1052 Citations
Analytical theory is proposed predicting remarkably large and fully electric-dipole-allowed circular dichroism CD in electronic ultraviolet-visible UV-vis absorbance spectroscopy of uniaxial surface assemblies Partial depolarization of the transmitted beam provides a pathway for surface-specific and chiral-specific dissymmetry parameters that are orders of magnitude greater than those from analogous measurements of isotropic systems Predictions of the model generated using ab initio quantum chemical calculations with no adjustable parameters agreed with UV-vis absorbance CD measurements of naproxen microcrystals prepared on hydrophilic substrates Notably these calculations correctly predicted i the key spectroscopic features ii the relative magnitudes of chiral-specific peaks in the CD ... More
Analytical theory is proposed predicting remarkably large and fully electric-dipole-allowed circular dichroism (CD) in electronic ultraviolet-visible (UV-vis) absorbance spectroscopy of uniaxial surface assemblies. Partial depolarization of the transmitted beam provides a pathway for surface-specific and chiral-specific dissymmetry parameters that are orders of magnitude greater than those from analogous measurements of isotropic systems. Predictions of the model generated using ab initio quantum chemical calculations with no adjustable parameters agreed with UV-vis absorbance CD measurements of naproxen microcrystals prepared on hydrophilic substrates. Notably, these calculations correctly predicted (i) the key spectroscopic features, (ii) the relative magnitudes of chiral-specific peaks in the CD spectrum, (iii) the absolute CD sign, and (iv) the reciprocal CD sign inversion arising from sample reorientation in the instrument. These results connect the molecular structure and orientation to large CD observable in oriented thin-film assemblies, with the potential for further extension to broad classes of chiral-specific spectral analyses. Less
In this study we integrate Ro s target evaluation SpectraView and DL-driven virtual screening HydraScreen tools alongside Strateos' robotic cloud labs high-throughput screening platform to accelerate target and hit identification Using SpectraView to select IRAK as the target we prospectively validate HydraScreen a structure-based deep learning model We demonstrate that HydraScreen could identify up to of all IRAK hits in the top of the ranked compounds simultaneously identifying the three most potent nanomolar scaffolds present in the library The three nanomolar scaffolds identified in our project are novel for IRAK and lend themselves for future development HydraScreen outperforms traditional virtual ... More
In this study, we integrate Ro5’s target evaluation SpectraView and DL-driven virtual screening HydraScreen tools alongside Strateos' robotic cloud labs high-throughput screening platform to accelerate target and hit identification. Using SpectraView to select IRAK1 as the target, we prospectively validate HydraScreen, a structure-based deep learning model. We demonstrate that HydraScreen could identify up to 23.8% of all IRAK1 hits in the top 1% of the ranked compounds, simultaneously identifying the three most potent (nanomolar) scaffolds present in the library. The three nanomolar scaffolds identified in our project are novel for IRAK1 and lend themselves for future development. HydraScreen outperforms traditional virtual screening methods in an unbiased prospective evaluation and offers advanced features such as ligand pose confidence scoring. Thus, SpectraView and HydraScreen are innovative tools which can aid and expedite early stages of drug discovery. Less
We have adapted the cell painting assay developed by Carpenter and colleagues on cultured U OS cells to human spermatozoa In Sperm Cell Painting SCP we assemble an image-based quantitative fingerprint of the functional state of sperm We use this assay to gain insight into the mechanism of action of compounds that modify sperm function and as a platform for contraceptive discovery
Isopentenyl diphosphate IDP and dimethylallyl diphosphate DMADP are synthesized as the final step of the -C-methyl-D-erythritol -phosphate MEP pathway by E - - hydroxy- -methylbut- -en- -yl diphosphate reductase HDR and serve as the fundamental precursors in the biosynthesis of isoprenoids Previous studies have determined distinct activities among HDR homologous originating from the same woody plants This study aims to via crystallization determine the structure for two Picea abies HDR isoforms to shed light on the observed variation in enzymatic activity Crystals for both of the HDr isoforms have been achieved in this study However time constraints have prevented any ... More
Isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) are synthesized
as the final step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway by (E)-4-
hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR) and serve as the fundamental
precursors in the biosynthesis of isoprenoids. Previous studies have determined distinct
activities among HDR homologous originating from the same woody plants. This study
aims to, via crystallization, determine the structure for two Picea abies HDR isoforms
to shed light on the observed variation in enzymatic activity. Crystals for both of the
HDr isoforms have been achieved in this study. However, time constraints have prevented
any further analysis, leaving their structures unresolved. Nonetheless, future endeavors
dedicated to exploring the HDR building upon these results are likely to result in solved
structures. Less
as the final step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway by (E)-4-
hydroxy-3-methylbut-2-en-1-yl diphosphate reductase (HDR) and serve as the fundamental
precursors in the biosynthesis of isoprenoids. Previous studies have determined distinct
activities among HDR homologous originating from the same woody plants. This study
aims to, via crystallization, determine the structure for two Picea abies HDR isoforms
to shed light on the observed variation in enzymatic activity. Crystals for both of the
HDr isoforms have been achieved in this study. However, time constraints have prevented
any further analysis, leaving their structures unresolved. Nonetheless, future endeavors
dedicated to exploring the HDR building upon these results are likely to result in solved
structures. Less
The COVID- pandemic caused by severe acute respiratory syndrome coronavirus SARS-CoV- virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV- variants or novel CoVs Here we describe small molecule inhibitors for SARS-CoV- Mac which counters ADP-ribosylation mediated innate immune responses The compounds inhibiting Mac were discovered through high-throughput screening HTS using a protein FRET-based competition assay and the best hit compound had an IC of M Three validated HTS hits have the same -amide- -methylester thiophene scaffold and the scaffold was selected for structure-activity relationship SAR studies through commercial and synthesized ... More
The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has made it clear that further development of antiviral therapies will be needed to combat additional SARS-CoV-2 variants or novel CoVs. Here, we describe small molecule inhibitors for SARS-CoV-2 Mac1, which counters ADP-ribosylation mediated innate immune responses. The compounds inhibiting Mac1 were discovered through high-throughput screening (HTS) using a protein FRET-based competition assay and the best hit compound had an IC50 of 14 µM. Three validated HTS hits have the same 2-amide-3-methylester thiophene scaffold and the scaffold was selected for structure-activity relationship (SAR) studies through commercial and synthesized analogs. We studied the compound binding mode in detail using X-ray crystallography and this allowed us to focus on specific features of the compound and design analogs. Compound 27 (MDOLL-0229) had an IC50 of 2.1 µM and was generally selective for CoV Mac1 proteins after profiling for activity against a panel of viral and human ADP-ribose binding proteins. The improved potency allowed testing of its effect on virus replication and indeed, 27 inhibited replication of a mouse hepatitis virus, a prototype CoV. Compound 27 is the first Mac1 targeted small molecule demonstrated to inhibit coronavirus replication in a cell model. This, together with its well-defined binding mode, makes 27 a good candidate for further hit/lead-optimization efforts. Less
RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF complex in cells RAF target engagement can be measured in the presence or absence of any mutant KRAS allele enabling the high-affinity state of RAF dimer inhibitors to be quantified in the cellular milieu The intracellular protomer selectivity of clinical-stage type II RAF inhibitors revealed that ARAF protomer engagement but not ... More
RAF dimer inhibitors offer therapeutic potential in RAF- and RAS-driven cancers. The utility of such drugs is predicated on their capacity to occupy both RAF protomers in the RAS-RAF signaling complex. Here we describe a method to conditionally quantify drug-target occupancy at selected RAF protomers within an active RAS-RAF complex in cells. RAF target engagement can be measured in the presence or absence of any mutant KRAS allele, enabling the high-affinity state of RAF dimer inhibitors to be quantified in the cellular milieu. The intracellular protomer selectivity of clinical-stage type II RAF inhibitors revealed that ARAF protomer engagement, but not engagement of BRAF or CRAF, is commensurate with inhibition of MAPK signaling in various mutant RAS cell lines. Our results support a fundamental role for ARAF in mutant RAS signaling and reveal poor ARAF protomer vulnerability for a cohort of RAF inhibitors undergoing clinical evaluation. Less
Biomolecular condensates are membrane-less cellular foci formed via liquid-liquid-phase separation LLPS of specific biological macromolecules to provide specialized compartments for regulating cellular functions Many viral proteins undergo LLPS to form such condensates to support viral replication and evade host antiviral responses and thus these condensates are potential targets for designing antivirals Human noroviruses HuNoV cause epidemic and sporadic gastroenteritis worldwide and are of significant health and economic burden Here we show that the RNA-dependent-RNA polymerase RdRp of the pandemic GII HuNoV which is essential for viral replication forms distinct condensates capable of recruiting other viral replication components Confocal microscopy and ... More
Biomolecular condensates are membrane-less cellular foci formed via liquid-liquid-phase separation (LLPS) of specific biological macromolecules to provide specialized compartments for regulating cellular functions. Many viral proteins undergo LLPS to form such condensates to support viral replication and evade host antiviral responses, and thus, these condensates are potential targets for designing antivirals. Human noroviruses (HuNoV) cause epidemic and sporadic gastroenteritis worldwide and are of significant health and economic burden. Here, we show that the RNA-dependent-RNA polymerase (RdRp) of the pandemic GII.4 HuNoV, which is essential for viral replication, forms distinct condensates capable of recruiting other viral replication components. Confocal microscopy and light scattering experiments show that RdRp phase separates into dynamic liquid-like condensates at physiological conditions. These condensates exhibit all the signature features of LLPS, including fluorescence recovery after photo-bleaching, droplet-fusion, surface wetting, and dripping in vitro and in live cells. More importantly, within these condensates, the RdRp exhibits a significant time-dependent increase in its enzymatic activity and recruits other components, such as RNA and the viral genome-linked protein (VPg), which are essential for viral replication. Such condensates, recognized by anti-RdRp antibodies, are observed in HuNoV-infected human intestinal enteroid cultures. Together, our studies demonstrate a hitherto unsuspected activity of HuNoV RdRp to form LLPS, which we suggest provides distinct cellular sites for efficient viral replication and its regulation. Less
Background Immunosuppression in sepsis is hypothesized to result from the increased expression of the immune checkpoint molecules programmed death- PD- and pro grammed death ligand- PD-L PD- and PD-L blockade therapies have been reported to increase survival in septic animals Currently the interleukin IL - within mesenchymal stem cell MSC secretome is known for its immunomodulatory capacity Objective To study the effect of IL- within MSC secretome on the expression of immune check points in the rat model of sepsis Methods We used male Rattus norvegicus rats in this research and divided them into four groups sham rats without sepsis ... More
Background: Immunosuppression in sepsis is hypothesized to result from the increased
expression of the immune checkpoint molecules programmed death-1 (PD-1) and pro
grammed death ligand-1 (PD-L1). PD-1 and PD-L1 blockade therapies have been reported
to increase survival in septic animals. Currently, the interleukin (IL)-10 within mesenchymal
stem cell (MSC) secretome is known for its immunomodulatory capacity. Objective: To
study the effect of IL-10 within MSC secretome on the expression of immune check
points in the rat model of sepsis. Methods: We used 48 male Rattus norvegicus rats
in this research and divided them into four groups: sham (rats without sepsis induction
and treatment), control (sepsis-induced rats without treatment), T1 (sepsis-induced rats
treated with 150 µL of secreted IL-10 from MSC), and T2 (sepsis-induced rats treated
with 300 µL of secreted IL-10 from MSC). Forty-eight hours after sepsis induction, we
terminated the rats and collected the blood to examine the PD-1 and PD-L1 expression
levels. Results: We found a decrease in the relative expression of PD-1 in the septic rat
group given 150 µL and 300 µL of secreted IL-10 from MSC compared to the control
group, but the decrease was not significant. We also found a decrease in the relative
expression of PD-L1 mRNA in the septic rat group given 150 µL and 300 µL of secreted
IL-10 from MSC compared to the control group. Conclusion: Administering secreted IL-10
from MSC reduces the expression of PD-1 and PD-L1 in sepsis. These findings suggest
that MSC secretome can improve the immunosuppression in sepsis. Less
expression of the immune checkpoint molecules programmed death-1 (PD-1) and pro
grammed death ligand-1 (PD-L1). PD-1 and PD-L1 blockade therapies have been reported
to increase survival in septic animals. Currently, the interleukin (IL)-10 within mesenchymal
stem cell (MSC) secretome is known for its immunomodulatory capacity. Objective: To
study the effect of IL-10 within MSC secretome on the expression of immune check
points in the rat model of sepsis. Methods: We used 48 male Rattus norvegicus rats
in this research and divided them into four groups: sham (rats without sepsis induction
and treatment), control (sepsis-induced rats without treatment), T1 (sepsis-induced rats
treated with 150 µL of secreted IL-10 from MSC), and T2 (sepsis-induced rats treated
with 300 µL of secreted IL-10 from MSC). Forty-eight hours after sepsis induction, we
terminated the rats and collected the blood to examine the PD-1 and PD-L1 expression
levels. Results: We found a decrease in the relative expression of PD-1 in the septic rat
group given 150 µL and 300 µL of secreted IL-10 from MSC compared to the control
group, but the decrease was not significant. We also found a decrease in the relative
expression of PD-L1 mRNA in the septic rat group given 150 µL and 300 µL of secreted
IL-10 from MSC compared to the control group. Conclusion: Administering secreted IL-10
from MSC reduces the expression of PD-1 and PD-L1 in sepsis. These findings suggest
that MSC secretome can improve the immunosuppression in sepsis. Less
In sepsis simultaneously elevated levels of pro-inflammatory cytokines and interleukin IL - indicate immune response dysregulation increasing the mortality of the host As mesenchymal stem cell MSC secretome is known to have immunomodulatory effects we aim to assess the role of MSC secretome in the inflammatory mediators NF- B p and p TNF- IL- and the survival rate of a rat model of sepsis In this study forty-eight male Rattus norvegicus rats were divided into one sham group and three groups with sepsis induction the control group and the sepsis-induced rat groups treated with L T and L T of ... More
In sepsis, simultaneously elevated levels of pro-inflammatory cytokines and interleukin (IL)-10 indicate immune response dysregulation, increasing the mortality of the host. As mesenchymal stem cell (MSC) secretome is known to have immunomodulatory effects, we aim to assess the role of MSC secretome in the inflammatory mediators (NF-κB p65 and p50, TNF-α, IL-10) and the survival rate of a rat model of sepsis. In this study, forty-eight male Rattus norvegicus rats were divided into one sham group and three groups with sepsis induction: the control group and the sepsis-induced rat groups treated with 150 μL (T1) and 300 μL (T2) of secretome. The survival rate was observed per 6 h for 48 h and plotted using the Kaplan–Meier method. Compared to the control group, T2 showed a significant decrease in the relative expression of NF-κB and the serum TNF-α level, and a significant increase in the serum IL-10 level. Meanwhile, T1 showed a significant decrease in the serum TNF-α level compared to the control group. The Kaplan–Meier Log Rank test did not show significance in the distribution of survival between T1, T2, and the control group. However, from the 18th to the 36th hour, the survival rate of T2 was lower than the survival rate of the control group and T1, with a noticeable difference between T2 and the control group, as well as T1 at the 36th hour. At the 42nd hour, the survival rate of T2 was the same as the control group and remained lower than T1. In conclusion, MSC secretome regulated the inflammatory mediators in rat model of sepsis, with a dose of 150 μL being more effective. Less
Next-generation sequencing NGS technology advancements continue to reduce the cost of high-throughput genome-wide genotyping for breeding and genetics research Skim sequencing which surveys the entire genome at low coverage has become feasible for quantitative trait locus QTL mapping and genomic selection in various crops However the genome complexity of allopolyploid crops such as wheat Triticum aestivum L still poses a significant challenge for genome-wide genotyping Targeted sequencing of the protein-coding regions i e exome reduces sequencing costs compared to whole genome re-sequencing and can be used for marker discovery and genotyping We developed a method called skim exome capture SEC ... More
Next-generation sequencing (NGS) technology advancements continue to reduce the cost of high-throughput genome-wide genotyping for breeding and genetics research. Skim sequencing, which surveys the entire genome at low coverage, has become feasible for quantitative trait locus (QTL) mapping and genomic selection in various crops. However, the genome complexity of allopolyploid crops such as wheat (Triticum aestivum L.) still poses a significant challenge for genome-wide genotyping. Targeted sequencing of the protein-coding regions (i.e., exome) reduces sequencing costs compared to whole genome re-sequencing and can be used for marker discovery and genotyping. We developed a method called skim exome capture (SEC) that combines the strengths of these existing technologies and produces targeted genotyping data while decreasing the cost on a per-sample basis compared to traditional exome capture. Specifically, we fragmented genomic DNA using a tagmentation approach, then enriched those fragments for the low-copy genic portion of the genome using commercial wheat exome baits and multiplexed the sequencing at different levels to achieve desired coverage. We demonstrated that for a library of 48 samples, ∼7–8× target coverage was sufficient for high-quality variant detection. For higher multiplexing levels of 528 and 1056 samples per library, we achieved an average coverage of 0.76× and 0.32×, respectively. Combining these lower coverage SEC sequencing data with genotype imputation using a customized wheat practical haplotype graph database that we developed, we identified hundreds of thousands of high-quality genic variants across the genome. The SEC method can be used for high-resolution QTL mapping, genome-wide association studies, genomic selection, and other downstream applications. Less
Single-cell analysis has clearly established itself in biology and biomedical fields as an invaluable tool that allows one to comprehensively understand the relationship between cells including their types states transitions trajectories and spatial position Scientific methods such as fluorescence labeling nanoscale super-resolution microscopy advances in single cell RNAseq and proteomics technologies provide more detailed information about biological processes which were not evident with the analysis of bulk material This new era of single-cell biology provides a better understanding of such complex biological systems as cancer inflammation immunity mechanism and aging processes and opens the door into the field of drug ... More
Single-cell analysis has clearly established itself in biology and biomedical fields as an invaluable tool that allows one to comprehensively understand the relationship between cells, including their types, states, transitions, trajectories, and spatial position. Scientific methods such as fluorescence labeling, nanoscale super-resolution microscopy, advances in single cell RNAseq and proteomics technologies, provide more detailed information about biological processes which were not evident with the analysis of bulk material. This new era of single-cell biology provides a better understanding of such complex biological systems as cancer, inflammation, immunity mechanism and aging processes, and opens the door into the field of drug response heterogeneity. The latest discoveries of cellular heterogeneity gives us an unique understanding of complex biological processes, such as disease mechanism, and will lead to new strategies for better and personalized treatment strategies. Recently, single-cell proteomics techniques that allow quantification of thousands of proteins from single mammalian cells have been introduced. Here we present an improved single-cell mass spectrometry-based proteomics platform called SCREEN (Single Cell pRotEomE aNalysis) for deep and high-throughput single-cell proteome coverage with high efficiency, less turnaround time and with an improved ability for protein quantitation across more cells than previously achieved. We applied this new platform to analyze the single-cell proteomic landscape under different drug treatment over time to uncover heterogeneity in cancer cell response, which for the first time, to our knowledge, has been achieved by mass spectrometry based analytical methods. We discuss challenges in single-cell proteomics, future improvements and general trends with the goal to encourage forthcoming technical developments. Less
Schistosomiasis is caused by parasites of the genus Schistosoma which infect more than million people While praziquantel PZQ has been the main drug for controlling schistosomiasis for over four decades PZQ drug resistant strains have already been reported highlighting the need to search for new schistosomicidal drugs S mansoni survival relies on the redox enzyme thioredoxin glutathione reductase Sm TGR a validated target for the development of new anti-schistosomal drugs Here we report a fragment screening campaign of compounds against Sm TGR using X-ray crystallography and our efforts to optimize the hits found into potent inhibitors We observed binding events ... More
Schistosomiasis is caused by parasites of the genus Schistosoma , which infect more than 200 million people. While praziquantel (PZQ) has been the main drug for controlling schistosomiasis for over four decades, PZQ drug resistant strains have already been reported, highlighting the need to search for new schistosomicidal drugs. S. mansoni survival relies on the redox enzyme thioredoxin glutathione reductase ( Sm TGR), a validated target for the development of new anti-schistosomal drugs. Here we report a fragment screening campaign of 768 compounds against Sm TGR using X-ray crystallography and our efforts to optimize the hits found into potent inhibitors. We observed 49 binding events involving 35 distinct molecular fragments which were found to be distributed across 16 binding sites. Most sites are described for the first time within Sm TGR, a noteworthy exception being the “door stop pocket” near the NADPH binding site. Fragments binding to the latter were prioritized to undergo a “SAR by catalog” strategy for optimization into potential inhibitors. A search for compounds containing any of the prioritized fragments as a substructure was made in commercial databases. The ability of these compounds to inhibit Sm TGR was predicted based on a binary ML classification model, followed by an analysis of the putative binding mode by molecular docking. The 38 best ranked compounds were purchased and experimentally evaluated for Sm TGR inhibition. Compound 14 inhibited 63.6% of enzyme activity at 100 µM and presented an estimated IC 50 of 33 µM against Sm TGR. Less
Receptor-interacting protein kinases and RIPK and RIPK are considered attractive therapeutic enzyme targets for the treatment of a multitude of inflammatory diseases and cancers In this study we developed three interrelated series of novel quinazoline-based derivatives to investigate the effects of extensive modifications of positions and of the central core on the inhibitory activity and the selectivity against these RIPKs The design of the derivatives was inspired by analyses of available literary knowledge on both RIPK and RIPK in complex with known quinazoline or quinoline inhibitors Enzymatic investigations for bioactivity of the prepared molecules against purified RIPKs RIPK - shed ... More
Receptor-interacting protein kinases 2 and 3 (RIPK2 and RIPK3) are considered attractive therapeutic enzyme targets for the treatment of a multitude of inflammatory diseases and cancers. In this study, we developed three interrelated series of novel quinazoline-based derivatives to investigate the effects of extensive modifications of positions 6 and 7 of the central core on the inhibitory activity and the selectivity against these RIPKs. The design of the derivatives was inspired by analyses of available literary knowledge on both RIPK2 and RIPK3 in complex with known quinazoline or quinoline inhibitors. Enzymatic investigations for bioactivity of the prepared molecules against purified RIPKs (RIPK1-4) shed light on multiple potent and selective RIPK2 and dual RIPK2/3 inhibitors. Furthermore, evaluations in living cells against the RIPK2-NOD1/2-mediated signaling pathways, identified as the potential primary targets, demonstrated nanomolar inhibition for a majority of the compounds. In addition, we have demonstrated overall good stability of various lead inhibitors in both human and mouse microsomes and plasma. Several of these compounds also were evaluated for selectivity across 58 human kinases other than RIPKs, exhibiting outstanding specificity profiles. We have thus clearly demonstrated that tuning appropriate substitutions at positions 6 and 7 of the developed quinazoline derivatives may lead to interesting potency and specificities against RIPK2 and RIPK3. This knowledge might therefore be employed for the targeted preparation of new, highly potent and selective tools against these RIPKs, which could be of utility in biological and clinical research. Less
Laboratory production of recombinant mammalian proteins particularly antibodies requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications Transient gene expression TGE in the suspension-adapted Chinese Hamster Ovary CHO cell lines has become the method of choice for this task The antibodies can be secreted into the media which facilitates subsequent purification and can be glycosylated However in general protein production in CHO cells is expensive and may provide variable outcomes namely in laboratories without previous experience While achievable yields may be influenced by the nucleotide sequence there are other aspects of the process which offer ... More
Laboratory production of recombinant mammalian proteins, particularly antibodies, requires an expression pipeline assuring sufficient yield and correct folding with appropriate posttranslational modifications. Transient gene expression (TGE) in the suspension-adapted Chinese Hamster Ovary (CHO) cell lines has become the method of choice for this task. The antibodies can be secreted into the media, which facilitates subsequent purification, and can be glycosylated. However, in general, protein production in CHO cells is expensive and may provide variable outcomes, namely in laboratories without previous experience. While achievable yields may be influenced by the nucleotide sequence, there are other aspects of the process which offer space for optimization, like gene delivery method, cultivation process or expression plasmid design. Polyethylenimine (PEI)-mediated gene delivery is frequently employed as a low-cost alternative to liposome-based methods. In this work, we are proposing a TGE platform for universal medium-scale production of antibodies and other proteins in CHO cells, with a novel expression vector allowing fast and flexible cloning of new genes and secretion of translated proteins. The production cost has been further reduced using recyclable labware. Nine days after transfection, we routinely obtain milligrams of antibody Fabs or human lactoferrin in a 25 mL culture volume. Potential of the platform is established based on the production and crystallization of antibody Fabs and their complexes. Less
The Dictyostelium discoideum dye-decolorizing peroxidase DdDyP is a newly discovered peroxidase which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily DdDyP catalyzes the H O -dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass holding promise for potential industrial and biotechnological applications To study the molecular mechanism of DdDyP highly pure and functional protein with a natively incorporated heme is required however obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors Alternatively ... More
The Dictyostelium discoideum dye-decolorizing peroxidase (DdDyP) is a newly discovered peroxidase, which belongs to a unique class of heme peroxidase family that lacks homology to the known members of plant peroxidase superfamily. DdDyP catalyzes the H2O2-dependent oxidation of a wide-spectrum of substrates ranging from polycyclic dyes to lignin biomass, holding promise for potential industrial and biotechnological applications. To study the molecular mechanism of DdDyP, highly pure and functional protein with a natively incorporated heme is required, however, obtaining a functional DyP-type peroxidase with a natively bound heme is challenging and often requires addition of expensive biosynthesis precursors. Alternatively, a heme in vitro reconstitution approach followed by a chromatographic purification step to remove the excess heme is often used. Here, we show that expressing the DdDyP peroxidase in ×2 YT enriched medium at low temperature (20°C), without adding heme supplement or biosynthetic precursors, allows for a correct native incorporation of heme into the apo-protein, giving rise to a stable protein with a strong Soret peak at 402 nm. Further, we crystallized and determined the native structure of DdDyP at a resolution of 1.95 Å, which verifies the correct heme binding and its geometry. The structural analysis also reveals a binding of two water molecules at the distal site of heme plane bridging the catalytic residues (Arg239 and Asp149) of the GXXDG motif to the heme-Fe(III) via hydrogen bonds. Our results provide new insights into the geometry of native DdDyP active site and its implication on DyP catalysis. Less
The use of antibiotics has undoubtedly been a boon for humanity in combating infections and microbial threats However their widespread utilization has contributed to the emergence and spread of antibiotic resistance which now poses a significant public health challenge Streptomyces bacterium produce diverse secondary metabolites with antibacterial antifungal antiviral antitumoral and immunosuppressant activities Among these compounds is echinomycin a nonribosomal peptide antibiotic synthesized by Streptomyces lasalocidi which inhibits DNA replication and transcription by intercalating the DNA duplex at CpG steps A gene called ecm was identified in the echinomycin biosynthetic gene cluster which provides echinomycin self-resistance Ecm recognizes DNA duplexes ... More
The use of antibiotics has undoubtedly been a boon for humanity in combating infections and microbial threats. However, their widespread utilization has contributed to the emergence and spread of antibiotic resistance, which now poses a significant public health challenge. Streptomyces bacterium, produce diverse secondary metabolites with antibacterial, antifungal, antiviral, antitumoral, and immunosuppressant activities. Among these compounds is echinomycin, a nonribosomal peptide antibiotic synthesized by Streptomyces lasalocidi, which inhibits DNA replication and transcription by intercalating the DNA duplex at CpG steps. A gene called ecm16 was identified in the echinomycin biosynthetic gene cluster, which provides echinomycin self-resistance. Ecm16 recognizes DNA duplexes that contain echinomycin, and neutralizes its toxicity through a yet undetermined mechanism. To shed light on this process, we are determining the crystal structure of Ecm16 containing ATP. We introduced strategic mutations in the nucleotide binding site of Ecm16 to prevent ATP hydrolysis. We have successfully cloned, expressed, and purified the recombinant Ecm16 E399Q,E708Q double mutant protein. We have solved the structure of this protein using X-ray Crystallography at a resolution of 2.07 Å. However, our crystal structure contained ADP instead of the expected ATP. We propose and alternative experimental strategies for structure determination of ATP-containing Ecm16. Less
Aim Type diabetes mellitus T DM is an autoimmune disease characterized by the chronic inflammation of the pancreatic islets of Langerhans Hyperglycaemia leads to suppressed antioxidant enzyme and increased inflammation in the pancreatic cell resul ting in pancreatic cell death Hypoxic secretome mesenchymal stem cells HS-MSCs are soluble molecules secreted by MSCS that have the antiinflammation ability by secreting various cytoki nes including IL- and TGF- which potent as a promising the rapeutic modality for T DM This study aims to investigate the role of HS-MSCs in regulating superoxide dismutase SOD and caspase- gene expression in T DM model Methods ... More
Aim Type 1 diabetes mellitus (T1DM) is an autoimmune disease
characterized by the chronic inflammation of the pancreatic islets
of Langerhans. Hyperglycaemia leads to suppressed antioxidant
enzyme and increased inflammation in the pancreatic cell, resul
ting in pancreatic cell death. Hypoxic secretome mesenchymal
stem cells (HS-MSCs) are soluble molecules secreted by MSCS
that have the antiinflammation ability by secreting various cytoki
nes including IL-10 and TGF-β which potent as a promising the
rapeutic modality for T1DM. This study aims to investigate the
role of HS-MSCs in regulating superoxide dismutase (SOD) and
caspase-3 gene expression in T1DM model.
Methods Twenty male Wistar rats (6 to 8 weeks old) were rando
mly divided into four groups (sham, control, HS-MSCs 0.5 mL
and HS-MSCs 1 mL intraperitoneal treatment group). Streptozo
tocin (STZ) 60mg/kgBB was conducted once on day 1, HS-MSCs
0.5mL (T1) and HS-MSCs 1 mL (T2) were administrated intrape
ritoneally on day 7, 14, and 21 after STZ administration. The rats
were sacrificed on day 28; the gene expression of SOD and IL-6
was analysed by qRT-PCR.
Results This study showed that the ratio of SOD significantly
increased in HS-MSCs treatment associated with suppression of
IL-6 gene expression.
Conclusion HS-MSCs administration suppresses oxidative stre
ss and inflammation by up regulating SOD and inhibiting IL-6 to
control T1DM. Less
characterized by the chronic inflammation of the pancreatic islets
of Langerhans. Hyperglycaemia leads to suppressed antioxidant
enzyme and increased inflammation in the pancreatic cell, resul
ting in pancreatic cell death. Hypoxic secretome mesenchymal
stem cells (HS-MSCs) are soluble molecules secreted by MSCS
that have the antiinflammation ability by secreting various cytoki
nes including IL-10 and TGF-β which potent as a promising the
rapeutic modality for T1DM. This study aims to investigate the
role of HS-MSCs in regulating superoxide dismutase (SOD) and
caspase-3 gene expression in T1DM model.
Methods Twenty male Wistar rats (6 to 8 weeks old) were rando
mly divided into four groups (sham, control, HS-MSCs 0.5 mL
and HS-MSCs 1 mL intraperitoneal treatment group). Streptozo
tocin (STZ) 60mg/kgBB was conducted once on day 1, HS-MSCs
0.5mL (T1) and HS-MSCs 1 mL (T2) were administrated intrape
ritoneally on day 7, 14, and 21 after STZ administration. The rats
were sacrificed on day 28; the gene expression of SOD and IL-6
was analysed by qRT-PCR.
Results This study showed that the ratio of SOD significantly
increased in HS-MSCs treatment associated with suppression of
IL-6 gene expression.
Conclusion HS-MSCs administration suppresses oxidative stre
ss and inflammation by up regulating SOD and inhibiting IL-6 to
control T1DM. Less
Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded ss DNA We utilized a yeast model to explore mutagenesis by glycidamide a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines The most frequent mutations in adenines occurred in the nAt nGt trinucleotide motif Base substitutions A G in this motif relied on Rev translesion polymerase activity Inactivating Rev did not alter the nAt trinucleotide preference suggesting it may be an intrinsic specificity of the chemical reaction ... More
Mutagens often prefer specific nucleotides or oligonucleotide motifs that can be revealed by studying the hypermutation spectra in single-stranded (ss) DNA. We utilized a yeast model to explore mutagenesis by glycidamide, a simple epoxide formed endogenously in humans from the environmental toxicant acrylamide. Glycidamide caused ssDNA hypermutation in yeast predominantly in cytosines and adenines. The most frequent mutations in adenines occurred in the nAt→nGt trinucleotide motif. Base substitutions A→G in this motif relied on Rev1 translesion polymerase activity. Inactivating Rev1 did not alter the nAt trinucleotide preference, suggesting it may be an intrinsic specificity of the chemical reaction between glycidamide and adenine in the ssDNA. We found this mutational motif enriched in published sequencing data from glycidamide-treated mouse cells and ubiquitous in human cancers. In cancers, this motif was positively correlated with the single base substitution (SBS) smoking-associated SBS4 signature, with the clock-like signatures SBS1, SBS5, and was strongly correlated with smoking history and with age of tumor donors. Clock-like feature of the motif was also revealed in cells of human skin and brain. Given its pervasiveness, we propose that this mutational motif reflects mutagenic lesions to adenines in ssDNA from a potentially broad range of endogenous and exogenous agents. Less
Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents However to fully realise the potential of these molecules selectivity remains a limiting challenge Herein we addressed the issue of selectivity in the design of CRL CRBN recruiting PROteolysis TArgeting Chimeras PROTACs Thalidomide derivatives used to generate CRL CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates such as GSPT Ikaros and Aiolos We leveraged structural insights from known CRL CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known ... More
Small molecules inducing protein degradation are important pharmacological tools to interrogate complex biology and are rapidly translating into clinical agents. However, to fully realise the potential of these molecules, selectivity remains a limiting challenge. Herein, we addressed the issue of selectivity in the design of CRL4CRBN recruiting PROteolysis TArgeting Chimeras (PROTACs). Thalidomide derivatives used to generate CRL4CRBN recruiting PROTACs have well described intrinsic monovalent degradation profiles by inducing the recruitment of neo-substrates, such as GSPT1, Ikaros and Aiolos. We leveraged structural insights from known CRL4CRBN neo-substrates to attenuate and indeed remove this monovalent degradation function in well-known CRL4CRBN molecular glues degraders, namely CC-885 and Pomalidomide. We then applied these design principles on a previously published BRD9 PROTAC (dBRD9-A) and generated an analogue with improved selectivity profile. Finally, we implemented a computational modelling pipeline to show that our degron blocking design does not impact PROTAC-induced ternary complex formation. We believe that the tools and principles presented in this work will be valuable to support the development of targeted protein degradation. Less
Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options Due to their chemical nature and physico-chemical properties delivery of polynucleic acids into target cells and subcellular compartments remains a significant challenge Adeno associated viruses AAV have gained a lot of interest for the efficient delivery of therapeutic single-stranded DNA ssDNA genomes over the past decades More than a hundred products have been tested in clinical settings and three products have received market authorization by the US FDA in recent years A lot of effort is being made to generate potent recombinant AAV rAAV vectors ... More
Gene therapies offer promising therapeutic alternatives for many disorders that currently lack efficient treatment options. Due to their chemical nature and physico-chemical properties, delivery of polynucleic acids into target cells and subcellular compartments remains a significant challenge. Adeno associated viruses (AAV) have gained a lot of interest for the efficient delivery of therapeutic single-stranded DNA (ssDNA) genomes over the past decades. More than a hundred products have been tested in clinical settings and three products have received
market authorization by the US FDA in recent years. A lot of effort is being made to generate potent recombinant AAV (rAAV) vectors that show favorable safety and immunogenicity profiles for either local or systemic administration. Manufacturing processes are gradually being optimized to deliver a consistently high product quality and to serve potential market needs beyond rare indications. In contrast to protein therapeutics, most rAAV products are still supplied as frozen liquids within rather simple formulation buffers to enable sufficient product shelf life, significantly hampering global distribution and access. In this review, we aim to outline the hurdles of rAAV drug product development and discuss critical formulation and composition aspects of rAAV
products under clinical evaluation. Further, we highlight recent development efforts in order to achieve stable liquid or lyophilized products. This review therefore provides a comprehensive overview on current state-of-the- art rAAV formulations and can further serve as a map for rational formulation development activities in the
future. Less
market authorization by the US FDA in recent years. A lot of effort is being made to generate potent recombinant AAV (rAAV) vectors that show favorable safety and immunogenicity profiles for either local or systemic administration. Manufacturing processes are gradually being optimized to deliver a consistently high product quality and to serve potential market needs beyond rare indications. In contrast to protein therapeutics, most rAAV products are still supplied as frozen liquids within rather simple formulation buffers to enable sufficient product shelf life, significantly hampering global distribution and access. In this review, we aim to outline the hurdles of rAAV drug product development and discuss critical formulation and composition aspects of rAAV
products under clinical evaluation. Further, we highlight recent development efforts in order to achieve stable liquid or lyophilized products. This review therefore provides a comprehensive overview on current state-of-the- art rAAV formulations and can further serve as a map for rational formulation development activities in the
future. Less
TP is the most frequently mutated gene in cancer yet key target genes for p -mediated tumor suppression remain unidentified Here we characterize a rare African-specific germline variant of TP in the DNA-binding domain Tyr His Y H Nuclear magnetic resonance and crystal structures reveal that Y H is structurally similar to wild-type p Consistent with this we find that Y H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p target genes this includes the epigenetic modifier PADI which deiminates arginine to the nonnatural amino acid citrulline Surprisingly we show ... More
TP53 is the most frequently mutated gene in cancer, yet key target genes for p53-mediated tumor suppression remain unidentified. Here, we characterize a rare, African-specific germline variant of TP53 in the DNA-binding domain Tyr107His (Y107H). Nuclear magnetic resonance and crystal structures reveal that Y107H is structurally similar to wild-type p53. Consistent with this, we find that Y107H can suppress tumor colony formation and is impaired for the transactivation of only a small subset of p53 target genes; this includes the epigenetic modifier PADI4, which deiminates arginine to the nonnatural amino acid citrulline. Surprisingly, we show that Y107H mice develop spontaneous cancers and metastases and that Y107H shows impaired tumor suppression in two other models. We show that PADI4 is itself tumor suppressive and that it requires an intact immune system for tumor suppression. We identify a p53–PADI4 gene signature that is predictive of survival and the efficacy of immune-checkpoint inhibitors. Less
The small size and flexibility of G protein-coupled receptors GPCRs have long posed a significant challenge to determining their structures for research and therapeutic applications Single particle cryogenic electron microscopy cryoEM is often out of reach due to the small size of the receptor without a signaling partner Crystallization of GPCRs in lipidic cubic phase LCP often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers Here we determine the previously unknown structure of the human vasopressin B receptor V BR ... More
The small size and flexibility of G protein-coupled receptors (GPCRs) have long posed a significant challenge to determining their structures for research and therapeutic applications. Single particle cryogenic electron microscopy (cryoEM) is often out of reach due to the small size of the receptor without a signaling partner. Crystallization of GPCRs in lipidic cubic phase (LCP) often results in crystals that may be too small and difficult to analyze using X-ray microcrystallography at synchrotron sources or even serial femtosecond crystallography at X-ray free electron lasers. Here, we determine the previously unknown structure of the human vasopressin 1B receptor (V1BR) using microcrystal electron diffraction (MicroED). To achieve this, we grew V1BR microcrystals in LCP and transferred the material directly onto electron microscopy grids. The protein was labeled with a fluorescent dye prior to crystallization to locate the microcrystals using cryogenic fluorescence microscopy, and then the surrounding material was removed using a plasma-focused ion beam to thin the sample to a thickness amenable to MicroED. MicroED data from 14 crystalline lamellae were used to determine the 3.2 Å structure of the receptor in the crystallographic space group P 1. These results demonstrate the use of MicroED to determine previously unknown GPCR structures that, despite significant effort, were not tractable by other methods. Less
Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp - HaDHR is a member of a small family of dehydrogenases containing a covalently attached FMN and the only member so far identified to date that does not exhibit substrate inhibition In this study we present the resolution crystal structure of HaDHR This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators Alanine was identified as the exit point of electrons from the Fe S cluster The enzyme was modified with a Ser Cys mutation to facilitate covalent attachment of a ferrocene moiety When modified ... More
Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators. Less
Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted compared to mesophilic orthologs In several cases the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation In the psychrophilic -amylase from an Antarctic bacterium the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature Here we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations ... More
Cold-adapted enzymes are characterized both by a higher catalytic activity at low temperatures and by having their temperature optimum down-shifted, compared to mesophilic orthologs. In several cases, the optimum does not coincide with the onset of protein melting but reflects some other type of inactivation. In the psychrophilic α-amylase from an Antarctic bacterium, the inactivation is thought to originate from a specific enzyme-substrate interaction that breaks around room temperature. Here, we report a computational redesign of this enzyme aimed at shifting its temperature optimum upward. A set of mutations designed to stabilize the enzyme-substrate interaction were predicted by computer simulations of the catalytic reaction at different temperatures. The predictions were verified by kinetic experiments and crystal structures of the redesigned α-amylase, showing that the temperature optimum is indeed markedly shifted upward and that the critical surface loop controlling the temperature dependence approaches the target conformation observed in a mesophilic ortholog. Less
Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation T- domain responsible for the delivery of the catalytic domain into the cell Here we use a combination of computational constant-pH Molecular Dynamics simulations and experimental NMR circular dichroism and fluorescence spectroscopy along with the X-ray crystallography approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H Q H Q double mutant This replacement suppresses the acid-induced transition resulting in the retention of a more stable protein structure in solutions at ... More
Protonation of key histidine residues has been long implicated in the acid-mediated cellular action of the diphtheria toxin translocation (T-) domain, responsible for the delivery of the catalytic domain into the cell. Here, we use a combination of computational (constant-pH Molecular Dynamics simulations) and experimental (NMR, circular dichroism, and fluorescence spectroscopy along with the X-ray crystallography) approaches to characterize the initial stages of conformational change happening in solution in the wild-type T-domain and in the H223Q/H257Q double mutant. This replacement suppresses the acid-induced transition, resulting in the retention of a more stable protein structure in solutions at pH 5.5 and, consequently, in reduced membrane-disrupting activity. Here, for the first time, we report the pKa values of the histidine residues of the T-domain, measured by NMR-monitored pH titrations. Most peaks in the histidine side chain spectral region are titrated with pKas ranging from 6.2 to 6.8. However, the two most up-field peaks display little change down to pH 6, which is a limiting pH for this protein in solution at concentrations required for NMR. These peaks are absent in the double mutant, suggesting they belong to H223 and H257. The constant-pH simulations indicate that for the T-domain in solution, the pKa values for histidine residues range from 3.0 to 6.5, with those most difficult to protonate being H251 and H257. Taken together, our experimental and computational data demonstrate that previously suggested cooperative protonation of all six histidines in the T-domain does not occur. Less
We recently converted the GAF domain of NpR cyanobacteriochrome into near-infrared NIR fluorescent proteins FPs Unlike cyanobacterichrome which incorporates phycocyanobilin tetrapyrrole engineered NIR FPs bind biliverdin abundant in mammalian cells thus being the smallest scaffold for it Here we determined the crystal structure of the brightest blue-shifted protein of the series miRFP nano at resolution characterized its chromophore environment and explained the molecular basis of its spectral properties Using the determined structure we have rationally designed a red-shifted NIR FP termed miRFP nano with excitation at nm and emission at nm miRFP nano exhibits a small size of kDa enhanced ... More
We recently converted the GAF domain of NpR3784 cyanobacteriochrome into near-infrared (NIR) fluorescent proteins (FPs). Unlike cyanobacterichrome, which incorporates phycocyanobilin tetrapyrrole, engineered NIR FPs bind biliverdin abundant in mammalian cells, thus being the smallest scaffold for it. Here, we determined the crystal structure of the brightest blue-shifted protein of the series, miRFP670nano3, at 1.8 Å resolution, characterized its chromophore environment and explained the molecular basis of its spectral properties. Using the determined structure, we have rationally designed a red-shifted NIR FP, termed miRFP704nano, with excitation at 680 nm and emission at 704 nm. miRFP704nano exhibits a small size of 17 kDa, enhanced molecular brightness, photostability and pH-stability. miRFP704nano performs well in various protein fusions in live mammalian cells and should become a versatile genetically-encoded NIR probe for multiplexed imaging across spatial scales in different modalities. Less
The gut microbiome is complex raising questions about the role of individual strains in the community Here we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid -dehydroxylation niche Omitting Clostridium scindens Cs and Clostridium hylemonae Ch eliminates secondary bile acid production and reshapes the community in a highly specific manner eight strains change in relative abundance by -fold In single-strain dropout communities Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent However Clostridium sporogenes increases -fold in the Cs but ... More
The gut microbiome is complex, raising questions about the role of individual strains in the community. Here, we address this question by constructing variants of a complex defined community in which we eliminate strains that occupy the bile acid 7α-dehydroxylation niche. Omitting Clostridium scindens (Cs) and Clostridium hylemonae (Ch) eliminates secondary bile acid production and reshapes the community in a highly specific manner: eight strains change in relative abundance by >100-fold. In single-strain dropout communities, Cs and Ch reach the same relative abundance and dehydroxylate bile acids to a similar extent. However, Clostridium sporogenes increases >1,000-fold in the ΔCs but not ΔCh dropout, reshaping the pool of microbiome-derived phenylalanine metabolites. Thus, strains that are functionally redundant within a niche can have widely varying impacts outside the niche, and a strain swap can ripple through the community in an unpredictable manner, resulting in a large impact on an unrelated community-level phenotype. Less
Genome analysis of Pyrobaculum calidifontis revealed the presence of -glucosidase Pcal gene Structural analysis affirmed the presence of signature sequences of Type II -glucosidases in Pcal We have heterologously expressed the gene and produced recombinant Pcal in Escherichia coli Biochemical characteristics of the recombinant enzyme resembled to that of Type I -glucosidases instead of Type II Recombinant Pcal existed in a tetrameric form in solution and displayed highest activity at C and pH independent of any metal ions A short heat-treatment at C resulted in a increase in enzyme activity A slight structural shift was observed by CD spectrometry at ... More
Genome analysis of Pyrobaculum calidifontis revealed the presence of α-glucosidase (Pcal_0917) gene. Structural analysis affirmed the presence of signature sequences of Type II α-glucosidases in Pcal_0917. We have heterologously expressed the gene and produced recombinant Pcal_0917 in Escherichia coli. Biochemical characteristics of the recombinant enzyme resembled to that of Type I α-glucosidases, instead of Type II. Recombinant Pcal_0917 existed in a tetrameric form in solution and displayed highest activity at 95 °C and pH 6.0, independent of any metal ions. A short heat-treatment at 90 °C resulted in a 35 % increase in enzyme activity. A slight structural shift was observed by CD spectrometry at this temperature. Half-life of the enzyme was >7 h at 90 °C. Pcal_0917 exhibited apparent Vmax values of 1190 ± 5 and 3.9 ± 0.1 U/mg against p-nitrophenyl α-D-glucopyranoside and maltose, respectively. To the best of our knowledge, Pcal_0917 displayed the highest ever reported p-nitrophenyl α-D-glucopyranosidase activity among the characterized counterparts. Moreover, Pcal_0917 displayed transglycosylation activity in addition to α-glucosidase activity. Furthermore, in combination with α-amylase, Pcal_0917 was capable of producing glucose syrup from starch with >40 % glucose content. These properties make Pcal_0917 a potential candidate for starch hydrolyzing industry. Less
We have used a cohort of human induced pluripotent stem cell hiPSC lines to develop a laboratory-based drug screening platform to predict variable drug responses of potential clinical relevance Our approach is based on the findings that hiPSC lines reflect the genetic identity of the donor and that pluripotent hiPSC lines express a broad repertoire of gene transcripts and proteins We demonstrate that a cohort of hiPSC lines from different donors can be screened efficiently in their pluripotent state using high-throughput cell painting assays allowing detection of variable phenotypic responses to a wide range of clinically approved drugs across multiple ... More
We have used a cohort of human induced pluripotent stem cell (hiPSC) lines to develop a laboratory-based drug screening platform to predict variable drug responses of potential clinical relevance. Our approach is based on the findings that hiPSC lines reflect the genetic identity of the donor and that pluripotent hiPSC lines express a broad repertoire of gene transcripts and proteins. We demonstrate that a cohort of hiPSC lines from different donors can be screened efficiently in their pluripotent state using high-throughput cell painting assays, allowing detection of variable phenotypic responses to a wide range of clinically approved drugs, across multiple disease areas. Furthermore, we provide information on mechanisms of drug-cell interactions underlying the observed variable responses by using quantitative proteomic analysis to compare sets of hiPSC lines that had been stratified objectively using cell painting data. We propose that information derived from comparative drug screening using curated libraries of hiPSC lines can help to increase the success rate of drug development pipelines and improve the delivery of safe new drugs suitable for a broader ethnic and gender diversity within human populations. Less
To address the limitation associated with degron based systems we have developed iTAG a synthetic tag based on IMiDs CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs CeLMoDs based tags Using structural and sequence analysis we systematically explored native and chimeric degron containing domains DCDs and evaluated their ability to induce degradation We identified the optimal chimeric iTAG DCD aa that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented hook effect of PROTAC-based systems We showed that iTAG can also induce target degradation by ... More
To address the limitation associated with degron based systems, we have developed iTAG, a synthetic tag based on IMiDs/CELMoDs mechanism of action that improves and addresses the limitations of both PROTAC and previous IMiDs/CeLMoDs based tags. Using structural and sequence analysis, we systematically explored native and chimeric degron containing domains (DCDs) and evaluated their ability to induce degradation. We identified the optimal chimeric iTAG(DCD23 60aa) that elicits robust degradation of targets across cell types and subcellular localizations without exhibiting the well documented “hook effect” of PROTAC-based systems. We showed that iTAG can also induce target degradation by murine CRBN and enabled the exploration of natural neo-substrates that can be degraded by murine CRBN. Hence, the iTAG system constitutes a versatile tool to degrade targets across the human and murine proteome. Less
The high morbidity and mortality associated with SARS-CoV- infection the etiological agent of COVID- has had a major impact on global public health Significant progress has been made in the development of an array of vaccines and biologics however the emergence of SARS-CoV- variants and breakthrough infections are an ongoing major concern Furthermore there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV- and any emerging and re-emerging coronaviruses We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent ... More
The high morbidity and mortality associated with SARS-CoV-2 infection, the etiological agent of COVID-19, has had a major impact on global public health. Significant progress has been made in the development of an array of vaccines and biologics, however, the emergence of SARS-CoV-2 variants and breakthrough infections are an ongoing major concern. Furthermore, there is an existing paucity of small-molecule host and virus-directed therapeutics and prophylactics that can be used to counter the spread of SARS-CoV-2, and any emerging and re-emerging coronaviruses. We describe herein our efforts to address this urgent need by focusing on the structure-guided design of potent broad-spectrum inhibitors of SARS-CoV-2 3C-like protease (3CLpro or Main protease), an enzyme essential for viral replication. The inhibitors exploit the directional effects associated with the presence of a gem-dimethyl group that allow the inhibitors to optimally interact with the S4 subsite of the enzyme. Several compounds were found to potently inhibit SARS-CoV-2 and MERS-CoV 3CL proteases in biochemical and cell-based assays. Specifically, the EC50 values of aldehyde 1c and its corresponding bisulfite adduct 1d against SARS-CoV-2 were found to be 12 and 10 nM, respectively, and their CC50 values were >50 μM. Furthermore, deuteration of these compounds yielded compounds 2c/2d with EC50 values 11 and 12 nM, respectively. Replacement of the aldehyde warhead with a nitrile (CN) or an α-ketoamide warhead or its corresponding bisulfite adduct yielded compounds 1g, 1e and 1f with EC50 values 60, 50 and 70 nM, respectively. High-resolution cocrystal structures have identified the structural determinants associated with the binding of the inhibitors to the active site of the enzyme and, furthermore, have illuminated the mechanism of action of the inhibitors. Overall, the high Safety Index (SI) (SI=CC50/EC50) displayed by these compounds suggests that they are well-suited to conducting further preclinical studies. Less
The Gs protein-coupled adenosine A A receptor A AAR represents an emerging drug target for cancer immunotherapy The clinical candidate Etrumadenant was developed as an A AAR antagonist with ancillary blockade of the A BAR subtype It constitutes a unique chemotype featuring a poly-substituted -amino- -phenyl- -triazolylpyrimidine core structure Herein we report two crystal structures of the A AAR in complex with Etrumadenant obtained with differently thermostabilized A AAR constructs This led to the discovery of an unprecedented interaction a hydrogen bond of T with the cyano group of Etrumadenant T is mutated in most A AAR constructs used for ... More
The Gs protein-coupled adenosine A2A receptor (A2AAR) represents an emerging drug target for cancer immunotherapy. The clinical candidate Etrumadenant was developed as an A2AAR antagonist with ancillary blockade of the A2BAR subtype. It constitutes a unique chemotype featuring a poly-substituted 2-amino-4-phenyl-6-triazolylpyrimidine core structure. Herein, we report two crystal structures of the A2AAR in complex with Etrumadenant, obtained with differently thermostabilized A2AAR constructs. This led to the discovery of an unprecedented interaction, a hydrogen bond of T883.36 with the cyano group of Etrumadenant. T883.36 is mutated in most A2AAR constructs used for crystallization, which has prevented the discovery of its interactions. In-vitro characterization of Etrumadenant indicated low selectivity versus the A1AR subtype, which can be rationalized by the structural data. These results will facilitate the future design of AR antagonists with desired selectivity. Moreover, they highlight the advantages of the employed A2AAR crystallization construct that is devoid of ligand binding site mutations. Less
Arc is an immediate early gene that regulates synaptic plasticity in glutamatergic neurons The formation of new long-term memories requires functioning Arc protein Arc is both a protein interaction hub at the dendritic spines and is able to encapsulate its own mRNA in virus-like capsids that transfect nearby cells Relatively little is known about the structure of the mammalian Arc protein It consists of mainly -helical structures that make up the N- and Cterminal domain which are connected by a flexible linker and flanked by flexible N- and Cterminal tails Arc is found in many oligomeric states ranging from dimers ... More
Arc is an immediate early gene that regulates synaptic plasticity in glutamatergic neurons. The
formation of new long-term memories requires functioning Arc protein. Arc is both a protein
interaction hub at the dendritic spines and is able to encapsulate its own mRNA in virus-like
capsids that transfect nearby cells. Relatively little is known about the structure of the
mammalian Arc protein. It consists of mainly α-helical structures that make up the N- and Cterminal
domain, which are connected by a flexible linker and flanked by flexible N- and Cterminal
tails. Arc is found in many oligomeric states ranging from dimers to the predicted 140-
subunit capsids. This study aimed to solve the full-length structure of dimeric mammalian Arc
using X-ray crystallography and single-particle cryo-EM. Nanobodies that bind to Arc with
high affinity were used to stabilize and enlarge the dimeric complex. Structural information
about the Arc-nanobody complex was gathered using SAXS and compared with structures
predicted using AlphaFold. The results show that AlphaFold struggles to predict structures that
match the low-resolution structures of Arc in solution obtained from SAXS, likely due to Arc’s
structural flexibility and tendency to oligomerize. The study has also provided insight into the
binding dynamics of these nanobodies to Arc and highlighted their many uses in addition to
structural chaperones. Optimization of the sample preparation and data collection were
performed for the use in single-particle cryo-EM to solve the dimeric structure of full-length
Arc, although the data collection and processing have not been finished as of this moment. This
data could provide valuable new structural information about Arc, which will help better the
understanding of Arc’s functions and roles in disease. Less
formation of new long-term memories requires functioning Arc protein. Arc is both a protein
interaction hub at the dendritic spines and is able to encapsulate its own mRNA in virus-like
capsids that transfect nearby cells. Relatively little is known about the structure of the
mammalian Arc protein. It consists of mainly α-helical structures that make up the N- and Cterminal
domain, which are connected by a flexible linker and flanked by flexible N- and Cterminal
tails. Arc is found in many oligomeric states ranging from dimers to the predicted 140-
subunit capsids. This study aimed to solve the full-length structure of dimeric mammalian Arc
using X-ray crystallography and single-particle cryo-EM. Nanobodies that bind to Arc with
high affinity were used to stabilize and enlarge the dimeric complex. Structural information
about the Arc-nanobody complex was gathered using SAXS and compared with structures
predicted using AlphaFold. The results show that AlphaFold struggles to predict structures that
match the low-resolution structures of Arc in solution obtained from SAXS, likely due to Arc’s
structural flexibility and tendency to oligomerize. The study has also provided insight into the
binding dynamics of these nanobodies to Arc and highlighted their many uses in addition to
structural chaperones. Optimization of the sample preparation and data collection were
performed for the use in single-particle cryo-EM to solve the dimeric structure of full-length
Arc, although the data collection and processing have not been finished as of this moment. This
data could provide valuable new structural information about Arc, which will help better the
understanding of Arc’s functions and roles in disease. Less
Extrachromosomal DNA amplifications are common in cancer and are associated with decreased patient survival A key feature of extrachromosomal circular DNA is its ability to be randomly mis-segregated to daughter cells promoting rapid intercellular heterogeneity Understanding how extrachromosomal circular DNA dynamics contribute to intercellular heterogeneity remains crucial to better understand its role in tumor evolution and adaptation to therapy Here we introduce scEC T-seq s ingle c ell e xtrachromosomal c ircular DNA and t ranscriptomic seq uencing a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cancer cells In this protocol a single cell ... More
Extrachromosomal DNA amplifications are common in cancer and are associated with decreased patient survival. A key feature of extrachromosomal circular DNA is its ability to be randomly mis-segregated to daughter cells promoting rapid intercellular heterogeneity. Understanding how extrachromosomal circular DNA dynamics contribute to intercellular heterogeneity remains crucial to better understand its role in tumor evolution and adaptation to therapy. Here, we introduce scEC&T-seq ( s ingle c ell e xtrachromosomal c ircular DNA and t ranscriptomic seq uencing), a method for parallel detection of extrachromosomal circular DNAs and full-length mRNA in single cancer cells. In this protocol, a single cell’s DNA is separated from its polyadenylated RNA as described by Macaulay et al. (2015) 1 . This is followed by removal of linear DNA through exonuclease digestion and further enrichment of circular DNA by rolling circle amplification with φ29 polymerase 2-4 . The separated mRNA from the same cell is processed using on-bead Smart-seq2 1 . The duration of the entire procedure from cell sorting to library preparation is approximately 8 days. Our scEC&T-seq protocol has been validated in single cancer cells from neuroblastoma cell lines and primary tumors, and in normal single T-cells isolated from patient’s blood. Besides identifying large, oncogene-containing circular DNAs in cancer cells, our method also captures other smaller circular DNAs, which have been previously described in both cancer and non-malignant cells 5 . We envision that our method may enable the analysis of yet unknown prerequisites for the maintenance of both small and large circular DNA in cancers, but also in the context of other diseases and normal cellular development. Less
Spatial tissue proteomics combining microscopy-based cell phenotyping with ultra-sensitive mass spectrometry MS -based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease However optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue are currently lacking Here we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed paraffin-embedded FFPE material Benchmarking in the murine liver resulted in up to quantified proteins from single hepatocyte contours and nearly proteins from -cell ... More
Spatial tissue proteomics combining microscopy-based cell phenotyping with ultra-sensitive mass spectrometry (MS)-based proteomics is an emerging and powerful concept for the study of cell function and heterogeneity in health and disease. However, optimized workflows that preserve morphological information for image-based phenotype discovery and maximize proteome coverage of few or even single cells from laser microdissected archival tissue, are currently lacking. Here, we report a robust and scalable workflow for the proteomic analysis of ultra-low input formalin-fixed, paraffin-embedded (FFPE) material. Benchmarking in the murine liver resulted in up to 2,000 quantified proteins from single hepatocyte contours and nearly 5,000 proteins from 50-cell regions with high quantitative reproducibility. Applied to human tonsil, we profiled 146 microregions including spatially defined T and B lymphocyte niches and quantified cell type specific markers, cytokines, immune cell regulators and transcription factors. These rich data also highlighted proteome dynamics in spatially defined zones of activated germinal centers, illuminating sites undergoing active B-cell proliferation and somatic hypermutation. Our results demonstrate the power of spatially-resolved proteomics for tissue phenotyping by integrating high-content imaging, laser microdissection, and ultra-sensitive mass spectrometry. This approach has broad implications for a wide range of biomedical applications, including early disease profiling, drug target discovery and biomarker research. Less
Cytotoxic-T-lymphocyte CTL mediated control of HIV- is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I HLA-I However the extent to which the presenting HLA allele contributes to this process is unknown Here we examine the CTL response to QW a highly networked epitope presented by the disease-protective HLA-B and disease-neutral HLA-B Despite robust targeting of QW in persons expressing either allele T cell receptor TCR cross-recognition of the naturally occurring variant QW S T is consistently reduced when presented by HLA-B but not by HLA-B Crystal structures show substantial conformational changes from QW -HLA to QW S T-HLA ... More
Cytotoxic-T-lymphocyte (CTL) mediated control of HIV-1 is enhanced by targeting highly networked epitopes in complex with human-leukocyte-antigen-class-I (HLA-I). However, the extent to which the presenting HLA allele contributes to this process is unknown. Here we examine the CTL response to QW9, a highly networked epitope presented by the disease-protective HLA-B57 and disease-neutral HLA-B53. Despite robust targeting of QW9 in persons expressing either allele, T cell receptor (TCR) cross-recognition of the naturally occurring variant QW9_S3T is consistently reduced when presented by HLA-B53 but not by HLA-B57. Crystal structures show substantial conformational changes from QW9-HLA to QW9_S3T-HLA by both alleles. The TCR-QW9-B53 ternary complex structure manifests how the QW9-B53 can elicit effective CTLs and suggests sterically hindered cross-recognition by QW9_S3T-B53. We observe populations of cross-reactive TCRs for B57, but not B53 and also find greater peptide-HLA stability for B57 in comparison to B53. These data demonstrate differential impacts of HLAs on TCR cross-recognition and antigen presentation of a naturally arising variant, with important implications for vaccine design. Less
The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool Room-temperature data collection also enables time-resolved experiments In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines room-temperature methodology is less mature Here the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described and a highly efficient pipeline from protein sample to ... More
The utility of X-ray crystal structures determined under ambient-temperature conditions is becoming increasingly recognized. Such experiments can allow protein dynamics to be characterized and are particularly well suited to challenging protein targets that may form fragile crystals that are difficult to cryo-cool. Room-temperature data collection also enables time-resolved experiments. In contrast to the high-throughput highly automated pipelines for determination of structures at cryogenic temperatures widely available at synchrotron beamlines, room-temperature methodology is less mature. Here, the current status of the fully automated ambient-temperature beamline VMXi at Diamond Light Source is described, and a highly efficient pipeline from protein sample to final multi-crystal data analysis and structure determination is shown. The capability of the pipeline is illustrated using a range of user case studies representing different challenges, and from high and lower symmetry space groups and varied crystal sizes. It is also demonstrated that very rapid structure determination from crystals in situ within crystallization plates is now routine with minimal user intervention. Less
The hourglass model describes the convergence of species within the same phylum to a similar body plan during development however the molecular mechanisms underlying this phenomenon in mammals remain poorly described Here we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days and and compared the species using a framework for time-resolved single-cell differentiation-flows analysis We find convergence toward similar cell-state compositions at E supported by the quantitatively conserved expression of transcription factors despite divergence in surrounding trophoblast and hypoblast signaling However we ... More
The hourglass model describes the convergence of species within the same phylum to a similar body plan during development; however, the molecular mechanisms underlying this phenomenon in mammals remain poorly described. Here, we compare rabbit and mouse time-resolved differentiation trajectories to revisit this model at single-cell resolution. We modeled gastrulation dynamics using hundreds of embryos sampled between gestation days 6.0 and 8.5 and compared the species using a framework for time-resolved single-cell differentiation-flows analysis. We find convergence toward similar cell-state compositions at E7.5, supported by the quantitatively conserved expression of 76 transcription factors, despite divergence in surrounding trophoblast and hypoblast signaling. However, we observed noticeable changes in specification timing of some lineages and divergence of primordial germ cell programs, which in the rabbit do not activate mesoderm genes. Comparative analysis of temporal differentiation models provides a basis for studying the evolution of gastrulation dynamics across mammals. Less
Nowadays the vastly increasing demand for novel biotechnological products is supported by the continuous development of biocatalytic applications that provide sustainable green alternatives to chemical processes The success of a biocatalytic application is critically dependent on how quickly we can identify and characterize enzyme variants fitting the conditions of industrial processes While miniaturization and parallelization have dramatically increased the throughput of next-generation sequencing systems the subsequent characterization of the obtained candidates is still a limiting process in identifying the desired biocatalysts Only a few commercial microfluidic systems for enzyme analysis are currently available and the transformation of numerous published prototypes ... More
Nowadays, the vastly increasing demand for novel biotechnological products is supported by the continuous development of biocatalytic applications that provide sustainable green alternatives to chemical processes. The success of a biocatalytic application is critically dependent on how quickly we can identify and characterize enzyme variants fitting the conditions of industrial processes. While miniaturization and parallelization have dramatically increased the throughput of next-generation sequencing systems, the subsequent characterization of the obtained candidates is still a limiting process in identifying the desired biocatalysts. Only a few commercial microfluidic systems for enzyme analysis are currently available, and the transformation of numerous published prototypes into commercial platforms is still to be streamlined. This review presents the state-of-the-art, recent trends, and perspectives in applying microfluidic tools in the functional and structural analysis of biocatalysts. We discuss the advantages and disadvantages of available technologies, their reproducibility and robustness, and readiness for routine laboratory use. We also highlight the unexplored potential of microfluidics to leverage the power of machine learning for biocatalyst development. Less
Wastewater-based SARS-CoV- epidemiology WBE has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants However for a timely transmission of results sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis comparative quality control of the analytical procedures is essential to report reliable and comparable results In this study we performed an interlaboratory comparison at laboratories ... More
Wastewater-based SARS-CoV-2 epidemiology (WBE) has proven as an excellent tool to monitor pandemic dynamics supporting individual testing strategies. WBE can also be used as an early warning system for monitoring the emergence of novel pathogens or viral variants. However, for a timely transmission of results, sophisticated sample logistics and analytics performed in decentralized laboratories close to the sampling sites are required. Since multiple decentralized laboratories commonly use custom in-house workflows for sample purification and PCR-analysis, comparative quality control of the analytical procedures is essential to report reliable and comparable results.In this study, we performed an interlaboratory comparison at laboratories specialized for PCR and high-throughput-sequencing (HTS)-based WBE analysis. Frozen reserve samples from low COVID-19 incidence periods were spiked with different inactivated authentic SARS-CoV-2 variants in graduated concentrations and ratios. Samples were sent to the participating laboratories for analysis using laboratory specific methods and the reported viral genome copy numbers and the detection of viral variants were compared with the expected values.Despite the different procedures, a high concordance regarding the SARS-CoV-2 PCR quantification could be achieved with low variation between the workflows. PCR-based genotyping was, in dependence of the underlying PCR-assay performance, able to predict the relative amount of variant specific substitutions even in samples with low spike-in amount. The identification of variants by HTS, however, required >100 copies/mL wastewater and had limited predictive value when analyzing at a genome coverage below 60%.This interlaboratory test demonstrates that despite different extraction and analysis methods, a high agreement of the SARS-CoV-2 genome copy equivalents could be achieved. Hence, decentralized SARS-CoV-2 wastewater monitoring is feasible to generate comparable analysis results. However, since not all assays detected the correct variant, prior evaluation of PCR and sequencing workflows as well as sustained quality control such as interlaboratory comparisons are mandatory for correct variant detection. Less
Latrophilins ADGRLs are conserved adhesion-type G protein-coupled receptors associated with early embryonic morphogenesis defects lethality and sterility across multiple model organisms However their mechanistic roles in embryogenesis and the identity of their binding ligands remain unknown Here we identified a cell-surface receptor TOL- the sole Toll-like receptor in C elegans as a novel ligand for the C elegans Latrophilin LAT- The extracellular lectin domain of LAT- directly binds to the second leucine-rich repeat domain of TOL- The highresolution crystal structure and the cryo-EM density map of the LAT- TOL- ectodomain complex reveal a previously-unobserved mode of one-to-one interaction enabled by ... More
Latrophilins/ADGRLs are conserved adhesion-type G protein-coupled receptors associated with early embryonic morphogenesis defects, lethality, and sterility across multiple model organisms. However, their mechanistic roles in embryogenesis and the identity of their binding ligands remain unknown. Here, we identified a cell-surface receptor, TOL-1, the sole Toll-like receptor in C. elegans, as a novel ligand for the C. elegans Latrophilin, LAT-1. The extracellular lectin domain of LAT-1 directly binds to the second leucine-rich repeat domain of TOL-1. The highresolution crystal structure and the cryo-EM density map of the LAT-1–TOL-1 ectodomain complex reveal a previously-unobserved mode of one-to-one interaction enabled by a large interface. CRISPR/Cas9-mediated mutation of key interface residues selectively disrupted the endogenous LAT-1–TOL-1 interaction in C. elegans, leading to partial sterility, lethality, and malformed embryos. Thus, TOL-1 binding to LAT-1 represents a receptor-ligand axis essential for animal morphogenesis. Less
Despite advances in high-dimensional cellular analysis the molecular profiling of dynamic behaviors of cells in their native environment remains a major challenge We present a method that allows us to couple the physiological behaviors of cells in an intact murine 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 injury in murine airways
In the DarTG toxin-antitoxin system the DarT toxin ADP-ribosylates single-stranded DNA ssDNA which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG DarG also binds DarT but the role of these interactions in DarT neutralization is unknown Here we report that the C-terminal domain of DarG DarG toxin-binding domain DarGTBD interacts with DarT to form a stoichiometric heterodimeric complex We determined the resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex The comparative structural analysis reveals that DarGTBD interacts with ... More
In the DarTG toxin-antitoxin system, the DarT toxin ADP-ribosylates single-stranded DNA (ssDNA), which stalls DNA replication and plays a crucial role in controlling bacterial growth and bacteriophage infection. This toxic activity is reversed by the N-terminal macrodomain of the cognate antitoxin DarG. DarG also binds DarT, but the role of these interactions in DarT neutralization is unknown. Here, we report that the C-terminal domain of DarG (DarG toxin-binding domain [DarGTBD]) interacts with DarT to form a 1:1 stoichiometric heterodimeric complex. We determined the 2.2 Å resolution crystal structure of the Mycobacterium tuberculosis DarT-DarGTBD complex. The comparative structural analysis reveals that DarGTBD interacts with DarT at the DarT/ssDNA interaction interface, thus sterically occluding substrate ssDNA binding and consequently inactivating toxin by direct protein-protein interactions. Our data support a unique two-layered DarT toxin neutralization mechanism of DarG, which is important in keeping the toxin molecules in check under normal growth conditions. Less
Training artificial intelligence AI systems to perform autonomous experiments would vastly increase the throughput of microbiology however few microbes have large enough datasets for training such a system In the present study we introduce BacterAI an automated science platform that maps microbial metabolism but requires no prior knowledge BacterAI learns by converting scientific questions into simple games that it plays with laboratory robots The agent then distils its findings into logical rules that can be interpreted by human scientists We use BacterAI to learn the amino acid requirements for two oral streptococci Streptococcus gordonii and Streptococcus sanguinis We then show ... More
Training artificial intelligence (AI) systems to perform autonomous experiments would vastly increase the throughput of microbiology; however, few microbes have large enough datasets for training such a system. In the present study, we introduce BacterAI, an automated science platform that maps microbial metabolism but requires no prior knowledge. BacterAI learns by converting scientific questions into simple games that it plays with laboratory robots. The agent then distils its findings into logical rules that can be interpreted by human scientists. We use BacterAI to learn the amino acid requirements for two oral streptococci: Streptococcus gordonii and Streptococcus sanguinis. We then show how transfer learning can accelerate BacterAI when investigating new environments or larger media with up to 39 ingredients. Scientific gameplay and BacterAI enable the unbiased, autonomous study of organisms for which no training data exist. Less
Proteorhodopsins PRs bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins they play a significant role in life on the Earth A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name mirror proteorhodopsins from Sphingomonas paucimobilis SpaR shows cavity gate architecture of ... More
Proteorhodopsins (PRs), bacterial light-driven outward proton pumps comprise the first discovered and largest family of rhodopsins, they play a significant role in life on the Earth. A big remaining mystery was that up-to-date there was no described bacterial rhodopsins pumping protons at acidic pH despite the fact that bacteria live in different pH environment. Here we describe conceptually new bacterial rhodopsins which are operating as outward proton pumps at acidic pH. A comprehensive function-structure study of a representative of a new clade of proton pumping rhodopsins which we name “mirror proteorhodopsins”, from Sphingomonas paucimobilis (SpaR) shows cavity/gate architecture of the proton translocation pathway rather resembling channelrhodopsins than the known rhodopsin proton pumps. Another unique property of mirror proteorhodopsins is that proton pumping is inhibited by a millimolar concentration of zinc. We also show that mirror proteorhodopsins are extensively represented in opportunistic multidrug resistant human pathogens, plant growth-promoting and zinc solubilizing bacteria. They may be of optogenetic interest. Less
SARS-CoV- and MERS-CoV president the human coronaviruses and zoonotic coronaviruses due to tremendous repercussions to wellbeing of the world population Investigational efforts focusing on the disease pathology and viral lifecycle have led to identifying multiple druggable targets of both viruses including CL protease PL protease and RNA dependent RNA polymerase This dissertation summarizes the research findings related to CL protease inhibitors of SARS-CoV- and MERS-CoV A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor- CL protease complexes The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency Multiple inhibitors ... More
SARS-CoV-2 and MERS-CoV president the human coronaviruses and zoonotic coronaviruses due to tremendous repercussions to wellbeing of the world population. Investigational efforts focusing on the disease pathology and viral lifecycle have led to identifying multiple druggable targets of both viruses including 3CL protease, PL protease and RNA dependent RNA polymerase.
This dissertation summarizes the research findings related to 3CL protease inhibitors of SARS-CoV-2 and MERS-CoV. A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor-3CL protease complexes. The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency. Multiple inhibitors were found to be highly potent (IC50 < 100 nM) against 3CL proteases of SARS-CoV-2 and MERS-CoV. Inhibitors with dramatically improved antiviral activity in cell-based assays (EC50 = 11-13 nM) against SARS-CoV-2 (chapter 5, compounds 5c/d and 11c/d) were developed. Importantly, these inhibitors specifically target viral lifecycle without causing harmful effects on healthy cells as evidenced by having high safety indices (CC50/EC50 > 1000).
Bioisosteric replacement of metabolically vulnerable protons of GC376 with deuterium has led to identification of compound 2 (chapter 1) which showed significantly enhanced survival of SARS-CoV-2 infected K18-hACE2 mice 83 – 100 % compared to 0 % when untreated. Several other drug candidates suitable for conducting further preclinical studies have been identified Less
This dissertation summarizes the research findings related to 3CL protease inhibitors of SARS-CoV-2 and MERS-CoV. A structure-guided design approach was used with the aid of X-ray crystallography of inhibitor-3CL protease complexes. The iterative optimization of potency in enzyme assays and cell-based assays yielded inhibitors with nanomolar potency. Multiple inhibitors were found to be highly potent (IC50 < 100 nM) against 3CL proteases of SARS-CoV-2 and MERS-CoV. Inhibitors with dramatically improved antiviral activity in cell-based assays (EC50 = 11-13 nM) against SARS-CoV-2 (chapter 5, compounds 5c/d and 11c/d) were developed. Importantly, these inhibitors specifically target viral lifecycle without causing harmful effects on healthy cells as evidenced by having high safety indices (CC50/EC50 > 1000).
Bioisosteric replacement of metabolically vulnerable protons of GC376 with deuterium has led to identification of compound 2 (chapter 1) which showed significantly enhanced survival of SARS-CoV-2 infected K18-hACE2 mice 83 – 100 % compared to 0 % when untreated. Several other drug candidates suitable for conducting further preclinical studies have been identified Less
Influenza virus IV causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars Several influenza pandemics have occurred during the last century and estimated to have caused million deaths There are four genera of IV A IVA B IVB C IVC and D IVD with IVA being the most virulent to the human population Hemagglutinin HA is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell Here we have characterised the high-resolution structures of seven IVA HAs with one ... More
Influenza virus (IV) causes several outbreaks of the flu each year resulting in an economic burden to the healthcare system in the billions of dollars. Several influenza pandemics have occurred during the last century and estimated to have caused 100 million deaths. There are four genera of IV, A (IVA), B (IVB), C (IVC), and D (IVD), with IVA being the most virulent to the human population. Hemagglutinin (HA) is an IVA surface protein that allows the virus to attach to host cell receptors and enter the cell. Here we have characterised the high-resolution structures of seven IVA HAs, with one in complex with the anti-influenza head-binding antibody C05. Our analysis revealed conserved receptor binding residues in all structures, as seen in previously characterised IV HAs. Amino acid conservation is more prevalent on the stalk than the receptor binding domain (RBD; also called the head domain), allowing the virus to escape from antibodies targeting the RBD. The equivalent site of C05 antibody binding to A/Denver/57 HA appears hypervariable in the other H1N1 IV HAs. Modifications within this region appear to disrupt binding of the C05 antibody, as these HAs no longer bind the C05 antibody by analytical SEC. Our study brings new insights into the structural and functional recognition of IV HA proteins and can contribute to further development of anti-influenza vaccines. Less
The microbial guild coupling anammox and nitrite nitrate-dependent anaerobic methane oxidation n-DAMO is an innovative process to achieve energy-efficient nitrogen removal with the beneficial use of methane in biogas or in anaerobically treated wastewater Here metagenomics and metatranscriptomics were used to reveal the microbial ecology of two biofilm systems which incorporate anammox and n-DAMO for high-level nitrogen removal in low-strength domestic sewage and high-strength sidestream wastewater respectively We find that different nitrogen loadings i e vs kg N m d lead to different combinations of anammox bacteria and anaerobic methanotrophs Candidatus Methanoperedens and Candidatus Methylomirabilis which play primary roles for ... More
The microbial guild coupling anammox and nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is an innovative process to achieve energy-efficient nitrogen removal with the beneficial use of methane in biogas or in anaerobically treated wastewater. Here, metagenomics and metatranscriptomics were used to reveal the microbial ecology of two biofilm systems, which incorporate anammox and n-DAMO for high-level nitrogen removal in low-strength domestic sewage and high-strength sidestream wastewater, respectively. We find that different nitrogen loadings (i.e., 0.1 vs. 1.0 kg N/m3/d) lead to different combinations of anammox bacteria and anaerobic methanotrophs (“Candidatus Methanoperedens” and “Candidatus Methylomirabilis”), which play primary roles for carbon and nitrogen transformations therein. Despite methane being the only exogenous organic carbon supplied, heterotrophic populations (e.g., Verrucomicrobiota and Bacteroidota) co-exist and actively perform partial denitrification or dissimilatory nitrate reduction to ammonium (DNRA), likely using organic intermediates from the breakdown of methane and biomass as carbon sources. More importantly, two novel genomes belonging to “Ca. Methylomirabilis” are recovered, while one surprisingly expresses nitrate reductases, which we designate as “Ca. Methylomirabilis nitratireducens” representing its inferred capability in performing nitrate-dependent anaerobic methane oxidation. This finding not only suggests a previously neglected possibility of “Ca. Methylomirabilis” bacteria in performing methane-dependent nitrate reduction, and also challenges the previous understanding that the methane-dependent complete denitrification from nitrate to dinitrogen gas is carried out by the consortium of bacteria and archaea. Less
Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes It has two different enzymatic activities microbial cell walls destruction muramidase activity and dissolution of the stabilized fibrin isopeptidase activity Both activities are known to be inhibited by sodium chloride at near physiological concentrations but the structural basis remains unknown Here we present two crystal structures of destabilase including a -resolution structure in complex with sodium ion Our structures reveal the location of sodium ion between Glu Asp residues which were previously recognized as a glycosidase active site While sodium coordination with these amino acids may ... More
Destabilase from the medical leech Hirudo medicinalis belongs to the family of i-type lysozymes. It has two different enzymatic activities: microbial cell walls destruction (muramidase activity), and dissolution of the stabilized fibrin (isopeptidase activity). Both activities are known to be inhibited by sodium chloride at near physiological concentrations, but the structural basis remains unknown. Here we present two crystal structures of destabilase, including a 1.1 Å-resolution structure in complex with sodium ion. Our structures reveal the location of sodium ion between Glu34/Asp46 residues, which were previously recognized as a glycosidase active site. While sodium coordination with these amino acids may explain inhibition of the muramidase activity, its influence on previously suggested Ser49/Lys58 isopeptidase activity dyad is unclear. We revise the Ser49/Lys58 hypothesis and compare sequences of i-type lysozymes with confirmed destabilase activity. We suggest that the general base for the isopeptidase activity is His112 rather than Lys58. pKa calculations of these amino acids, assessed through the 1 μs molecular dynamics simulation, confirm the hypothesis. Our findings highlight the ambiguity of destabilase catalytic residues identification and build foundations for further research of structure–activity relationship of isopeptidase activity as well as structure-based protein design for potential anticoagulant drug development. Less
Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes A new challenge for the field is to use these designs directly in cells to intervene in biological process and augment natural systems The bottom-up design of self-assembled objects like microcompartments and membraneless organelles is one such challenge which also presents opportunities for chemical and synthetic biology Here we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli E coli To do this we combine de novo -helical sequences intrinsically disordered linkers and client proteins in ... More
Recent advances in de novo protein design have delivered a diversity of discrete de novo protein structures and complexes. A new challenge for the field is to use these designs directly in cells to intervene in biological process and augment natural systems. The bottom-up design of self-assembled objects like microcompartments and membraneless organelles is one such challenge, which also presents opportunities for chemical and synthetic biology. Here, we describe the design of genetically encoded polypeptides that form membraneless organelles in Escherichia coli (E. coli). To do this, we combine de novo α-helical sequences, intrinsically disordered linkers, and client proteins in single-polypeptide constructs. We tailor the properties of the helical regions to shift protein assembly from diffusion-limited assemblies to dynamic condensates. The designs are characterised in cells and in vitro using biophysical and soft-matter physics methods. Finally, we use the designed polypeptide to co-compartmentalise a functional enzyme pair in E. coli. Less