1181 Citations
The receptor binding domain of spike protein of SARS-CoV- binds angitensin coverting enzyme on the surface of epithelial cells leading to fusion and entry of virus into the cell Based on our experience we described a work flow for expressing and purifying proteins and screening conditions for generating diffraction quality crystals of the complex Production and crystallization of protein complex take abut twelve days from construction of vectors to harvesting and freezing crystals for data collection
De novo-designed receptor transmembrane domains TMDs present opportunities for precise control of cellular receptor functions We developed a de novo design strategy for generating programmed membrane proteins proMPs single-pass -helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor CAR that we expressed in mouse primary T cells and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD from monomers up to tetramers All programmed CARs stimulated substantially ... More
De novo-designed receptor transmembrane domains (TMDs) present opportunities for precise control of cellular receptor functions. We developed a de novo design strategy for generating programmed membrane proteins (proMPs): single-pass α-helical TMDs that self-assemble through computationally defined and crystallographically validated interfaces. We used these proMPs to program specific oligomeric interactions into a chimeric antigen receptor (CAR) that we expressed in mouse primary T cells and found that both in vitro CAR T cell cytokine release and in vivo antitumor activity scaled linearly with the oligomeric state encoded by the receptor TMD, from monomers up to tetramers. All programmed CARs stimulated substantially lower T cell cytokine release relative to the commonly used CD28 TMD, which we show elevated cytokine release through lateral recruitment of the endogenous T cell costimulatory receptor CD28. Precise design using orthogonal and modular TMDs thus provides a new way to program receptor structure and predictably tune activity for basic or applied synthetic biology. Less
Broadly HIV- neutralizing VRC -class antibodies bind the CD -binding site of Env and contain VH - derived heavy chains paired with light chains expressing five amino acid long CDRL s Their unmutated germline forms do not recognize HIV- Env and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding na ve B cells by the vaccine immunogens To address this point we examined Env-specific B cell receptor sequences from participants in the HVTN clinical trial Of all the sequences analyzed only one displayed homology to VRC -class antibodies but ... More
Broadly HIV-1–neutralizing VRC01-class antibodies bind the CD4-binding site of Env and contain VH1-2*02–derived heavy chains paired with light chains expressing five–amino acid–long CDRL3s. Their unmutated germline forms do not recognize HIV-1 Env, and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding naïve B cells by the vaccine immunogens. To address this point, we examined Env-specific B cell receptor sequences from participants in the HVTN 100 clinical trial. Of all the sequences analyzed, only one displayed homology to VRC01-class antibodies, but the corresponding antibody (FH1) recognized the C1C2 gp120 domain. For FH1 to switch epitope recognition to the CD4-binding site, alterations in the CDRH3 and CDRL3 were necessary. Only germ line–targeting Env immunogens efficiently activated VRC01 B cells, even in the presence of FH1 B cells. Our findings support the use of these immunogens to activate VRC01 B cells in humans. Less
The dysregulation of the PRC complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype CBX a key component of the canonical PRC complex is an epigenetic reader recognizing trimethylated lysine on histone H K me and is overexpressed in metastatic neuroendocrine prostate cancer We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX binding to chromatin Construct design and phosphorylation state of CBX were critical for successful implementation and execution of an HTS library screen A rigorous screening funnel including counter and selectivity assays allowed us to ... More
The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively. Less
Chemically induced dimerization CID is used to induce proximity and result in artificial complex formation between a pair of proteins involved in biological processes in cells to investigate and regulate these processes The induced heterodimerization of FKBP fusion proteins by rapamycin and FK has been extensively exploited as a chemically induced dimerization system to regulate and understand highly dynamic cellular processes Here we report the crystal structure of the AtFKBP FKBD in complex with rapamycin The crystal packing reveals an unusual feature whereby two rapamycin molecules appear to mediate homodimerization of the FKBD The triene arm of rapamycin appears to ... More
Chemically induced dimerization (CID) is used to induce proximity and result in artificial complex formation between a pair of proteins involved in biological processes in cells to investigate and regulate these processes. The induced heterodimerization of FKBP fusion proteins by rapamycin and FK506 has been extensively exploited as a chemically induced dimerization system to regulate and understand highly dynamic cellular processes. Here, we report the crystal structure of the AtFKBP53 FKBD in complex with rapamycin. The crystal packing reveals an unusual feature whereby two rapamycin molecules appear to mediate homodimerization of the FKBD. The triene arm of rapamycin appears to play a significant role in forming this dimer. This forms the first structural report of rapamycin-mediated homodimerization of an FKBP. The structural information on the rapamycin-mediated FKBD dimerization may be employed to design and synthesize covalently linked dimeric rapamycin, which may subsequently serve as a chemically induced dimerization system for the regulation and characterization of cellular processes. Less
In the last decade zebrafish have accompanied the mouse as a robust animal model for cancer research The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable However the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive easy and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition ... More
In the last decade, zebrafish have accompanied the mouse as a robust animal model for cancer research. The possibility of screening small-molecule inhibitors in a large number of zebrafish embryos makes this model particularly valuable. However, the dynamic visualization of fluorescently labeled tumor cells needs to be complemented by a more sensitive, easy, and rapid mode for evaluating tumor growth in vivo to enable high-throughput screening of clinically relevant drugs. In this study we proposed and validated a pre-clinical screening model for drug discovery by utilizing bioluminescence as our readout for the determination of transplanted cancer cell growth and inhibition in zebrafish embryos. For this purpose, we used NanoLuc luciferase, which ensured rapid cancer cell growth quantification in vivo with high sensitivity and low background when compared to conventional fluorescence measurements. This allowed us large-scale evaluation of in vivo drug responses of 180 kinase inhibitors in zebrafish. Our bioluminescent screening platform could facilitate identification of new small-molecules for targeted cancer therapy as well as for drug repurposing. Less
Although monoclonal antibodies mAbs have been shown to be extremely effective in treating a number of diseases they often suffer from poor developability attributes such as high viscosity and low solubility at elevated concentrations Since experimental candidate screening is often materials and labor intensive there is substantial interest in developing in silico tools for expediting mAb design Here we present a strategy using machine learning-based QSAR models for the a priori estimation of mAb solubility The extrapolated protein solubilities of a set of antibodies in a histidine buffer were determined using a high throughput PEG precipitation assay D homology models ... More
Although monoclonal antibodies (mAbs) have been shown to be extremely effective in treating a number of diseases, they often suffer from poor developability attributes, such as high viscosity and low solubility at elevated concentrations. Since experimental candidate screening is often materials and labor intensive, there is substantial interest in developing in silico tools for expediting mAb design. Here, we present a strategy using machine learning-based QSAR models for the a priori estimation of mAb solubility. The extrapolated protein solubilities of a set of 111 antibodies in a histidine buffer were determined using a high throughput PEG precipitation assay. 3D homology models of the antibodies were determined, and a large set of in house and commercially available molecular descriptors were then calculated. The resulting experimental and descriptor data were then used for the development of QSAR models of mAb solubilities. After feature selection and training with different machine learning algorithms, the models were evaluated with external test sets. The resulting regression models were able to estimate the solubility values of external test set data with R2 of 0.81 and 0.85 for the two regression models developed. In addition, three class and binary classification models were developed and shown to be good estimators of mAb solubility behavior, with overall test set accuracies of 0.70 and 0.95, respectively. The analysis of the selected molecular descriptors in these models was also found to be informative and suggested that several charge-based descriptors and isotype may play important roles in mAb solubility. The combination of high throughput relative solubility experimental techniques in concert with efficient machine learning QSAR models offers an opportunity to rapidly screen potential mAb candidates and to design therapeutics with improved solubility characteristics. Less
Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications Bilirubin oxidase BOD is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials BOD has been extensively investigated over the last few decades While the enzyme s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated a crystal structure of BOD from bacterial origin has never been determined Here we present the first crystal structure of BOD from Bacillus pumilus BpBOD at resolution Overall BpBOD shows high homology with the fungal enzymes however it holds a ... More
Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications. Bilirubin oxidase (BOD) is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials. BOD has been extensively investigated over the last few decades. While the enzyme’s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated, a crystal structure of BOD from bacterial origin has never been determined. Here we present the first crystal structure of BOD from Bacillus pumilus (BpBOD) at 3.5 Å resolution. Overall, BpBOD shows high homology with the fungal enzymes; however, it holds a unique surface-exposed disulfide bond between Cys229 and Cys322 residues. We present methodologies to orient the T1 site towards the electrode by coupling the reduced disulfide bond with maleimide moiety on the electrodes. The developed configurations were further investigated and revealed improved direct electron transfer rates with the electrodes. The work presented here may contribute to the construction of rationally designed bioanodes or biocathode configurations that are based on redox-active enzymes. Less
Understanding macrophage heterogeneity in tissue repair is a major challenge Here we describe a protocol that combines isolation of immune cells from skin wounds with subsequent flow-cytometry-based sorting of wound macrophages and single-cell RNA sequencing We use a modified version of the original Smart-seq protocol to increase speed and accuracy This protocol is useful for analyzing the pronounced heterogeneity of activation phenotypes in wound macrophages and might be adapted to other experimental models of skin inflammation
In this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins MPs at a molecular level High pressure atmospheres of argon and krypton were used to produce noble gas derivatives of crystals of three well studied MPs two different proton pumps and a sodium light-driven ion pump The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs a surface usually accommodating hydrophobic chains of annular lipids which are known structural and functional determinants for MPs In conformity ... More
In this work we examine how small hydrophobic molecules such as inert gases interact with membrane proteins (MPs) at a molecular level. High pressure atmospheres of argon and krypton were used to produce noble gas derivatives of crystals of three well studied MPs (two different proton pumps and a sodium light-driven ion pump). The structures obtained using X-ray crystallography showed that the vast majority of argon and krypton binding sites were located on the outer hydrophobic surface of the MPs – a surface usually accommodating hydrophobic chains of annular lipids (which are known structural and functional determinants for MPs). In conformity with these results, supplementary in silico molecular dynamics (MD) analysis predicted even greater numbers of argon and krypton binding positions on MP surface within the bilayer. These results indicate a potential importance of such interactions, particularly as related to the phenomenon of noble gas-induced anaesthesia. Less
Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past years The most current and widespread of these is SARS-CoV- The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection We previously identified a neutralizing monoclonal antibody CV - that binds to the SARS-CoV- spike neutralizes the SARS-CoV- Beta variant comparably to the ancestral Wuhan Hu- ... More
Three betacoronaviruses have crossed the species barrier and established human-to-human transmission causing significant morbidity and mortality in the past 20 years. The most current and widespread of these is SARS-CoV-2. The identification of CoVs with zoonotic potential in animal reservoirs suggests that additional outbreaks could occur. Monoclonal antibodies targeting conserved neutralizing epitopes on diverse CoVs can form the basis for prophylaxis and therapeutic treatments and enable the design of vaccines aimed at providing pan-CoV protection. We previously identified a neutralizing monoclonal antibody, CV3-25 that binds to the SARS-CoV-2 spike, neutralizes the SARS-CoV-2 Beta variant comparably to the ancestral Wuhan Hu-1 strain, cross neutralizes SARS-CoV-1 and binds to recombinant proteins derived from the spike-ectodomains of HCoV-OC43 and HCoV-HKU1. Here, we show that the neutralizing activity of CV3-25 is maintained against the Alpha, Delta, Gamma and Omicron variants of concern as well as a SARS-CoV-like bat coronavirus with zoonotic potential by binding to a conserved linear peptide in the stem-helix region. Negative stain electron microscopy and a 1.74 Å crystal structure of a CV3-25/peptide complex demonstrates that CV3-25 binds to the base of the stem helix at the HR2 boundary to an epitope that is distinct from other stem-helix directed neutralizing mAbs. Less
Synthetic biology efforts for cannabinoid research have seen a rapid expansion in recent years This is in response to the increasing awareness and legalization of the secondary metabolites from Cannabis sativa dubbed the green rush In transgenic synthetic biology applications NphB is a promiscuous prenyltransferase from Streptomyces sp often used as a replacement in the prenylation step producing the cannabinoid cannabigerolic acid CBGA the key precursor to many other cannabinoids However its application as a CBGA synthase replacement is limited by its nonspecific regioselectivity in producing a side product along with CBGA Herein we demonstrated a detailed and extensive computational ... More
Synthetic biology efforts for cannabinoid research have seen a rapid expansion in recent years. This is in response to the increasing awareness and legalization of the secondary metabolites from Cannabis sativa, dubbed the green rush. In transgenic synthetic biology applications, NphB is a promiscuous prenyltransferase from Streptomyces sp. often used as a replacement in the prenylation step producing the cannabinoid cannabigerolic acid (CBGA), the key precursor to many other cannabinoids. However, its application as a CBGA synthase replacement is limited by its nonspecific regioselectivity in producing a side product along with CBGA. Herein, we demonstrated a detailed and extensive computational structure-guided approach in identifying target residues of mutation for engineering NphB for optimal CBGA production. Our comprehensive computational workflow has led to the discovery of several highly regiospecific variants that produce CBGA exclusively, with the best-performing V49W/Y288P variant having a 13.6-fold yield improvement, outperforming all previous work on NphB enzyme engineering. We subsequently investigated the effects of these mutations by X-ray crystallographic studies of the mutant variants and performed molecular dynamics simulations to uncover an interplay of a H-bonding network and an optimal ligand orientation that favors the CBGA production over the side product. Collectively, this study not only recapitulates the utility of computational tools in informing and accelerating experimental design but also contributes to a better understanding of molecular mechanisms that govern enzyme regioselectivity and readily aids in cannabinoid synthetic biology production for future research into maximizing their therapeutic potential. Less
Continuous flow injection is a key technology for serial crystallography measurements of protein crystals suspended in the lipidic cubic phase LCP To date there has been little discussion in the literature regarding the impact of the injection process itself on the structure of the lipidic phase This is despite the fact that the phase of the injection matrix is critical for the flow properties of the stream and potentially for sample stability Here we report small-angle X-ray scattering measurements of a monoolein water mixture during continuous delivery using a high viscosity injector We observe both an alignment and modification of ... More
Continuous flow injection is a key technology for serial crystallography measurements of protein crystals suspended in the lipidic cubic phase (LCP). To date, there has been little discussion in the literature regarding the impact of the injection process itself on the structure of the lipidic phase. This is despite the fact that the phase of the injection matrix is critical for the flow properties of the stream and potentially for sample stability. Here we report small-angle X-ray scattering measurements of a monoolein:water mixture during continuous delivery using a high viscosity injector. We observe both an alignment and modification of the LCP as a direct result of the injection process. The orientation of the cubic lattice with respect to the beam was estimated based on the anisotropy of the diffraction pattern and does not correspond to a single low order zone axis. The solvent fraction was also observed to impact the stability of the cubic phase during injection. In addition, depending on the distance traveled by the lipid after exiting the needle, the phase is observed to transition from a pure diamond phase () to a mixture containing both gyriod () and lamellar () phases. Finite element modelling of the observed phase behaviour during injection indicates that the pressure exerted on the lipid stream during extrusion accounts for the variations in the phase composition of the monoolein:water mixture. Less
Detailed description of the mechanism of action of the therapeutic antibodies is essential for the functional characterization and future optimization of potential clinical agents We recently developed KD a fully human antibody targeting vascular endothelial growth factor receptor VEGFR KD blocked VEGF-A and VEGF-C-mediated VEGFR activation as demonstrated by the in vitro binding and competition assays and functional cellular assays Here we report a computational model of the complex between the variable fragment of KD KD Fv and the domains and of the extracellular portion of VEGFR VEGFR D - Our modeling was guided by a priori experimental information including ... More
Detailed description of the mechanism of action of the therapeutic antibodies is essential for the functional characterization and future optimization of potential clinical agents. We recently developed KD035, a fully human antibody targeting vascular endothelial growth factor receptor 2 (VEGFR2). KD035 blocked VEGF-A, and VEGF-C-mediated VEGFR2 activation, as demonstrated by the in vitro binding and competition assays and functional cellular assays. Here, we report a computational model of the complex between the variable fragment of KD035 (KD035(Fv)) and the domains 2 and 3 of the extracellular portion of VEGFR2 (VEGFR2(D2-3)). Our modeling was guided by a priori experimental information including the X-ray structures of KD035 and related antibodies, binding assays, target domain mapping and comparison of KD035 affinity for VEGFR2 from different species. The accuracy of the model was assessed by molecular dynamics simulations, and subsequently validated by mutagenesis and binding analysis. Importantly, the steps followed during the generation of this model can set a precedent for future in silico efforts aimed at the accurate description of the antibody–antigen and more broadly protein–protein complexes. Less
The environmental microbiome harbors a vast repertoire of antibiotic resistance genes ARGs which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria or can be directly mobilized to pathogens in the presence of selection pressures Thus ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance Here we conduct a comprehensive functional analysis of the Antibiotic NAT family of aminoglycoside acetyltransferases We determined a pan-family antibiogram of Antibiotic NAT enzymes including derived from clinical isolates and from environmental metagenomic samples We find that environment-derived representatives confer high-level broad-spectrum resistance including against the atypical aminoglycoside ... More
The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an aac(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment. Less
Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and are considered promising drug targets for cancer and fibrotic diseases The catalytic ADP-ribosyltransferase domain of tankyrases contains a unique zinc-binding motif of unknown function Recently this motif was suggested to be involved in the catalytic activity of tankyrases In this work we set out to study the effect of the zinc-binding motif on the activity stability and structure of human tankyrases We generated mutants of human tankyrase TNKS and TNKS abolishing the zinc-binding capabilities and characterized the proteins biochemically and biophysically in vitro We further generated a ... More
Tankyrases are ADP-ribosylating enzymes that regulate many physiological processes in the cell and are considered promising drug targets for cancer and fibrotic diseases. The catalytic ADP-ribosyltransferase domain of tankyrases contains a unique zinc-binding motif of unknown function. Recently, this motif was suggested to be involved in the catalytic activity of tankyrases. In this work, we set out to study the effect of the zinc-binding motif on the activity, stability and structure of human tankyrases. We generated mutants of human tankyrase (TNKS) 1 and TNKS2, abolishing the zinc-binding capabilities, and characterized the proteins biochemically and biophysically in vitro. We further generated a crystal structure of TNKS2, in which the zinc ion was oxidatively removed. Our work shows that the zinc-binding motif in tankyrases is a crucial structural element which is particularly important for the structural integrity of the acceptor site. While mutation of the motif rendered TNKS1 inactive, probably due to introduction of major structural defects, the TNKS2 mutant remained active and displayed an altered activity profile compared to the wild-type. Less
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold They are generally described as electron carriers that rely on a T copper center coordinated by four ligands provided by the folded polypeptide The discovery of novel cupredoxins demonstrates the high diversity of this family with variations in term of copper-binding ligands copper center geometry redox potential as well as biological function AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans AcoP presents original features highly resistant to acidic pH it possesses a constrained green-type copper center of high ... More
Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper center coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in term of copper-binding ligands, copper center geometry, redox potential, as well as biological function. AcoP is a periplasmic protein belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans . AcoP presents original features: highly resistant to acidic pH, it possesses a constrained green-type copper center of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structure of AcoP at 1.65 Å resolution unveils a typical cupredoxin fold with extended loops, never observed in previously characterized cupredoxins, that might be involved in the interaction of AcoP with its physiological partners. Moreover, the structure shows that the green color of AcoP cannot be attributed to nonclassical copper ligands, its green-colored copper center raising from a long Cu-S (Cys) bond, determined by both X-ray diffraction and EXAFS. The crystal structures of two AcoP mutants confirm that the active center of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active center rigidity due to the presence of an extensive hydrogen bond network. Less
Gut inflammation directly impacts the growth and stability of commensal gut microbes and can lead to long-lasting changes in microbiota composition that can prolong or exacerbate disease states While mouse models are used extensively to investigate the interplay between microbes and the inflamed state the paucity of cultured mouse gut microbes has hindered efforts to determine causal relationships To address this issue we are assembling the Collection of Inflammation-Associated Mouse Intestinal Bacteria CIAMIB The initial release of this collection comprises isolates of unique bacterial species covering phyla and containing previously uncultivated isolates including novel family and novel genera The collection ... More
Gut inflammation directly impacts the growth and stability of commensal gut microbes and can lead to long-lasting changes in microbiota composition that can prolong or exacerbate disease states. While mouse models are used extensively to investigate the interplay between microbes and the inflamed state, the paucity of cultured mouse gut microbes has hindered efforts to determine causal relationships. To address this issue, we are assembling the Collection of Inflammation-Associated Mouse Intestinal Bacteria (CIAMIB). The initial release of this collection comprises 41 isolates of 39 unique bacterial species, covering 4 phyla and containing 10 previously uncultivated isolates, including 1 novel family and 7 novel genera. The collection significantly expands the number of available Muribaculaceae, Lachnospiraceae, and Coriobacteriaceae isolates and includes microbes from genera associated with inflammation, such as Prevotella and Klebsiella. We characterized the growth of CIAMIB isolates across a diverse range of nutritional conditions and predicted their metabolic potential and anaerobic fermentation capacity based on the genomes of these isolates. We also provide the first metabolic analysis of species within the genus Adlercreutzia, revealing these representatives to be nitrate-reducing and severely restricted in their ability to grow on carbohydrates. CIAMIB isolates are fully sequenced and available to the scientific community as a powerful tool to study host-microbiota interactions. Less
Intrinsically disordered proteins IDPs can coordinate often transient or weak interactions with multiple proteins to mediate complex signals within large reversible protein networks Among these the IDP hub protein G BP forms protein complexes with Caprin and USP and the resulting control of USP activity plays an important role in a pathogenic virulence system that targets CFTR endocytic recycling However while the identities of protein interactors are known for many of these 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 the ... More
Intrinsically disordered proteins (IDPs) can coordinate often transient or weak interactions with multiple proteins to mediate complex signals within large, reversible protein networks. Among these, the IDP hub protein G3BP1 forms protein complexes with Caprin1 and USP10, and the resulting control of USP10 activity plays an important role in a pathogenic virulence system that targets CFTR endocytic recycling. However, while the identities of protein interactors are known for many of these 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 the G3BP1 affinities, and show that substitution of G3BP1 residues F15 or F33 to tryptophan reduces affinity for both the USP10 and Caprin1 motif peptides. These same mutations significantly reduce formation of complexes by the full-length proteins. 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 the USP10 and Caprin1 peptides. An amino-acid scan of the USP10 and Caprin1 motif peptides reveals similarities and differences in the ability to substitute residues in the core motifs as well as specific mutations with the potential to create higher affinity peptides. Taken together, these data show that small changes in 1:1 binding affinity 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 emergence of several zoonotic viruses in the last twenty years especially the pandemic outbreak of SARS-CoV- has exposed a dearth of antiviral drug therapies for viruses with pandemic potential Developing a diverse drug portfolio will be critical for our ability to rapidly respond to novel coronaviruses CoVs and other viruses with pandemic potential Here we focus on the SARS-CoV- conserved macrodomain Mac a small domain of non-structural protein nsp Mac is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose MAR from target proteins protects the virus from the anti-viral effects of host ADP-ribosyltransferases and is critical for the replication and pathogenesis of ... More
The emergence of several zoonotic viruses in the last twenty years, especially the pandemic outbreak of SARS-CoV-2, has exposed a dearth of antiviral drug therapies for viruses with pandemic potential. Developing a diverse drug portfolio will be critical for our ability to rapidly respond to novel coronaviruses (CoVs) and other viruses with pandemic potential. Here we focus on the SARS-CoV-2 conserved macrodomain (Mac1), a small domain of non-structural protein 3 (nsp3). Mac1 is an ADP-ribosylhydrolase that cleaves mono-ADP-ribose (MAR) from target proteins, protects the virus from the anti-viral effects of host ADP-ribosyltransferases, and is critical for the replication and pathogenesis of CoVs. In this study, a luminescent-based high-throughput assay was used to screen ∼38,000 small molecules for those that could inhibit Mac1-ADP-ribose binding. We identified 5 compounds amongst 3 chemotypes that inhibit SARS-CoV-2 Mac1-ADP-ribose binding in multiple assays with IC50 values less than 100µM, inhibit ADP-ribosylhydrolase activity, and have evidence of direct Mac1 binding. These chemotypes are strong candidates for further derivatization into highly effective Mac1 inhibitors. Less
A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange altering the on off switch that forms the basis for their signaling functions Using X-ray crystallography nuclear magnetic resonance spectroscopy binding assays and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site ... More
A unifying feature of the RAS superfamily is a conserved GTPase cycle by which these proteins transition between active and inactive states. We demonstrate that autophosphorylation of some GTPases is an intrinsic regulatory mechanism that reduces nucleotide hydrolysis and enhances nucleotide exchange, altering the on/off switch that forms the basis for their signaling functions. Using X-ray crystallography, nuclear magnetic resonance spectroscopy, binding assays, and molecular dynamics on autophosphorylated mutants of H-RAS and K-RAS, we show that phosphoryl transfer from GTP requires dynamic movement of the switch II region and that autophosphorylation promotes nucleotide exchange by opening the active site and extracting the stabilizing Mg2+. Finally, we demonstrate that autophosphorylated K-RAS exhibits altered effector interactions, including a reduced affinity for RAF proteins in mammalian cells. Thus, autophosphorylation leads to altered active site dynamics and effector interaction properties, creating a pool of GTPases that are functionally distinct from their non-phosphorylated counterparts. Less
Over the last two decades fragment-based drug discovery FBDD has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods However it is also often used with great success as a primary screening technique In recent years technological advances at macromolecular crystallo graphy beamlines in terms of instrumentation beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography Here the development of ... More
Over the last two decades, fragment-based drug discovery (FBDD) has emerged as an effective and efficient method to identify new chemical scaffolds for the development of lead compounds. X-ray crystallography can be used in FBDD as a tool to validate and develop fragments identified as binders by other methods. However, it is also often used with great success as a primary screening technique. In recent years, technological advances at macromolecular crystallography beamlines in terms of instrumentation, beam intensity and robotics have enabled the development of dedicated platforms at synchrotron sources for FBDD using X-ray crystallography. Here, the development of the Fast Fragment and Compound Screening (FFCS) platform, an integrated next-generation pipeline for crystal soaking, handling and data collection which allows crystallography-based screening of protein crystals against hundreds of fragments and compounds, at the Swiss Light Source is reported. Less
L is a potent human monoclonal antibody mAb that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein PfCSP on malaria-infective sporozoites Understanding this mAb's ontogeny and mechanisms of binding PfCSP will facilitate vaccine development Here we isolate mAbs clonally related to L and show that this B cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity Pairing the L kappa light chain L with clonally related heavy chains results in chimeric mAbs that cross-link two NVDPs cross-react with NANP and more potently neutralize sporozoites in vivo ... More
L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAb's ontogeny and mechanisms of binding PfCSP will facilitate vaccine development. Here, we isolate mAbs clonally related to L9 and show that this B cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9κ) with clonally related heavy chains results in chimeric mAbs that cross-link two NVDPs, cross-react with NANP, and more potently neutralize sporozoites in vivo compared with their original light chain. Structural analyses reveal that the chimeric mAbs bound minor repeats in a type-1 β-turn seen in other repeat-specific antibodies. These data highlight the importance of L9κ in binding NVDP on PfCSP to neutralize sporozoites and suggest that PfCSP-based immunogens might be improved by presenting ≥2 NVDPs. Less
The human chemokines CCL and CCL bind to the G protein-coupled receptor GPCR CCR and play an important role in the trafficking of immune cells as well as cancer metastasis Conserved binding sites for sulfotyrosine residues on the receptor contribute significantly to the chemokine GPCR interaction and have been shown to provide promising targets for new drug-discovery efforts to disrupt the chemokine GPCR interaction and consequently tumor metastasis Here we report the first X-ray crystal structure of a truncated CCL residues at resolution revealing molecular details crucial for protein protein interactions Although the overall structure is similar to the previously ... More
The human chemokines CCL19 and CCL21 bind to the G protein-coupled receptor (GPCR) CCR7 and play an important role in the trafficking of immune cells as well as cancer metastasis. Conserved binding sites for sulfotyrosine residues on the receptor contribute significantly to the chemokine/GPCR interaction and have been shown to provide promising targets for new drug-discovery efforts to disrupt the chemokine/GPCR interaction and, consequently, tumor metastasis. Here, we report the first X-ray crystal structure of a truncated CCL19 (residues 7–70) at 2.50 Å resolution, revealing molecular details crucial for protein–protein interactions. Although the overall structure is similar to the previously determined NMR model, there are important variations, particularly near the N terminus and the so-called 30’s and 40’s loops. Computational analysis using the FTMap server indicates the potential importance of these areas in ligand binding and the differences in binding hotspots compared to CCL21. NMR titration experiments using a CCR7-derived peptide (residues 5–11, TDDYIGD) further demonstrate potential receptor recognition sites, such as those near the C terminus and 40’s loop, which consist of both positively charged and hydrophobic residues that may be important for receptor binding. Taken together, the X-ray, NMR, and computational analysis herein provide insights into the overall structure and molecular features of CCL19 and enables investigation into this chemokine’s function and inhibitor development. Less
Chromatin abnormalities are common hallmarks of cancer cells which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes These epigenetic patterns are partly established and maintained by UHRF ubiquitin-like PHD and RING finger domain-containing protein which senses existing methylation states through multiple reader domains and reinforces the modifications through recruitment of DNA methyltransferases Small molecule inhibitors of UHRF would be important tools to illuminate molecular functions yet no compounds capable of blocking UHRF -histone binding in the context of the full-length protein exist Here we report the discovery and mechanism of action of compounds that selectively inhibit ... More
Chromatin abnormalities are common hallmarks of cancer cells, which exhibit alterations in DNA methylation profiles that can silence tumor suppressor genes. These epigenetic patterns are partly established and maintained by UHRF1 (ubiquitin-like PHD and RING finger domain-containing protein 1), which senses existing methylation states through multiple reader domains, and reinforces the modifications through recruitment of DNA methyltransferases. Small molecule inhibitors of UHRF1 would be important tools to illuminate molecular functions, yet no compounds capable of blocking UHRF1-histone binding in the context of the full-length protein exist. Here, we report the discovery and mechanism of action of compounds that selectively inhibit the UHRF1-histone interaction with low micromolar potency. Biochemical analyses reveal that these molecules are the first inhibitors to target the PHD finger of UHRF1, specifically disrupting histone H3 arginine 2 interactions with the PHD finger. Importantly, this unique inhibition mechanism is sufficient to displace binding of full-length UHRF1 with histones in vitro and in cells. Together, our study provides insight into the critical role of the PHD finger in driving histone interactions, and demonstrates that targeting this domain through a specific binding pocket is a tractable strategy for UHRF1-histone inhibition. Less
Structural studies of G-protein-coupled receptors GPCRs are often limited by difficulties in obtaining well-diffracting crystals suitable for high-resolution structure determination During the past decade crystallization in lipidic cubic phase LCP has become the most successful and widely used technique for obtaining such crystals Despite often intense efforts many GPCRs remain refractory to crystallization even if receptors can be purified in sufficient amounts To address this issue we have developed a highly efficient screening and stabilization strategy for GPCRs based on a fluorescence thermal stability assay readout which seems to correlate particularly well with those GPCR constructs that remain native during ... More
Structural studies of G-protein-coupled receptors (GPCRs) are often limited by difficulties in obtaining well-diffracting crystals suitable for high-resolution structure determination. During the past decade, crystallization in lipidic cubic phase (LCP) has become the most successful and widely used technique for obtaining such crystals. Despite often intense efforts, many GPCRs remain refractory to crystallization, even if receptors can be purified in sufficient amounts. To address this issue, we have developed a highly efficient screening and stabilization strategy for GPCRs, based on a fluorescence thermal stability assay readout, which seems to correlate particularly well with those GPCR constructs that remain native during incorporation into the LCP. Detailed protocols are provided for rapid and cost-efficient mutant and construct generation using sequence- and ligation-independent cloning, high-throughput magnetic bead-based protein purification from small-scale expressions in mammalian cells, the screening and optimal combination of mutations for increased receptor thermostability and the rapid identification of suitable chimeric fusion protein constructs for successful crystallization in LCP. We exemplify the method on three receptors from two different classes: the neurokinin 1 receptor, the oxytocin receptor and the parathyroid hormone 1 receptor. Less
Macrophage migration inhibitory factor MIF is an inflammatory protein with various non-overlapping functions It is not only conserved in mammals but it is found in parasites fish and plants Human MIF is a homotrimer with an enzymatic cavity between two subunits with Pro as a catalytic base activates the receptors CD CXCR and CXCR has functional interactions in the cytosol and is reported to be a nuclease There is a solvent channel down its -fold axis with a recently identified gating residue as an allosteric site important for regulating to different extents the enzymatic activity and CD binding and signaling ... More
Macrophage migration inhibitory factor (MIF) is an inflammatory protein with various non-overlapping functions. It is not only conserved in mammals, but it is found in parasites, fish, and plants. Human MIF is a homotrimer with an enzymatic cavity between two subunits with Pro1 as a catalytic base, activates the receptors CD74, CXCR2, and CXCR4, has functional interactions in the cytosol, and is reported to be a nuclease. There is a solvent channel down its 3-fold axis with a recently identified gating residue as an allosteric site important for regulating, to different extents, the enzymatic activity and CD74 binding and signaling. In this study we explore the consequence of converting the allosteric residue Tyr99 to cysteine (Y99C) and characterize its crystallographic structure, NMR dynamics, stability, CD74 function, and enzymatic activity. In addition to the homotrimeric variant, we develop strategies for expressing and purifying a heterotrimeric variant consisting of mixed wild type and Y99C for characterization of the allosteric site to provide more insight. Less
Many animal species are susceptible to severe acute respiratory syndrome coronavirus SARS-CoV- infection and could act as reservoirs however transmission in free-living animals has not been documented White-tailed deer the predominant cervid in North America are susceptible to SARS-CoV- infection and experimentally infected fawns can transmit the virus To test the hypothesis that SARS-CoV- is circulating in deer retropharyngeal lymph node RPLN samples collected from free-living and captive deer in Iowa from April through January of were assayed for the presence of SARS-CoV- RNA Ninety-four of the deer samples were positive for SARS-CoV- RNA as assessed by RT-PCR Notably following ... More
Many animal species are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and could act as reservoirs; however, transmission in free-living animals has not been documented. White-tailed deer, the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 is circulating in deer, 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through January of 2021 were assayed for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 (33.2%) deer samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, following the November 2020 peak of human cases in Iowa, and coinciding with the onset of winter and the peak deer hunting season, SARS-CoV-2 RNA was detected in 80 of 97 (82.5%) RPLN samples collected over a 7-wk period. Whole genome sequencing of all 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%) and B.1.311 (n = 19; 20%) accounting for ∼75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple human-to-deer transmission events followed by subsequent deer-to-deer spread. These discoveries have important implications for the long-term persistence of the SARS-CoV-2 pandemic. Our findings highlight an urgent need for a robust and proactive “One Health” approach to obtain enhanced understanding of the ecology, molecular evolution, and dissemination of SARS-CoV-2. Less
Tousled-like kinases TLKs are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants A major function of TLKs is to phosphorylate the histone chaperone proteins ASF a and ASF b to facilitate DNA replication-coupled nucleosome assembly but how TLKs selectively target these critical substrates is unknown Here we show that TLK selectivity towards ASF substrates is achieved in two ways First the TLK catalytic domain recognizes consensus phosphorylation site motifs in the ASF C-terminal tail Second a short sequence at the TLK N-terminus docks onto the ASF a globular N-terminal domain in a manner ... More
Tousled-like kinases (TLKs) are nuclear serine-threonine kinases essential for genome maintenance and proper cell division in animals and plants. A major function of TLKs is to phosphorylate the histone chaperone proteins ASF1a and ASF1b to facilitate DNA replication-coupled nucleosome assembly, but how TLKs selectively target these critical substrates is unknown. Here, we show that TLK2 selectivity towards ASF1 substrates is achieved in two ways. First, the TLK2 catalytic domain recognizes consensus phosphorylation site motifs in the ASF1 C-terminal tail. Second, a short sequence at the TLK2 N-terminus docks onto the ASF1a globular N-terminal domain in a manner that mimics its histone H3 client. Disrupting either catalytic or non-catalytic interactions through mutagenesis hampers ASF1 phosphorylation by TLK2 and cell growth. Our results suggest that the stringent selectivity of TLKs for ASF1 is enforced by an unusual interaction mode involving mutual recognition of a short sequence motifs by both kinase and substrate. Less
Genetic variants of severe acute respiratory syndrome coronavirus SARS-CoV- have repeatedly altered the course of the coronavirus disease COVID- pandemic Delta variants are now the focus of intense international attention because they are causing widespread COVID- globally and are associated with vaccine breakthrough cases We sequenced SARS-CoV- genomes from samples acquired March through September in the Houston Methodist hospital system This sample represents of all Methodist system COVID- patients during the study period Delta variants increased rapidly from late April onward to cause of all COVID- cases and spread throughout the Houston metroplex Compared with all other variants combined Delta ... More
Genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have repeatedly altered the course of the coronavirus disease 2019 (COVID-19) pandemic. Delta variants are now the focus of intense international attention because they are causing widespread COVID-19 globally and are associated with vaccine breakthrough cases. We sequenced 16,965 SARS-CoV-2 genomes from samples acquired March 15, 2021, through September 20, 2021, in the Houston Methodist hospital system. This sample represents 91% of all Methodist system COVID-19 patients during the study period. Delta variants increased rapidly from late April onward to cause 99.9% of all COVID-19 cases and spread throughout the Houston metroplex. Compared with all other variants combined, Delta caused a significantly higher rate of vaccine breakthrough cases (23.7% for Delta compared with 6.6% for all other variants combined). Importantly, significantly fewer fully vaccinated individuals required hospitalization. Vaccine breakthrough cases caused by Delta had a low median PCR cycle threshold value (a proxy for high virus load). This value was similar to the median cycle threshold value for unvaccinated patients with COVID-19 caused by Delta variants, suggesting that fully vaccinated individuals can transmit SARS-CoV-2 to others. Patients infected with Alpha and Delta variants had several significant differences. The integrated analysis indicates that vaccines used in the United States are highly effective in decreasing severe COVID-19, hospitalizations, and deaths. Less
The metabolic activities of microbial communities play a defining role in the evolution and persistence of life on Earth driving redox reactions that give rise to global biogeochemical cycles Community metabolism emerges from a hierarchy of processes including gene expression ecological interactions and environmental factors In wild communities gene content is correlated with environmental context but predicting metabolite dynamics from genomes remains elusive Here we show for the process of denitrification that metabolite dynamics of a community are predictable from the genes each member of the community possesses A simple linear regression reveals a sparse and generalizable mapping from gene ... More
The metabolic activities of microbial communities play a defining role in the evolution and persistence of life on Earth, driving redox reactions that give rise to global biogeochemical cycles. Community metabolism emerges from a hierarchy of processes, including gene expression, ecological interactions, and environmental factors. In wild communities, gene content is correlated with environmental context, but predicting metabolite dynamics from genomes remains elusive. Here, we show, for the process of denitrification, that metabolite dynamics of a community are predictable from the genes each member of the community possesses. A simple linear regression reveals a sparse and generalizable mapping from gene content to metabolite dynamics for genomically diverse bacteria. A consumer-resource model correctly predicts community metabolite dynamics from single-strain phenotypes. Our results demonstrate that the conserved impacts of metabolic genes can predict community metabolite dynamics, enabling the prediction of metabolite dynamics from metagenomes, designing denitrifying communities, and discovering how genome evolution impacts metabolism. Less
The nonpolymorphic class Ib molecule HLA-E primarily presents peptides from HLA class Ia leader peptides providing an inhibitory signal to NK cells via CD NKG interactions Although peptides of pathogenic origin can also be presented by HLA-E to T cells the molecular basis underpinning their role in antigen surveillance is largely unknown Here we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide pHLA-E from bacterial Mycobacterium tuberculosis origin and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral HIV origin The structures provided a molecular foundation to develop a novel method to introduce ... More
The nonpolymorphic class Ib molecule, HLA-E, primarily presents peptides from HLA class Ia leader peptides, providing an inhibitory signal to NK cells via CD94/NKG2 interactions.
Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral (HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes while maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E-specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs. Less
Although peptides of pathogenic origin can also be presented by HLA-E to T cells, the molecular basis underpinning their role in antigen surveillance is largely unknown. Here, we solved a co-complex crystal structure of a TCR with an HLA-E presented peptide (pHLA-E) from bacterial (Mycobacterium tuberculosis) origin, and the first TCR-pHLA-E complex with a noncanonically presented peptide from viral (HIV) origin. The structures provided a molecular foundation to develop a novel method to introduce cysteine traps using non-natural amino acid chemistry that stabilized pHLA-E complexes while maintaining native interface contacts between the TCRs and different pHLA-E complexes. These pHLA-E monomers could be used to isolate pHLA-E-specific T cells, with obvious utility for studying pHLA-E restricted T cells, and for the identification of putative therapeutic TCRs. Less
Pathogen effectors are crucial players during plant colonisation and infection Plant resistance mostly relies on effector recognition to activate defence responses Understanding how effector proteins escape from plant surveillance is important for plant breeding and resistance deployment Here we examined the role of genetic diversity of the stem rust Puccinia graminis f sp tritici Pgt AvrSr gene in determining recognition by the corresponding wheat Sr resistance gene We solved the crystal structure of a natural variant of AvrSr and used site-directed mutagenesis and transient expression assays to dissect the molecular mechanisms explaining gain of virulence We report that AvrSr can ... More
Pathogen effectors are crucial players during plant colonisation and infection. Plant resistance mostly relies on effector recognition to activate defence responses. Understanding how effector proteins escape from plant surveillance is important for plant breeding and resistance deployment. Here we examined the role of genetic diversity of the stem rust (Puccinia graminis f. sp. tritici (Pgt)) AvrSr50 gene in determining recognition by the corresponding wheat Sr50 resistance gene. We solved the crystal structure of a natural variant of AvrSr50 and used site-directed mutagenesis and transient expression assays to dissect the molecular mechanisms explaining gain of virulence. We report that AvrSr50 can escape recognition by Sr50 through different mechanisms including DNA insertion, stop codon loss or by amino-acid variation involving a single substitution of the AvrSr50 surface-exposed residue Q121. We also report structural homology of AvrSr50 to cupin superfamily members and carbohydrate-binding modules indicating a potential role in binding sugar moieties. This study identifies key polymorphic sites present in AvrSr50 alleles from natural stem rust populations that play important roles to escape from Sr50 recognition. This constitutes an important step to better understand Pgt effector evolution and to monitor AvrSr50 variants in natural rust populations. Less
Despite advances in spatial transcriptomics the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge We present a method termed behavioral transcriptomics that allows us to couple physiological behaviors of single cells in an intact tissue to deep molecular profiling of individual cells This method enabled us to establish a novel molecular signature for a striking migratory cellular behavior following tissue injury
Here we describe a protocol to set up a screening assay for ADP-ribosyl binding proteins including proteins that possess O-glycosidase or N-glycosidase activities The FRET-based assay measures the interaction of any ADP-ribosyl binding protein fused to CFP with a cysteine-ADP-ribosylated GAP-tag fused to YFP Recombinant PtxS and PARP are used to mono-ADP-ribosylate and poly-ADP-ribosylate the GAP-tag The protocol does not require specialized compounds or substrates making it accessible and easy to adapt in any laboratory or for other proteins of interest
Squamous cell carcinoma SCC is a common type of skin cancer that typically arises from premalignant precursor lesions named actinic keratoses AK Chronic inflammation is a well-known promoter of skin cancer progression AK and SCC have been associated with an overabundance of the bacterium Staphylococcus aureus S aureus Certain secreted products from S aureus are known to promote cutaneous pro-inflammatory responses however not all S aureus strains produce these As inflammation plays a key role in SCC development we investigated the pro-inflammatory potential and toxin secretion profiles of skin-cancer associated S aureus Sterile culture supernatants secretomes of S aureus clinical ... More
Squamous cell carcinoma (SCC) is a common type of skin cancer that typically arises from premalignant precursor lesions named actinic keratoses (AK). Chronic inflammation is a well-known promoter of skin cancer progression. AK and SCC have been associated with an overabundance of the bacterium Staphylococcus aureus (S. aureus). Certain secreted products from S. aureus are known to promote cutaneous pro-inflammatory responses; however, not all S. aureus strains produce these. As inflammation plays a key role in SCC development, we investigated the pro-inflammatory potential and toxin secretion profiles of skin-cancer associated S. aureus. Sterile culture supernatants (“secretomes”) of S. aureus clinical strains isolated from AK and SCC were applied to human keratinocytes in vitro. Some S. aureus secretomes induced keratinocytes to overexpress inflammatory mediators that have been linked to skin carcinogenesis, including IL-6, IL-8, and TNFα. A large phenotypic variation between the tested clinical strains was observed. Strains that are highly pro-inflammatory in vitro also caused more pronounced skin inflammation in mice. Proteomic characterization of S. aureus secretomes using mass spectrometry established that specific S. aureus enzymes and cytolytic toxins, including hemolysins, phenol-soluble modulins, and serine proteases, as well as currently uncharacterized proteins, correlate with the pro-inflammatory S. aureus phenotype. This study is the first to describe the toxin secretion profiles of AK and SCC-associated S. aureus, and their potential to induce a pro-inflammatory environment in the skin. Further studies are needed to establish whether these S. aureus products promote SCC development by mediating chronic inflammation. Less
Retroviral elements from endogenous retroviruses have functions in mammalian physiology The best-known examples are the envelope proteins that function in placenta development and immune suppression Porcine endogenous retroviruses PERVs are an understudied class of endogenous retroviruses that infect cultured human cells raising concern regarding porcine xenografts The PERV envelope glycoprotein has also been proposed as a possible swine syncytin with a role in placental development Despite the growing interest in PERVs their envelope glycoproteins remain poorly characterized Here we successfully determined the postfusion crystal structure of the PERV core fusion ectodomain The PERV fusion protein structure reveals a conserved class ... More
Retroviral elements from endogenous retroviruses have functions in mammalian physiology. The best-known examples are the envelope proteins that function in placenta development and immune suppression. Porcine endogenous retroviruses (PERVs) are an understudied class of endogenous retroviruses that infect cultured human cells, raising concern regarding porcine xenografts. The PERV envelope glycoprotein has also been proposed as a possible swine syncytin with a role in placental development. Despite the growing interest in PERVs, their envelope glycoproteins remain poorly characterized. Here, we successfully determined the postfusion crystal structure of the PERV core fusion ectodomain. The PERV fusion protein structure reveals a conserved class I viral fusion protein six-helix bundle. Biophysical experiments demonstrated that the thermodynamic stability of the PERV fusion protein secondary structure was the same at physiological and acidic pHs. A conserved surface analysis highlights the high degree of sequence conservation among retroviral fusogens in the chain reversal region that facilitates the large-scale conformational change required for membrane fusion. Further structural alignment of class I viral fusogens revealed a phylogenetic clustering that shows evolution into various lineages that correlate with virus mechanisms of cell entry. Our work indicates that structural dendrograms can be used to qualitatively infer insights into the fusion mechanisms of newly discovered class I viral fusogen structures. Less
-adrenergic receptors ARs are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems The therapeutic potential of ARs however is largely unexploited and hampered by the scarcity of subtype-selective ligands Moreover several aminergic drugs either show off-target binding to ARs or fail to interact with the desired subtype Here we report the crystal structure of human BAR bound to the inverse agonist -cyclazosin enabled by the fusion to a DARPin crystallization chaperone The BAR structure allows the identification of two unique secondary binding pockets By structural comparison of BAR with ARs and by constructing BAR- CAR ... More
α-adrenergic receptors (αARs) are G protein-coupled receptors that regulate vital functions of the cardiovascular and nervous systems. The therapeutic potential of αARs, however, is largely unexploited and hampered by the scarcity of subtype-selective ligands. Moreover, several aminergic drugs either show off-target binding to αARs or fail to interact with the desired subtype. Here, we report the crystal structure of human α1BAR bound to the inverse agonist (+)-cyclazosin, enabled by the fusion to a DARPin crystallization chaperone. The α1BAR structure allows the identification of two unique secondary binding pockets. By structural comparison of α1BAR with α2ARs, and by constructing α1BAR-α2CAR chimeras, we identify residues 3.29 and 6.55 as key determinants of ligand selectivity. Our findings provide a basis for discovery of α1BAR-selective ligands and may guide the optimization of aminergic drugs to prevent off-target binding to αARs, or to elicit a selective interaction with the desired subtype. Less
Alphaviruses such as Ross River virus RRV chikungunya virus CHIKV Sindbis virus SINV and Venezuelan equine encephalitis virus VEEV are mosquito-borne pathogens that can cause arthritis or encephalitis diseases Nonstructural protein nsP of alphaviruses possesses RNA-dependent RNA polymerase RdRp activity essential for viral RNA replication No D structure has been available for nsP of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs Here we report crystal structures of the RdRp domain of nsP from both RRV and SINV determined at resolutions of and The structure of the alphavirus RdRp domain appears ... More
Alphaviruses such as Ross River virus (RRV), chikungunya virus (CHIKV), Sindbis virus (SINV), and Venezuelan equine encephalitis virus (VEEV) are mosquito-borne pathogens that can cause arthritis or encephalitis diseases. Nonstructural protein 4 (nsP4) of alphaviruses possesses RNA-dependent RNA polymerase (RdRp) activity essential for viral RNA replication. No 3D structure has been available for nsP4 of any alphaviruses despite its importance for understanding alphaviral RNA replication and for the design of antiviral drugs. Here, we report crystal structures of the RdRp domain of nsP4 from both RRV and SINV determined at resolutions of 2.6 Å and 1.9 Å. The structure of the alphavirus RdRp domain appears most closely related to RdRps from pestiviruses, noroviruses, and picornaviruses. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) and nuclear magnetic resonance (NMR) methods showed that in solution, nsP4 is highly dynamic with an intrinsically disordered N-terminal domain. Both full-length nsP4 and the RdRp domain were capable to catalyze RNA polymerization. Structure-guided mutagenesis using a trans-replicase system identified nsP4 regions critical for viral RNA replication. Less
Protein homeostasis proteostasis refers to the dynamic regulation of a stable and functional proteome The extensive proteostasis network includes integrated cellular mechanisms that control biogenesis folding trafficking and degradation of proteins Posttranslational modification and protein degradation are key pathways that minimize homeostatic perturbations however disease occurs when these processes become dysregulated Loss of protein homeostasis such as by an increase in misfolded proteins contributes to the pathology of many disorders including cancers to neurodegenerative diseases Maintaining the integrity of the proteome is essential for viability but cells continuously face extracellular and intracellular stresses that destabilize protein homeostasis Collaborations among several ... More
Protein homeostasis (proteostasis) refers to the dynamic regulation of a
stable and functional proteome. The extensive proteostasis network includes
integrated cellular mechanisms that control biogenesis, folding, trafficking, and
degradation of proteins. Posttranslational modification and protein degradation are
key pathways that minimize homeostatic perturbations; however, disease occurs
when these processes become dysregulated. Loss of protein homeostasis such
as by an increase in misfolded proteins contributes to the pathology of many
disorders including cancers to neurodegenerative diseases. Maintaining the
integrity of the proteome is essential for viability, but cells continuously face
extracellular and intracellular stresses that destabilize protein homeostasis.
Collaborations among several laboratories at Dartmouth led to the
discovery of virulence factor secreted from Pseudomonas aeruginosa that
dysregulates the homeostasis of the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR). The virulence factor, named the CFTR inhibitory
factor (Cif), decreases the number of CFTR channels on the plasma membrane by
manipulating the host ubiquitination system. Cif causes a complex to form between
G3BP1 and USP10, which renders USP10’s unable to perform its deubiquitinase
activity. Unfortunately, the mechanism of Cif-mediated G3BP1:USP10 complex
formation remains unknown. Cif causes protein dyshomeostasis resulting in
decreased CFTR level which ultimately aids bacterial colonization of the
compromised host.
The work detailed within expands our working model of Cif virulence to
include additional proteins and cellular phenomena. Caprin1, which was not included in previous investigations, competes with USP10 to bind G3BP1. We
provide a detailed stereochemical investigation of the G3BP1:USP10 and
G3BP1:Caprin1 complexes. Further, we describe discrete proteins mutations and
their effects on protein homeostasis. In parallel, we develop molecular tools to
determine the roles of G3BP1, USP10, and Caprin1 in the Cif virulence pathway.
These results offer new insights into the mechanism of Cif-mediated protein
dyshomeostasis. Less
stable and functional proteome. The extensive proteostasis network includes
integrated cellular mechanisms that control biogenesis, folding, trafficking, and
degradation of proteins. Posttranslational modification and protein degradation are
key pathways that minimize homeostatic perturbations; however, disease occurs
when these processes become dysregulated. Loss of protein homeostasis such
as by an increase in misfolded proteins contributes to the pathology of many
disorders including cancers to neurodegenerative diseases. Maintaining the
integrity of the proteome is essential for viability, but cells continuously face
extracellular and intracellular stresses that destabilize protein homeostasis.
Collaborations among several laboratories at Dartmouth led to the
discovery of virulence factor secreted from Pseudomonas aeruginosa that
dysregulates the homeostasis of the Cystic Fibrosis Transmembrane
Conductance Regulator (CFTR). The virulence factor, named the CFTR inhibitory
factor (Cif), decreases the number of CFTR channels on the plasma membrane by
manipulating the host ubiquitination system. Cif causes a complex to form between
G3BP1 and USP10, which renders USP10’s unable to perform its deubiquitinase
activity. Unfortunately, the mechanism of Cif-mediated G3BP1:USP10 complex
formation remains unknown. Cif causes protein dyshomeostasis resulting in
decreased CFTR level which ultimately aids bacterial colonization of the
compromised host.
The work detailed within expands our working model of Cif virulence to
include additional proteins and cellular phenomena. Caprin1, which was not included in previous investigations, competes with USP10 to bind G3BP1. We
provide a detailed stereochemical investigation of the G3BP1:USP10 and
G3BP1:Caprin1 complexes. Further, we describe discrete proteins mutations and
their effects on protein homeostasis. In parallel, we develop molecular tools to
determine the roles of G3BP1, USP10, and Caprin1 in the Cif virulence pathway.
These results offer new insights into the mechanism of Cif-mediated protein
dyshomeostasis. Less
Activity-regulated cytoskeleton-associated protein Arc is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals However the mechanistic and structural basis of Arc function is little understood Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands In addition the NTD and CTD both function in Arc oligomerization including assembly of retrovirus-like capsid involved in intercellular signaling We produced and characterised six ultra-high-affinity anti-Arc nanobodies Nb The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs ... More
Activity-regulated cytoskeleton-associated protein (Arc) is a multidomain protein of retroviral origin with a vital role in the regulation of synaptic plasticity and memory formation in mammals. However, the mechanistic and structural basis of Arc function is little understood. Arc has an NTD involved in membrane binding and a CTD which binds postsynaptic protein ligands. In addition, the NTD and CTD both function in Arc oligomerization, including assembly of retrovirus-like capsid involved in intercellular signaling. We produced and characterised six ultra-high-affinity anti-Arc nanobodies (Nb). The CTD of both rat and human Arc could be crystallised in ternary complexes with two Nbs simultaneously bound (H11 and C11). H11 binding deep into the stargazing-binding pocket of Arc CTD suggested competitive binding with Arc ligand peptides, which was confirmed in vitro. This indicates that the H11 Nb could serve as a genetically-encoded tool for inhibition of endogenous Arc N-lobe interactions in study of neuronal function and plasticity. The crystallisation of the human Arc CTD in two different conformations, accompanied by SAXS data and molecular dynamics simulations, paints a dynamic picture of the mammalian Arc CTD. Dynamics were affected by mutations known to inhibit capsid formation, implying a role for Arc CTD dynamics in oligomerisation. Dimerisation of the NTD, together with structural dynamics of the CTD, suggest a mechanism, by which structural dynamics of the CTD may promote capsomer formation, and dimerisation of the NTD links capsomers, facilitating the formation of capsids. The described recombinant ultrahigh-affinity anti-Arc Nbs are versatile tools that can be further developed for studying mammalian Arc structure and function in vitro and in vivo. Less
Identification of selective deubiquitinase DUB inhibitors is critical for probe development to further understand and explore DUB biological function Here we detail the optimization and deployment of an in vitro fluorogenic ubiquitin-rhodamine assay to conduct high-throughput screening of a small molecule library against a panel of DUBs In screening the compound library against multiple DUBs in parallel we describe an approach for identifying selective DUB inhibitors and provide a roadmap for enabling selective DUB inhibitor discovery
Deubiquitinating enzymes DUBs are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors Despite growing interest in DUB biological function and potential as therapeutic targets few selective small-molecule inhibitors and no approved drugs currently exist To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs spanning three well-characterized DUB families Promising hit compounds were validated in a series of counter-screens and orthogonal assays as well as ... More
Deubiquitinating enzymes (DUBs) are a class of isopeptidases that regulate ubiquitin dynamics through catalytic cleavage of ubiquitin from protein substrates and ubiquitin precursors. Despite growing interest in DUB biological function and potential as therapeutic targets, few selective small-molecule inhibitors and no approved drugs currently exist. To identify chemical scaffolds targeting specific DUBs and establish a broader framework for future inhibitor development across the gene family, we performed high-throughput screening of a chemically diverse small-molecule library against eight different DUBs, spanning three well-characterized DUB families. Promising hit compounds were validated in a series of counter-screens and orthogonal assays, as well as further assessed for selectivity across expanded panels of DUBs. Through these efforts, we have identified multiple highly selective DUB inhibitors and developed a roadmap for rapidly identifying and validating selective inhibitors of related enzymes. Less
Base excision repair BER is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species BER is initiated by DNA glycosylases each of which repairs a particular class of base damage NTHL a bifunctional DNA glycosylase possesses both glycolytic and -lytic activities with a preference for oxidized pyrimidine substrates Defects in human NTHL drive a class of polyposis colorectal cancer We report the first X-ray crystal structure of hNTHL revealing an open conformation not previously observed in the bacterial orthologs In this conformation the six-helical barrel domain comprising the helix-hairpin-helix HhH DNA binding motif ... More
Base excision repair (BER) is the main pathway protecting cells from the continuous damage to DNA inflicted by reactive oxygen species. BER is initiated by DNA glycosylases, each of which repairs a particular class of base damage. NTHL1, a bifunctional DNA glycosylase, possesses both glycolytic and β-lytic activities with a preference for oxidized pyrimidine substrates. Defects in human NTHL1 drive a class of polyposis colorectal cancer. We report the first X-ray crystal structure of hNTHL1, revealing an open conformation not previously observed in the bacterial orthologs. In this conformation, the six-helical barrel domain comprising the helix-hairpin-helix (HhH) DNA binding motif is tipped away from the iron sulphur cluster-containing domain, requiring a conformational change to assemble a catalytic site upon DNA binding. We found that the flexibility of hNTHL1 and its ability to adopt an open configuration can be attributed to an interdomain linker. Swapping the human linker sequence for that of Escherichia coli yielded a protein chimera that crystallized in a closed conformation and had a reduced activity on lesion-containing DNA. This large scale interdomain rearrangement during catalysis is unprecedented for a HhH superfamily DNA glycosylase and provides important insight into the molecular mechanism of hNTHL1. Less
Single-cell proteomics is a novel application area of bioanalysis aiming to characterize proteomes of isolated single cells which in contrast to bulk cell analysis has the potential to reveal a more detailed heterogeneity of cell populations Although several antibody-based targeted approaches have been readily available for single-cell analysis so far only the mass spectrometry methodology can offer unbiased proteome profiling While this strategy has only recently emerged it has already demonstrated unparalleled analytical power quantifying proteins in single cells Several applications of a general isobaric labeling scheme for multiplexed sample preparation and data acquisition have been outlined using various cell ... More
Single-cell proteomics is a novel application area of bioanalysis aiming to characterize proteomes of isolated single cells, which in contrast to bulk cell analysis has the potential to reveal a more detailed heterogeneity of cell populations. Although several antibody-based targeted approaches have been readily available for single-cell analysis, so far only the mass spectrometry methodology can offer unbiased proteome profiling. While this strategy has only recently emerged, it has already demonstrated unparalleled analytical power quantifying >1000 proteins in single cells. Several applications of a general isobaric labeling scheme for multiplexed sample preparation and data acquisition have been outlined using various cell types and instrumentation. This chapter provides a typical example of mass spectrometry-based single-cell proteomics workflow with details about the critical steps of analysis and alternative methods useful for optimization purposes. Less
The cancer-associated fibroblast CAF marker podoplanin PDPN is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target Despite its biomedical relevance basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized thus challenging drug development Here we utilize a high throughput platform to elucidate the PDPN cell surface interactome and uncover the neutrophil protein CD as a new binding partner Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling growth and actomyosin contractility among other processes Moreover cellular assays demonstrate ... More
The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment. Less
Over the past years single-cell sequencing has become very popular For this reason many laboratories of different biological disciplines that span from neurobiology to developmental biology from immunology to tumor biology have been approaching this technique For someone new to this field that wants to investigate heterogeneity in what appears to be a single-cell population the choice of the best protocol can be difficult due to the high abundance of available protocols instruments and options For this reason here we describe the Smart-seq protocol for full-length mRNA sequencing of single cell This protocol can be easily optimized in every molecular ... More
Over the past 7 years, single-cell sequencing has become very popular. For this reason, many laboratories of different biological disciplines that span from neurobiology to developmental biology from immunology to tumor biology have been approaching this technique. For someone new to this field that wants to investigate heterogeneity in what appears to be a single-cell population, the choice of the best protocol can be difficult, due to the high abundance of available protocols, instruments, and options. For this reason, here we describe the Smart-seq2 protocol for full-length mRNA sequencing of single cell. This protocol can be easily optimized in every molecular biology laboratory provided with standard laboratory equipment. The protocol is suitable for many different cell types, and the cost per cell is relatively small, allowing a good balance between costs and transcript coverage. Less
The COVID- pandemic is having a major impact on public health worldwide and there is an urgent need for the creation of an armamentarium of effective therapeutics including vaccines biologics and small-molecule therapeutics to combat SARS-CoV- and emerging variants Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus SARS-CoV- C-like protease CLpro an enzyme essential for viral replication is an attractive target for therapeutic intervention and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV- -specific antivirals We describe herein the results ... More
The COVID-19 pandemic is having a major impact on public health worldwide, and there is an urgent need for the creation of an armamentarium of effective therapeutics, including vaccines, biologics, and small-molecule therapeutics, to combat SARS-CoV-2 and emerging variants. Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is an attractive target for therapeutic intervention, and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV-2-specific antivirals. We describe herein the results of our studies related to the application of X-ray crystallography, the Thorpe–Ingold effect, deuteration, and stereochemistry in the design of highly potent and nontoxic inhibitors of SARS-CoV-2 3CLpro. Less
Ongoing antibiotic drug discovery is vital as antimicrobial resistance continues to be a significant issue faced in the clinic Natural products have long been a highly productive source to mine for new antimicrobials While it has been challenging to discover new and unique antimicrobial natural products numerous drugs have been derived from studying how natural products function as secondary metabolites Previous studies suggested that screening natural product extract fraction libraries for antimicrobials can be more productive than screening crude extracts alone These studies from large industrial enterprises are generally not directly portable to an academic setting due to significant infrastructure ... More
Ongoing antibiotic drug discovery is vital as antimicrobial resistance continues to be a significant issue faced in the clinic. Natural products have long been a highly productive source to mine for new antimicrobials. While it has been challenging to discover new and unique antimicrobial natural products, numerous drugs have been derived from studying how natural products function as secondary metabolites. Previous studies suggested that screening natural product extract fraction libraries for antimicrobials can be more productive than screening crude extracts alone. These studies from large industrial enterprises are generally not directly portable to an academic setting due to significant infrastructure costs. We developed a screening platform consisting of low pressure reversed-phase chromatographic separation of methanolic extracts of bacteria and fungi to generate a prefractionated natural product library. This platform is suitable for academic labs to screen for antimicrobial compounds. A large growth inhibitor screen against multiple pathogens and lab strains of microbes was conducted to assess the validity of the advantages of screening fraction libraries versus crude extract libraries and to search for potential new drug-like compounds. Hits were investigated for reproducibility, isolated, and purified. One compound was discovered in an antifungal screen which may be a novel lipopeptide. Less
Ubiquitination is a highly abundant post-translation modification that is involved in the control of a large number of cellular processes Target ubiquitination is achieved through the action of three separate enzymes the E ubiquitin-activating enzyme the E ubiquitin-conjugating enzyme and the E ubiquitin ligase TRIM E ligases are the largest family of RING-type E ligases and are classified by a N-terminal tripartite motif consisting of the catalytic RING domain one or two B-box domains B and B and a coiled-coil domain In addition most TRIMs possess a C-terminal substrate-binding domain which classifies them into one of eleven TRIM classes The ... More
Ubiquitination is a highly abundant post-translation modification that is involved in the control of a large number of cellular processes. Target ubiquitination is achieved through the action of three separate enzymes; the E1, ubiquitin-activating enzyme, the E2, ubiquitin-conjugating enzyme and the E3, ubiquitin ligase. TRIM E3 ligases are the largest family of RING-type E3 ligases and are classified by a N-terminal tripartite motif consisting of the catalytic RING domain, one or two B-box domains, B1 and B2, and a coiled-coil domain. In addition, most TRIMs possess a C-terminal substrate-binding domain, which classifies them into one of eleven TRIM classes. The PRYSPRY domain is the most common substrate-binding domain in humans and links class IV TRIMs to roles in cellular innate immunity. TRIM22 and TRIM6, are Class IV TRIMs that share high sequence identity with the well-studied HIV restriction factor, TRIM5, and have also been implicated in the anti-viral response. TRIM22 is reported to function directly as a viral restriction factor against RNA viruses such as, IAV, HCV and EMCV. While TRIM6 functions to activate the innate immune signalling pathway through activation of the immune signalling factor, IKK. Aspects of TRIM22 and TRIM6 function remain understudied, including their biochemical and biophysical properties and this is the focus of this study. The results described herein outline key differences in the self-association properties of these proteins in comparison to TRIM5. Furthermore, they highlight discrepancies between the ubiquitination profiles of TRIM22 and TRIM6 presented in the literature and the activity observed in this study. Overall, this emphasizes the need for further study of the roles of TRIM22 and TRIM6, to verify current proposed functions, as well as identify potential additional functions within the cell. Less