knowledge of 3D protein structures, which has recently been further augmented by revolutionary advances in cryoEM. However, a single structural conformation identified at cryogenic temperatures may introduce a fictitious structure as a result of cryogenic cooling artefacts, limiting the overview of inherent protein physiological dynamics, which play a critical role in the biological functions of proteins. Here, a room-temperature X-ray crystallographic method using temperature as a trigger to record movie-like structural snapshots has been developed. The method has been used to show how TL00150, a 175.15 Da fragment, undergoes binding-mode changes in endothiapepsin. A surprising fragment-binding discrepancy was observed between the cryo-cooled and physiological temperature structures, and multiple binding poses and their interplay with DMSO were captured. The observations here open up new promising prospects for structure determination and interpretation at physiological temperatures with implications for structure-based drug discovery. Less
A series of amino acid based H-pyrrolo d pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV- nsp macrodomain Mac an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis Structure activity studies identified compound c as a low-micromolar inhibitor of Mac in two ADP-ribose binding assays This compound also demonstrated inhibition in an enzymatic assay of Mac and displayed a thermal shift comparable to ADPr in the melting temperature of Mac supporting binding to the target protein A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network ... More
A series of amino acid based 7H-pyrrolo[2,3–d]pyrimidines were designed and synthesized to discern the structure activity relationships against the SARS-CoV-2 nsp3 macrodomain (Mac1), an ADP-ribosylhydrolase that is critical for coronavirus replication and pathogenesis. Structure activity studies identified compound 15c as a low-micromolar inhibitor of Mac1 in two ADP-ribose binding assays. This compound also demonstrated inhibition in an enzymatic assay of Mac1 and displayed a thermal shift comparable to ADPr in the melting temperature of Mac1 supporting binding to the target protein. A structural model reproducibly predicted a binding mode where the pyrrolo pyrimidine forms a hydrogen bonding network with Asp22 and the amide backbone NH of Ile23 in the adenosine binding pocket and the carboxylate forms hydrogen bonds to the amide backbone of Phe157 and Asp156, part of the oxyanion subsite of Mac1. Compound 15c also demonstrated notable selectivity for coronavirus macrodomains when tested against a panel of ADP-ribose binding proteins. Together, this study identified several low MW, low µM Mac1 inhibitors to use as small molecule chemical probes for this potential anti-viral target and offers starting points for further optimization. Less
N-Methylated amino acids N-MeAAs are privileged residues of naturally occurring peptides critical to bioactivity However de novo discovery from ribosome display is limited by poor incorporation of N-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the N-methyl-aminoacyl-tRNA By reconfiguring the tRNA s T-stem region to compensate and tune the EF-Tu affinity we conducted Random nonstandard Peptides Integrated Discovery RaPID display of a macrocyclic peptide MCP library containing six different N-MeAAs We have here devised a pool-and-split enrichment strategy using the RaPID display and identified N-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate ... More
N-Methylated amino acids (N-MeAAs) are privileged residues of naturally occurring peptides critical to bioactivity. However, de novo discovery from ribosome display is limited by poor incorporation of N-methylated amino acids into the nascent peptide chain attributed to a poor EF-Tu affinity for the N-methyl-aminoacyl-tRNA. By reconfiguring the tRNA’s T-stem region to compensate and tune the EF-Tu affinity, we conducted Random nonstandard Peptides Integrated Discovery (RaPID) display of a macrocyclic peptide (MCP) library containing six different N-MeAAs. We have here devised a “pool-and-split” enrichment strategy using the RaPID display and identified N-methylated MCPs against three species of prokaryotic metal-ion-dependent phosphoglycerate mutases. The enriched MCPs reached 57% N-methylation with up to three consecutively incorporated N-MeAAs, rivaling natural products. Potent nanomolar inhibitors ranging in ortholog selectivity, strongly mediated by N-methylation, were identified. Co-crystal structures reveal an architecturally related Ce-2 Ipglycermide active-site metal-ion-coordinating Cys lariat MCP, functionally dependent on two cisN-MeAAs with broadened iPGM species selectivity over the original nematode-selective MCPs. Furthermore, the isolation of a novel metal-ion-independent Staphylococcus aureus iPGM inhibitor utilizing a phosphoglycerate mimetic mechanism illustrates the diversity of possible chemotypes encoded by the N-MeAA MCP library. Less
The SARS-CoV- infection cycle is a multistage process that relies on functional interactions between the host and the pathogen Here we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA '-O-ribose cap needed for viral immune escape We find that the host cap '-O-ribose methyltransferase MTr can compensate for loss of viral NSP methyltransferase in facilitating virus replication Concomitant inhibition of MTr and NSP efficiently suppresses SARS-CoV- replication Using in silico target-based drug screening we identify a bispecific MTr NSP inhibitor with anti-SARS-CoV- activity in vitro and in vivo but with unfavorable ... More
The SARS-CoV-2 infection cycle is a multistage process that relies on functional interactions between the host and the pathogen. Here, we repurposed antiviral drugs against both viral and host enzymes to pharmaceutically block methylation of the viral RNA 2'-O-ribose cap needed for viral immune escape. We find that the host cap 2'-O-ribose methyltransferase MTr1 can compensate for loss of viral NSP16 methyltransferase in facilitating virus replication. Concomitant inhibition of MTr1 and NSP16 efficiently suppresses SARS-CoV-2 replication. Using in silico target-based drug screening, we identify a bispecific MTr1/NSP16 inhibitor with anti-SARS-CoV-2 activity in vitro and in vivo but with unfavorable side effects. We further show antiviral activity of inhibitors that target independent stages of the host SAM cycle providing the methyltransferase co-substrate. In particular, the adenosylhomocysteinase (AHCY) inhibitor DZNep is antiviral in in vitro, in ex vivo, and in a mouse infection model and synergizes with existing COVID-19 treatments. Moreover, DZNep exhibits a strong immunomodulatory effect curbing infection-induced hyperinflammation and reduces lung fibrosis markers ex vivo. Thus, multispecific and metabolic MTase inhibitors constitute yet unexplored treatment options against COVID-19. Less
Microbial rhodopsins are light-sensitive transmembrane proteins evolutionary adapted by various organisms like archaea bacteria simple eukaryote and viruses to utilize solar energy for their survival A complete understanding of functional mechanisms of these proteins is not possible without the knowledge of their high-resolution structures which can be primarily obtained by X-ray crystallography This technique however requires high-quality crystals growing of which is a great challenge especially in case of membrane proteins In this chapter we summarize methods applied for crystallization of microbial rhodopsins with the emphasis on crystallization in lipidic mesophases also known as in meso approach In particular we ... More
Microbial rhodopsins are light-sensitive transmembrane proteins, evolutionary adapted by various organisms like archaea, bacteria, simple eukaryote, and viruses to utilize solar energy for their survival. A complete understanding of functional mechanisms of these proteins is not possible without the knowledge of their high-resolution structures, which can be primarily obtained by X-ray crystallography. This technique, however, requires high-quality crystals, growing of which is a great challenge especially in case of membrane proteins. In this chapter, we summarize methods applied for crystallization of microbial rhodopsins with the emphasis on crystallization in lipidic mesophases, also known as in meso approach. In particular, we describe in detail the methods of crystallization using lipidic cubic phase to grow both large crystals optimized for traditional crystallographic data collection and microcrystals for serial crystallography. Less
T-cell receptor TCR immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials The safety of patients is paramount so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor ie TCR specificity and cross-reactivity We recently reported a TCR engineering campaign and selectivity profiling assay X-scan targeting a melanoma antigen gene MAGE -A peptide This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation allowing one to be advanced to T-cell immunotherapeutic clinical trials Here we present three-dimensional structural information on ... More
T-cell receptor (TCR) immunotherapy is becoming a viable modality in cancer treatment with efficacy in clinical trials. The safety of patients is paramount, so innovative cell engineering methods are being employed to exploit adaptive immunity while controlling the factors governing antigen receptor (ie, TCR) specificity and cross-reactivity. We recently reported a TCR engineering campaign and selectivity profiling assay (X-scan) targeting a melanoma antigen gene (MAGE)-A10 peptide. This helped to distinguish between two well-performing TCRs based on cross-reactivity potential during preclinical drug evaluation, allowing one to be advanced to T-cell immunotherapeutic clinical trials. Here, we present three-dimensional structural information on those TCRs, highlighting engineering improvements and molecular mechanisms likely underpinning differential selectivity. Less
Heavy-atom soaking has been a major method for experimental phasing but it has been difficult for membrane proteins partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding The lipid cubic phase LCP has proven to be a successful method for membrane protein crystallization but experimental phasing with LCP-grown crystals remains difficult and so far only such structures were phased experimentally Here the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion SAD phasing of crystals grown in LCP Using a single crystal the structure of the glycerol -phosphate acyltransferase ... More
Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for membrane protein crystallization, but experimental phasing with LCP-grown crystals remains difficult, and so far, only 68 such structures were phased experimentally. Here, the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion (SAD) phasing of crystals grown in LCP. Using a single crystal, the structure of the glycerol 3-phosphate acyltransferase (PlsY, ~21 kDa), a very hydrophobic enzyme with 80% membrane-embedded residues, was solved. Remarkably, a total of 15 Se sites were found in the two monomers of PlsY, translating to one selenourea-binding site per every six residues in the accessible extramembrane protein. Structure analysis reveals that surface-exposed selenourea sites are mostly contributed by mainchain amides and carbonyls. This low-specificity binding pattern may explain its high loading ratio. Importantly, both the crystal diffraction quality and the LCP integrity were unaffected by selenourea soaking. Taken together, selenourea presents a promising and generally useful reagent for heavy-atom soaking of membrane protein crystals grown in LCP. Less
Clonogenic assays evaluate the ability of single cells to proliferate and form colonies This process approximates the regrowth and recurrence of tumors after treatment with radiation or chemotherapy and thereby provides a drug discovery platform for compounds that block this process However because of their labor-intensive and cumbersome nature adapting canonical clonogenic assays for high throughput screening HTS has been challenging We overcame these barriers by developing an integrated system that automates cell- and liquid-handling irradiation dosimetry drug administration and incubation Further we developed a fluorescent live-cell based automated colony scoring methodology that identifies and counts colonies precisely based upon ... More
Clonogenic assays evaluate the ability of single cells to proliferate and form colonies. This process approximates the regrowth and recurrence of tumors after treatment with radiation or chemotherapy, and thereby provides a drug discovery platform for compounds that block this process. However, because of their labor-intensive and cumbersome nature, adapting canonical clonogenic assays for high throughput screening (HTS) has been challenging. We overcame these barriers by developing an integrated system that automates cell- and liquid-handling, irradiation, dosimetry, drug administration, and incubation. Further, we developed a fluorescent live-cell based automated colony scoring methodology that identifies and counts colonies precisely based upon actual nuclei number rather than colony area, thereby eliminating errors in colony counts caused by radiation induced changes in colony morphology. We identified 13 cell lines from 7 cancer types, where radiation is a standard treatment module, that exhibit identical radiation and chemoradiation response regardless of well format and are amenable to miniaturization into small-well HTS formats. We performed pilot screens through a 1584 compound NCI Diversity Set library using two cell lines representing different cancer indications. Radiation modulators identified in the pilot screens were validated in traditional clonogenic assays, providing proof-of-concept for the screen. The integrated methodology, hereafter ‘clonogenic HTS’, exhibits excellent robustness (Z’ values >0.5) and shows high reproducibility (>95%). We propose that clonogenic HTS we developed can function as a drug discovery platform to identify compounds that inhibit tumor regrowth following radiation therapy, to identify new efficacious pair-wise combinations of known oncologic therapies, or to identify novel modulators of approved therapies. Less
Buffalo flies Haematobia irritans exigua are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas most commonly on the medial canthus of the eye along the lateral and ventral neck and on the abdomen of cattle For closely related horn flies Haematobia irritans irritans Staphylococcus aureus has been suggested as a contributing factor in the development of lesions To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions swabs were taken from ... More
Buffalo flies (Haematobia irritans exigua) are hematophagous ectoparasites of cattle causing production and welfare impacts in northern Australian herds. Skin lesions associated with buffalo fly infestation and Stephanofilaria nematode infection are manifested as focal dermatitis or ulcerated areas, most commonly on the medial canthus of the eye, along the lateral and ventral neck, and on the abdomen of cattle. For closely related horn flies (Haematobia irritans irritans), Staphylococcus aureus has been suggested as a contributing factor in the development of lesions. To investigate the potential role of bacterial infection in the pathogenesis of buffalo fly lesions, swabs were taken from lesions and normal skin, and bacteria were also isolated from surface washings of buffalo flies and surface-sterilized homogenized flies. Bacterial identification was conducted by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) and strain typing by repetitive sequence-based PCR (rep-PCR) and DNA sequencing to determine species similarity and virulence factors. Of 50 bacterial isolates collected from lesions, 38 were identified as Staphylococcus agnetis and 12 as Staphylococcus hyicus, whereas four isolates from normal skin were S. hyicus and one was Mammaliicoccus sciuri. Of the Staphylococcus isolates isolated from buffalo flies, five were identified as S. agnetis and three as S. hyicus. Fifty percent of the buffalo fly isolates had rep-PCR genotypic patterns identical to those of the lesion isolates. Genome sequencing of 16 S. agnetis and four S. hyicus isolates revealed closely similar virulence factor profiles, with all isolates possessing exfoliative toxin A and C genes. The findings from this study suggest the involvement of S. agnetis and S. hyicus in buffalo fly lesion pathogenesis. This should be taken into account in the development of effective treatment and control strategies for lesions. Less
The technique of cryogenic-electron microscopy cryo-EM has revolutionized the field of membrane protein structure and function with a focus on the dominantly observed molecular species This report describes the structural characterization of a fully active human apelin receptor APJR complexed with heterotrimeric G protein observed in both and stoichiometric ratios We use cryo-EM single-particle analysis to determine the structural details of both species from the same sample preparation Protein preparations in the presence of the endogenous peptide ligand ELA or a synthetic small molecule both demonstrate these mixed stoichiometric states Structural differences in G protein engagement between dimeric and monomeric ... More
The technique of cryogenic-electron microscopy (cryo-EM) has revolutionized the field of membrane protein structure and function with a focus on the dominantly observed molecular species. This report describes the structural characterization of a fully active human apelin receptor (APJR) complexed with heterotrimeric G protein observed in both 2:1 and 1:1 stoichiometric ratios. We use cryo-EM single-particle analysis to determine the structural details of both species from the same sample preparation. Protein preparations, in the presence of the endogenous peptide ligand ELA or a synthetic small molecule, both demonstrate these mixed stoichiometric states. Structural differences in G protein engagement between dimeric and monomeric APJR suggest a role for the stoichiometry of G protein-coupled receptor- (GPCR-)G protein coupling on downstream signaling and receptor pharmacology. Furthermore, a small, hydrophobic dimer interface provides a starting framework for additional class A GPCR dimerization studies. Together, these findings uncover a mechanism of versatile regulation through oligomerization by which GPCRs can modulate their signaling. Less
Candida Als family adhesins mediate adhesion to biological and abiotic substrates as well as fungal cell aggregation fungal-bacterial co-aggregation and biofilm formation The activity of at least two family members Als and Als is dependent on amyloid-like protein aggregation that is initiated by shear force Each Als adhesin has a -residue N-terminal Ig-like invasin region The following -residue low complexity threonine-rich T domain unfolds under shear force to expose a critical amyloid-forming segment SNGIVIVATTRTV at the interface between the Ig-like invasin domain and the T domain of Candida albicans Als Amyloid prediction programs identified six potential amyloidogenic sequences in the ... More
Candida Als family adhesins mediate adhesion to biological and abiotic substrates, as well as fungal cell aggregation, fungal-bacterial co-aggregation and biofilm formation. The activity of at least two family members, Als5 and Als1, is dependent on amyloid-like protein aggregation that is initiated by shear force. Each Als adhesin has a ∼300-residue N-terminal Ig-like/invasin region. The following 108-residue, low complexity, threonine-rich (T) domain unfolds under shear force to expose a critical amyloid-forming segment 322SNGIVIVATTRTV334 at the interface between the Ig-like/invasin domain 2 and the T domain of Candida albicans Als5. Amyloid prediction programs identified six potential amyloidogenic sequences in the Ig-like/invasin region and three others in the T domain of C. albicans Als5. Peptides derived from four of these sequences formed fibrils that bound thioflavin T, the amyloid indicator dye, and three of these revealed atomic-resolution structures of cross-β spines. These are the first atomic-level structures for fungal adhesins. One of these segments, from the T domain, revealed kinked β-sheets, similarly to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. Based on the cross-β structures in Als proteins, we use evolutionary arguments to identify functional amyloidogenic sequences in other fungal adhesins, including adhesins from Candida auris. Thus, cross-β structures are often involved in fungal pathogenesis and potentially in antifungal therapy. Less
G protein-coupled receptors GPCRs play vital roles in human physiology and pathophysiology This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics However the production and crystallization of GPCRs for structure determination comes with many challenges In this chapter we provide a comprehensive protocol for expressing and purifying the thromboxane A receptor TPR an attractive therapeutic target for use in structure studies Guidelines for crystallizing the TPR are also included Together these procedures provide a template for generating crystal structures of the TPR and indeed other ... More
G protein-coupled receptors (GPCRs) play vital roles in human physiology and pathophysiology. This makes the elucidation of the high-resolution blueprints of these high value membrane proteins of crucial importance for the structure-based design of novel therapeutics. However, the production and crystallization of GPCRs for structure determination comes with many challenges.
In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands. Less
In this chapter, we provide a comprehensive protocol for expressing and purifying the thromboxane A2 receptor (TPR), an attractive therapeutic target, for use in structure studies. Guidelines for crystallizing the TPR are also included. Together, these procedures provide a template for generating crystal structures of the TPR and indeed other GPCRs in complex with pharmacologically interesting ligands. Less
Background The consequences of past coronavirus disease COVID- infection for personal and population health are emerging but accurately identifying distant infection is a challenge Anti-spike antibodies rise after both vaccination and infection and anti-nucleocapsid antibodies rapidly decline Methods We evaluated anti-membrane antibodies in COVID- naive vaccinated and convalescent subjects to determine if they persist and accurately detect distant infection Results We found that anti-membrane antibodies persist for at least year and are a sensitive and specific marker of past COVID- infection Conclusions Thus anti-membrane and anti-spike antibodies together can differentiate between COVID- convalescent vaccinated and naive states to advance public ... More
Background The consequences of past coronavirus disease 2019 (COVID-19) infection for personal and population health are emerging, but accurately identifying distant infection is a challenge. Anti-spike antibodies rise after both vaccination and infection and anti-nucleocapsid antibodies rapidly decline. Methods We evaluated anti-membrane antibodies in COVID-19 naive, vaccinated, and convalescent subjects to determine if they persist and accurately detect distant infection. Results We found that anti-membrane antibodies persist for at least 1 year and are a sensitive and specific marker of past COVID-19 infection. Conclusions Thus, anti-membrane and anti-spike antibodies together can differentiate between COVID-19 convalescent, vaccinated, and naive states to advance public health and research. Less
As discussed in previous chapters the methylation of specific arginine and lysine side chains is carried out by two families of histone methyltransferases the Protein Arginine Methyltransferase PRMT family for arginine and the SET domain family for lysine The methylation of H K by Dot is a notable outlier In all cases X-ray crystallography has been a powerful technique that has provided the framework for understanding the enzyme mechanism kinetics regulation and specificity of these enzymes and is now a platform for the design of compounds aimed to inhibit their activity either to further understand their function or in a ... More
As discussed in previous chapters, the methylation of specific arginine and lysine side chains is carried out by two families of histone methyltransferases, the Protein Arginine Methyltransferase (PRMT) family for arginine, and the SET domain family for lysine. The methylation of H3K79 by Dot1 is a notable outlier. In all cases, X-ray crystallography has been a powerful technique that has provided the framework for understanding the enzyme mechanism, kinetics, regulation and specificity of these enzymes and is now a platform for the design of compounds aimed to inhibit their activity either to further understand their function or in a therapeutic setting. Notably, in combination with the structures of the complementary recognition domains that recognize their products, these structures have provided an important insight into how integral the number of methyl groups added to the acceptor amine is to making histone methylation a key process in epigenetic regulation of gene transcription. Here the concepts applied to determine their structure by X-ray crystallography are outlined, with particular emphasis on lysine methylation by the SET domain. Less
Chemical proteomics studies the effects of drugs upon a cellular proteome Due to the complexity and diversity of tumors the response of cancer cells to drugs is also heterogeneous and thus proteome analysis at the single-cell level is needed Here we demonstrate that single-cell proteomics techniques have become quantitative enough to tackle the drug effects on target proteins enabling single-cell chemical proteomics SCCP Using SCCP we studied here the time-resolved response of individual adenocarcinoma A cells to anticancer drugs methotrexate camptothecin and tomudex revealing the early emergence of cellular subpopulations committed and uncommitted to death As a novel and useful ... More
Chemical proteomics studies the effects of drugs upon a cellular proteome. Due to the complexity and diversity of tumors, the response of cancer cells to drugs is also heterogeneous, and thus, proteome analysis at the single-cell level is needed. Here, we demonstrate that single-cell proteomics techniques have become quantitative enough to tackle the drug effects on target proteins, enabling single-cell chemical proteomics (SCCP). Using SCCP, we studied here the time-resolved response of individual adenocarcinoma A549 cells to anticancer drugs methotrexate, camptothecin, and tomudex, revealing the early emergence of cellular subpopulations committed and uncommitted to death. As a novel and useful approach to exploring the heterogeneous response to drugs of cancer cells, SCCP may prove to be a breakthrough application for single-cell proteomics. Less
Advancements in technology and communication have revolutionised the twenty-first century with the introduction of mobile phones and smartphones These phones are known to be platforms harbouring microbes with recent research shedding light on the abundance and broad spectrum of organisms they harbour Mobile phone use in the community and in professional sectors including health care settings is a potential source of microbial dissemination To identify the diversity of microbial genetic signature present on mobile phones owned by hospital medical staff Twenty-six mobile phones of health care staff were swabbed DNA extraction for downstream next generation sequencing shotgun metagenomic microbial profiling ... More
Advancements in technology and communication have revolutionised the twenty-first century with the introduction of mobile phones and smartphones. These phones are known to be platforms harbouring microbes with recent research shedding light on the abundance and broad spectrum of organisms they harbour. Mobile phone use in the community and in professional sectors including health care settings is a potential source of microbial dissemination. To identify the diversity of microbial genetic signature present on mobile phones owned by hospital medical staff. Twenty-six mobile phones of health care staff were swabbed. DNA extraction for downstream next generation sequencing shotgun metagenomic microbial profiling was performed. Survey questionnaires were handed to the staff to collect information on mobile phone usage and users’ behaviours. Each of the 26 mobile phones of this study was contaminated with microbes with the detection of antibiotic resistance and virulent factors. Taken together the sum of microbes and genes added together across all 26 mobile phones totalised 11,163 organisms (5714 bacteria, 675 fungi, 93 protists, 228 viruses, 4453 bacteriophages) and 2096 genes coding for antibiotic resistance and virulent factors. The survey of medical staff showed that 46% (12/26) of the participants used their mobile phones in the bathroom. Mobile phones are vectors of microbes and can contribute to microbial dissemination and nosocomial diseases worldwide. As fomites, mobile phones that are not decontaminated may pose serious risks for public health and biosecurity. Less
The cysteine of HCD C in DYRK A is involved in disulfide bridge formation with a cysteine C in the DFGSSC sequence The purpose of this project was to investigate how the state of the disulfide bridge would affect enzyme catalytic and ligand binding properties of the protein kinase A mutant DYRK A C A was thus designed to eliminate the disulfide bridge The mutant was expressed and purified following the same protocol as for DYRK A wt including HisTrap purification TEV cleavage and size exclusion chromatography Crystallization trials were performed for both the wt and the mutant with the ... More
The cysteine of HCD (C286) in DYRK1A is involved in disulfide bridge formation with a cysteine (C312) in the DFGSSC sequence. The purpose of this project was to investigate how the state of the disulfide bridge would affect enzyme catalytic and ligand binding properties of the protein kinase. A mutant, DYRK1A C312A, was thus designed to eliminate the disulfide bridge. The mutant was expressed and purified following the same protocol as for DYRK1A wt, including HisTrap purification, TEV cleavage and size exclusion chromatography. Crystallization trials were performed for both the wt and the mutant with the kinase inhibitor Staurosporine. DYRK1A wt with STU crystallized and diffracted with at a resolution of 2.33 Å. The DYRK1A C312A mutant with STU crystallized and diffracted with a resolution of 2.59 Å. The structure was solved by molecular replacement in Molrep (CCP4) and refined by Refmac5 and Phenix. Molecular dynamics (MD) simulations (SCHRODINGER) were performed with the intent to compare diverse disulfide bridge states. Ligand binding and enzyme catalytic properties were analyzed using a combination of techniques, including activity assays, microscale thermophoresis, and isothermal calorimetry. The Thermofluor assay confirmed that both the wt and the mutant bind tightly to STU and AZ-191. It also showed that the mutant consistently has a slightly lower melting temperature than the wt, which would indicate that it is less stable. Solvent accessible surface area (SASA) analysis support the theory of accessibility to conserved cysteine residues. Less