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At least 415 records · Page 23

Discovery of multidrug efflux pump inhibitors with a novel chemical scaffold

Multidrug efflux is a major contributor to antibiotic resistance in Gram-negative bacterial pathogens. Inhibition of multidrug efflux pumps is a promising approach for reviving the efficacy of existing antibiotics. Previously, inhibitors targeting both the efflux transporter AcrB and the membrane fusion protein AcrA in the Escherichia coli AcrAB-TolC efflux pump were identified. In this work we use existing physicochemical property guidelines to generate a filtered library of compounds for computational docking. We then experimentally test the top candidate coumpounds using in vitro binding assays and in vivo potentiation assays in bacterial strains with controllable permeability barriers. We thus identify a new class of inhibitors of E. coli AcrAB-TolC. Six molecules with a shared scaffold were found to potentiate the antimicrobial activity of erythromycin and novobiocin in hyperporinated E. coli cells. Importantly, these six molecules were also active in wild-type strains of both Acinetobacter baumannii and Klebsiella pneumoniae, potentiating the activity of erythromycin and novobiocin up to 8-fold.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A novel polypeptide vaccine and adjuvant formulation of EV71

ABSTRACT Hand foot and mouth disease (HFMD) is an infectious disease mainly caused by Enterovirus 71 (EV 71). However, the effective treatment is limited currently. The aim of this study was to investigate the activity of the vaccine including the EV71 polypeptides mixed with a novel adjuvant containing CpG oligodeoxynucleotides (CpG ODNs). After collecting mouse sera, we determined the antibody concentration in serum by enzyme-linked immunosorbent assays (ELISA). Then, CD19+CD27+ B cells in the spleen were analysed by flow cytometry. The assay revealed that a substantial increase in antibody titers was achieved. This indicates a high level of immunogenicity for peptide vaccine and the good stability of adjuvant, also suggests that the combination of vaccine and adjuvant can stimulate the production of high-level antibodies and CD19+CD27+ B lymphocytes in mice. Furthermore, the antibody could effectively identify EV71 inactivated virus. The results demonstrated that the autonomous construction of EV71 polypeptide vaccine had a good immunogenicity. Moreover, the peptide vaccine injection with a novel adjuvant, which is easy to prepare, could cause a high antibody level of EV71 and shown a good application prospect.

Liu, Zhiang↗

A family of α/β hydrolases removes phytol from chlorophyll metabolites for tocopherol biosynthesis in Arabidopsis

Tocopherol synthesis requires phytyl diphosphate derived from phytol esterified to chlorophyll metabolites. The >600-member Arabidopsis thaliana α/β hydrolase (ABH) gene family contains 4 members that can release phytol from chlorophyll metabolites in vitro; however, only pheophytinase (PPH) affects tocopherol synthesis when mutated, reducing seed tocopherols by 5%. We report the biochemical analysis of 2 previously uncharacterized ABHs, chlorophyll dephytylase 2 (CLD2) and CLD3, and their respective mutants singly and in combinations with pph and cld1 alleles. While all CLDs localized to the thylakoid and could hydrolyze phytol from chlorophylls and Pheophytin a in vitro, CLD3 had the highest in vitro activity and the largest effect on tocopherol synthesis in vivo. The 3 CLDs acted cooperatively to provide phytol for 31% of tocopherols synthesized in light-grown leaf tissue. Dark-induced leaf senescence assays showed PPH is required for 18% of the tocopherols synthesized. Though the cld123 triple mutant had no impact on dark-induced tocopherol content, cld123 in the pph background reduced tocopherol levels by an additional 18%. In seeds, pph and cld123 each reduced tocopherol content by 5% and by 15% in the cld123pph quadruple mutant. VTE7 ( ViTamin E7 ) is an envelope-localized ABH that specifically affects chlorophyll biosynthetic intermediates in vivo and is required for 55% of seed tocopherol synthesis. The introduction of cld123pph into the vte7 background further reduced seed tocopherol levels to 23% of that of the wild type. Our findings demonstrate that phytol provision for tocopherol biosynthesis and homeostasis is a complex process involving the coordinated spatiotemporal expression of multiple ABH family members.

arabidopsis↗

FY25 Mid-Year Report: FNCL Enhancements Implementation

During the first half of FY25 the FNCL team has made consistent progress toward the completion of our project goals. The FNCL prototype panel design has been successfully applied to a fully instrumented 3-panel system which is actively under construction. The FNCL Demonstrator System contains solid scintillators instrumented with SiPMs, which operate on an updated CAEN digitizer, requires no high-voltage, and has a smaller overall footprint. The onboard software will include the LLNL-developed GMM-PSD signal processing. Later this year the system will be experimentally tested alongside the baseline FNCL instrument at LLNLs ISSA facility. In addition to a full systems test, the performance of a DD generator for active interrogation measurements compared to the standard AmLi source will be established for both systems. The data collected at the ISSA facility will be used to experimentally validate the FNCL-Fast Isotopic Fuel Assay’s (FIFA) capability to measure U-235 loading and to predict gadolinium poison content with passive interrogation. The FNCL-FIFA modal was benchmarked with simulation-based data and a user-friendly GUI was added earlier this year. Three separate codes have been submitted to the LLNL ESW system for review prior to their transfers. These include the Predictive Modeling Response toolkit, GMM-PSD firmware beta version, and the FNCL-FIFA analysis package with GUI and user documentation.

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗

Long-Range Allosteric Communication Modulated by Active Site Mn(II) Coordination Drives Catalysis in Xanthobacter autotrophicus Acetone Carboxylase

Acetone carboxylase (AC) from Xanthobacter autotrophicus is a 360 KDa α2β2γ2 heterohexamer that catalyzes the ATP-dependent formation of phosphorylated acetone and bicarbonate intermediates that react at Mn(II) metal active sites to form acetoacetate. Structural models of X. autotrophicus AC (XaAC) with and without nucleotides reveal that the binding and phosphorylation of the two substrates occurs ~40 Å from the Mn(II) active sites where acetoacetate is formed. Based on the crystal structures, a significant conformational change was proposed to open and close a tunnel that facilitates the passage of reaction intermediates between the sites for nucleotide binding and phosphorylation of substrates and Mn(II) sites of acetoacetate formation. We have employed electron paramagnetic resonance (EPR), kinetic assays, and hydrogen/deuterium exchange mass spectrometry (HDX-MS) of poised ligand-bound states and site-specific amino acid variants to complete an in-depth analysis of Mn(II) coordination and allosteric communication throughout the catalytic cycle. In contrast with the established paradigms for carboxylation, our analyses of XaAC suggested a carboxylate shift that couples both local and long-range structural transitions. Shifts in the coordination mode of a single carboxylic acid residue (αE89) mediate both catalysis proximal to a Mn(II) center and communication with an ATP active site in a separate subunit of a 180 kDa α2β2γ2 complex at a distance of 40 Å. This work demonstrates the power of combining structural models from X-ray crystallography with solution-phase spectroscopy and biophysical techniques to elucidate functional aspects of a multi-subunit enzyme.

Biochemistry & Molecular Biology↗

Development of a novel minigenome and recombinant VSV expressing Seoul hantavirus glycoprotein-based assays to identify anti-hantavirus therapeutics

Seoul virus (SEOV) is an emerging global health threat that can cause hemorrhagic fever with renal syndrome (HFRS), which results in case fatality rates of ~2%. There are no approved treatments for SEOV infections. We developed a cell-based assay system to identify potential antiviral compounds for SEOV and generated additional assays to characterize the mode of action of any promising antivirals. Here, to test if candidate antivirals targeted SEOV glycoprotein-mediated entry, we developed a recombinant reporter vesicular stomatitis virus expressing SEOV glycoproteins. To facilitate the identification of candidate antiviral compounds targeting viral transcription/replication, we successfully generated the first reported minigenome system for SEOV. This SEOV minigenome (SEOV-MG) screening assay will also serve as a prototype assay for discovery of small molecules inhibiting replication of other hantaviruses, including Andes and Sin Nombre viruses. Ours is a proof-of-concept study in which we tested several compounds previously reported to have activity against other negative-strand RNA viruses using our newly developed hantavirus antiviral screening systems. These systems can be used under lower biocontainment conditions than those needed for infectious viruses, and identified several compounds with robust anti-SEOV activity. Our findings have important implications for the development of anti-hantavirus therapeutics.

60 APPLIED LIFE SCIENCES↗

Structural, biophysical, and biochemical insights into C–S bond cleavage by dimethylsulfone monooxygenase

Sulfur is an essential element for life. Bacteria can obtain sulfur from inorganic sulfate; but in the sulfur starvation–induced response,Pseudomonadsemploy two-component flavin-dependent monooxygenases (TC-FMOs) from themsuandsfnoperons to assimilate sulfur from environmental compounds including alkanesulfonates and dialkylsulfones. Here, we report binding studies of oxidized FMN to enzymes involved within theP. fluorescensenzymatic pathway responsible for converting dimethylsulfone (DMSO 2 ) to sulfite. In this catabolic pathway, SfnG serves as the initial TC-FMO for sulfur assimilation, which is investigated in detail by solving the 2.6-Å resolution crystal structure of unliganded SfnG and the 1.75-Å resolution crystal structure of the SfnG ternary complex containing FMN and DMSO 2 . We find that SfnG adopts a (β/α) 8 barrel fold with a distinct quaternary configuration from other tetrameric class C TC-FMOs. To probe the unexpected tetramer arrangement, structural heterogeneity is assessed by chromatography and light scattering to confirm ligand binding correlates with a tetramer. Binding of FMN and DMSO 2 accompanies ordering of the active site, with DMSO 2 bound on thesi-face of the flavin. A previously unobserved protein backbone conformation is found within the oxygen-binding site on there-face of the flavin. Functional assays and the positioning of ligands with respect to the oxygen-binding site are consistent with use of an N5-(hydro)peroxyflavin pathway. Biochemical endpoint assays and docking studies reveal SfnG breaks the C–S bond of a range of dialkylsulfones.

Science & Technology - Other Topics↗

Crystal structure of MutYX: a novel clusterless adenine DNA glycosylase with a distinct C-terminal domain and 8-oxoguanine recognition sphere

Abstract The [4Fe–4S] cluster is an important cofactor of the base excision repair (BER) adenine DNA glycosylase MutY to prevent mutations associated with 8-oxoguanine (OG). Several MutYs lacking the [4Fe–4S] cofactor have been identified. Phylogenetic analysis shows that clusterless MutYs are distributed in two clades suggesting cofactor loss has occurred in multiple independent evolutionary events. Herein, we determined the first crystal structure of a clusterless MutY complexed with DNA. On the basis of the dramatic structural divergence from canonical MutYs, we refer to this as representative of a clusterless MutY subgroup “MutYX.” Interestingly, MutYX compensates for the missing [4Fe–4S] cofactor to maintain positioning of catalytic residues by expanding a pre-existing α-helix and acquisition of a new α-helix. Surprisingly, MutYX also acquired a new C-terminal domain that uniquely recognizes OG using residues Gln201 and Arg209. Adenine glycosylase assays and binding affinity measurements indicate that Arg209 is the primary residue responsible for OG:A lesion specificity, while Gln201 assists by bridging OG and Arg209. Surprisingly, replacement of Arg209 and Gln201 with Ala increased activity toward G:A mismatches. The MutYX structure serves as an example of devolution, capturing structural features required to retain function in the absence of a metal cofactor considered indispensable.

Trasviña-Arenas, Carlos H [University of Californi↗

A glycan receptor kinase facilitates intracellular accommodation of arbuscular mycorrhiza and symbiotic rhizobia in the legume Lotus japonicus

Receptors that distinguish the multitude of microbes surrounding plants in the environment enable dynamic responses to the biotic and abiotic conditions encountered. In this study, we identify and characterise a glycan receptor kinase, EPR3a, closely related to the exopolysaccharide receptor EPR3. Epr3a is up-regulated in roots colonised by arbuscular mycorrhizal (AM) fungi and is able to bind glucans with a branching pattern characteristic of surface-exposed fungal glucans. Expression studies with cellular resolution show localised activation of the Epr3a promoter in cortical root cells containing arbuscules. Fungal infection and intracellular arbuscule formation are reduced in epr3a mutants. In vitro , the EPR3a ectodomain binds cell wall glucans in affinity gel electrophoresis assays. In microscale thermophoresis (MST) assays, rhizobial exopolysaccharide binding is detected with affinities comparable to those observed for EPR3, and both EPR3a and EPR3 bind a well-defined β-1,3/β-1,6 decasaccharide derived from exopolysaccharides of endophytic and pathogenic fungi. Both EPR3a and EPR3 function in the intracellular accommodation of microbes. However, contrasting expression patterns and divergent ligand affinities result in distinct functions in AM colonisation and rhizobial infection in Lotus japonicus . The presence of Epr3a and Epr3 genes in both eudicot and monocot plant genomes suggest a conserved function of these receptor kinases in glycan perception.

59 BASIC BIOLOGICAL SCIENCES↗

Measurement of Volatile Compounds for Real-Time Analysis of Soil Microbial Metabolic Response to Simulated Snowmelt

Snowmelt dynamics are a significant determinant of microbial metabolism in soil and regulate global biogeochemical cycles of carbon and nutrients by creating seasonal variations in soil redox and nutrient pools. With an increasing concern that climate change accelerates both snowmelt timing and rate, obtaining an accurate characterization of microbial response to snowmelt is important for understanding biogeochemical cycles intertwined with soil. However, observing microbial metabolism and its dynamics non-destructively remains a major challenge for systems such as soil. Microbial volatile compounds (mVCs) emitted from soil represent information-dense signatures and when assayed non-destructively using state-of-the-art instrumentation such as Proton Transfer Reaction-Time of Flight-Mass Spectrometry (PTR-TOF-MS) provide time resolved insights into the metabolism of active microbiomes. In this study, we used PTR-TOF-MS to investigate the metabolic trajectory of microbiomes from a subalpine forest soil, and their response to a simulated wet-up event akin to snowmelt. Using an information theory approach based on the partitioning of mutual information, we identified mVC metabolite pairs with robust interactions, including those that were non-linear and with time lags. The biological context for these mVC interactions was evaluated by projecting the connections onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) network of known metabolic pathways. Simulated snowmelt resulted in a rapid increase in the production of trimethylamine (TMA) suggesting that anaerobic degradation of quaternary amine osmo/cryoprotectants, such as glycine betaine, may be important contributors to this resource pulse. Unique and synergistic connections between intermediates of methylotrophic pathways such as dimethylamine, formaldehyde and methanol were observed upon wet-up and indicate that the initial pulse of TMA was likely transformed into these intermediates by methylotrophs. Increases in ammonia oxidation signatures (transformation of hydroxylamine to nitrite) were observed in parallel, and while the relative role of nitrifiers or methylotrophs cannot be confirmed, the inferred connection to TMA oxidation suggests either a direct or indirect coupling between these processes. Overall, it appears that such mVC time-series from PTR-TOF-MS combined with causal inference represents an attractive approach to non-destructively observe soil microbial metabolism and its response to environmental perturbation.

59 BASIC BIOLOGICAL SCIENCES↗

A biophysical framework for double-drugging kinases

Selective orthosteric inhibition of kinases has been challenging due to the conserved active site architecture of kinases and emergence of resistance mutants. Simultaneous inhibition of distant orthosteric and allosteric sites, which we refer to as “double-drugging”, has recently been shown to be effective in overcoming drug resistance. However, detailed biophysical characterization of the cooperative nature between orthosteric and allosteric modulators has not been undertaken. Here, we provide a quantitative framework for double-drugging of kinases employing isothermal titration calorimetry, Förster resonance energy transfer, coupled-enzyme assays, and X-ray crystallography. We discern positive and negative cooperativity for Aurora A kinase (AurA) and Abelson kinase (Abl) with different combinations of orthosteric and allosteric modulators. We find that a conformational equilibrium shift is the main principle governing cooperativity. Notably, for both kinases, we find a synergistic decrease of the required orthosteric and allosteric drug dosages when used in combination to inhibit kinase activities to clinically relevant inhibition levels. X-ray crystal structures of the double-drugged kinase complexes reveal the molecular principles underlying the cooperative nature of double-drugging AurA and Abl with orthosteric and allosteric inhibitors. Finally, we observe a fully closed conformation of Abl when bound to a pair of positively cooperative orthosteric and allosteric modulators, shedding light on the puzzling abnormality of previously solved closed Abl structures. Collectively, our data provide mechanistic and structural insights into rational design and evaluation of double-drugging strategies.

59 BASIC BIOLOGICAL SCIENCES↗

Listeria monocytogenes loss of cultivability on carrot is associated with the formation of mesosome-like structures

Raw carrot is known to have antimicrobial activity against Listeria monocytogenes, but the mechanism of action has not been fully elucidated. In this study, we examined carrot antilisterial activity against several strains of Listeria species (including L. grayi, L. innocua, L. seeligeri, and L. welshimeri) and L. monocytogenes. A representative strain of L. monocytogenes was subsequently used for further characterizing carrot antilisterial activity. Exposure to fresh-cut carrot for 15 min resulted in a similar loss of cultivability, ranging from 2.5 to 4.7 log units, across all Listeria strains evaluated. L. monocytogenes recovered from the fresh-cut surface of different raw carrots was 1.6 to 4.1 log lower than levels obtained from paired boiled carrot samples with abolished antilisterial activity. L. monocytogenes levels recovered from fresh-cut carrot were 2.8 to 3.1 log lower when enumerated by culture-dependent methods than by the culture-independent method of PMAxx-qPCR, a qPCR assay that is performed using DNA pre-treated to selectively sequester DNA from cells with injured membranes. These results suggested that L. monocytogenes loss of cultivability on fresh-cut carrot was not associated with a loss of L. monocytogenes cell membrane integrity and putative cell viability. Transmission electron microscopy imaging revealed that L. monocytogenes rapidly formed mesosome-like structures upon exposure to carrot fresh-cut surface but not upon exposure to boiled carrot surface, suggesting there may be an association between the formation of these mesosome-like structures and a loss of cultivability in L. monocytogenes. As a result, further research is necessary to conclude the causality of this association.

59 BASIC BIOLOGICAL SCIENCES↗

Circadian immunometabolic states impart a temporal response to SARS-CoV-2 spike proteins in mammalian macrophages

Circadian rhythms, the 24-hour cycles that tune organismal physiology to the daily rhythms of light and dark, optimally organize cellular processes such as metabolism and mitochondrial function. In mammals, macrophage functions are regulated by these 24-hour circadian rhythms such that the immunometabolic response is coordinated across the day, consolidating macrophage physiology into temporally distinct phases to time the cellular immune response. However, while it is known that there are time-of-day specific responses to stress in a macrophage, little has been done to determine if circadian regulation coordinates the response of a macrophage to real-world pathogens. Importantly, key proteins in the response to viral infection have been found to be under circadian control, and time of day of application is known to affect the efficacy of vaccinations, including in the case of the COVID-19 virus. Therefore, to investigate if the circadian regulation of macrophage physiology imparted a time-of-day response to viral exposure, we exposed primary mouse and human macrophages to the SARS-CoV-1 and CoV-2 spike proteins at different times over the circadian day. To establish a time-of-day effect, we performed a multi-omics analysis and in vitro tissue culture assays examining macrophage responses over circadian time. We found that, conserved across the species, the timing of spike protein exposure dictated two distinct temporal responses which were characterized by hallmarks of immunometabolic suppression and modest inflammatory activation. However, these responses were primarily influenced by central metabolic and mitochondrial changes and not by classical immune activation.

Circadian Biology↗

Hypoxia-cultured mouse mesenchymal stromal cells from bone marrow and compact bone display different phenotypic traits

Highlights: • Mouse compact bone is richer in MSC compared to central bone marrow. • Derivation of MSC from mouse compact bone is associated with lower risk of contamination with hematopoietic cells. • MSC from compact bone display higher osteogenic differentiation capacity than bone marrow MSC. It has been suggested that the bone marrow microenvironment harbors two distinct populations of mesenchymal stromal cells (MSC), one with a perivascular location and other present in the endosteum. A better understanding of the biology of these MSC subsets has been pursued in order to refine its clinical application. However, most comparative characterizations of mouse MSC have been performed in normoxia. This can result in misleading interpretations since mouse MSC subsets with low/defective p53 activity are known to be selected during culture in normoxia. Here, we report a comprehensive in vitro characterization of mouse MSC isolated from bone marrow (BM-MSC) and compact bone (CB-MSC) expanded and assayed under hypoxia for their morphology, clonogenic efficiency and differentiation capacity. We found that, under hypoxia, compact bone is richer in absolute numbers of MSC and isolation of MSC from compact bone is associated with a reduced risk of hematopoietic cell carryover. In addition, CB-MSC have higher in vitro osteogenic capacity than BM-MSC, while adipogenic differentiation potential is similar. These findings reinforce the hypothesis of the existence of MSC in bone marrow and compact bone representing functionally distinct cell populations and highlight the compact bone as an efficient source of murine MSC under physiological oxygen concentrations.

60 APPLIED LIFE SCIENCES↗

Functional Impact of a Cancer-Related Variant in Human Δ 1 -Pyrroline-5-Carboxylate Reductase 1

Pyrroline-5-carboxylate reductase (PYCR) is a proline biosynthetic enzyme that catalyzes the NAD(P)H-dependent reduction of Δ 1 -pyrroline-5-carboxylate (P5C) to proline. Humans have three PYCR isoforms, with PYCR1 often upregulated in different types of cancers. Here, we studied the biochemical and structural properties of the Thr171Met variant of PYCR1, which is found in patients with malignant melanoma and lung adenocarcinoma. Although PYCR1 is strongly associated with cancer progression, characterization of a PYCR1 variant in cancer patients has not yet been reported. Thr171 is conserved in all three PYCR isozymes and is located near the P5C substrate binding site. We found that the amino acid replacement does not affect thermostability but has a profound effect on PYCR1 catalytic activity. The k cat of the PYCR1 variant T171M is 100- to 200-fold lower than wild-type PYCR1 when P5C is the variable substrate, and 10- to 25-fold lower when NAD(P)H is varied. A 1.84 Å resolution X-ray crystal structure of T171M reveals that the Met side chain invades the P5C substrate binding site, suggesting that the catalytic defect is due to steric clash preventing P5C from achieving the optimal pose for hydride transfer from NAD(P)H. These results suggest that any impact on PYCR1 function associated with T171M in cancer does not derive from increased catalytic activity.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

A tunable LIC1-adaptor interaction modulates dynein activity in a cargo-specific manner

Cytoplasmic dynein-1 (dynein) is the motor responsible for most retrograde transport of cargoes along microtubules in eukaryotic cells, including organelles, mRNA and viruses. Cargo selectivity and activation of processive motility depend on a group of so-called “activating adaptors” that link dynein to its general cofactor, dynactin, and cargoes. The mechanism by which these adaptors regulate dynein transport is poorly understood. Here, based on crystal structures, quantitative binding studies, and in vitro motility assays, we show that BICD2, CRACR2a, and HOOK3, representing three subfamilies of unrelated adaptors, interact with the same amphipathic helix of the dynein light intermediate chain-1 (LIC1). While the hydrophobic character of the interaction is conserved, the three adaptor subfamilies use different folds (coiled-coil, EF-hand, HOOK domain) and different surface contacts to bind the LIC1 helix with affinities ranging from 1.5 to 15.0 μM. We propose that a tunable LIC1-adaptor interaction modulates dynein’s motility in a cargo-specific manner.

59 BASIC BIOLOGICAL SCIENCES↗

The rate of microtubule breaking increases exponentially with curvature

Microtubules, cylindrical assemblies of tubulin proteins with a 25 nm diameter and micrometer lengths, are a central part of the cytoskeleton and also serve as building blocks for nanobiodevices. Microtubule breaking can result from the activity of severing enzymes and mechanical stress. Breaking can lead to a loss of structural integrity, or an increase in the numbers of microtubules. We observed breaking of taxol-stabilized microtubules in a gliding motility assay where microtubules are propelled by surface-adhered kinesin-1 motor proteins. We find that over 95% of all breaking events are associated with the strong bending following pinning events (where the leading tip of the microtubule becomes stuck). Furthermore, the breaking rate increased exponentially with increasing curvature. These observations are explained by a model accounting for the complex mechanochemistry of a microtubule. The presence of severing enzymes is not required to observe breaking at rates comparable to those measured previously in cells.

42 ENGINEERING↗