Structure and dynamics of a pentameric KCTD5/CUL3/Gßγ E3 ubiquitin ligase complex.

Heterotrimeric G proteins can be regulated by posttranslational modifications, including ubiquitylation. KCTD5, a pentameric substrate receptor protein consisting of an N-terminal BTB domain and a C-terminal domain, engages CUL3 to form the central scaffold of a cullin-RING E3 ligase complex (CRL3KCTD5) that ubiquitylates Gßγ and reduces Gßγ protein levels in cells. The cryo-EM structure of a 5:5:5 KCTD5/CUL3NTD/Gß1γ2 assembly reveals a highly dynamic complex with rotations of over 60° between the KCTD5BTB/CUL3NTD and KCTD5CTD/Gßγ moieties of the structure. CRL3KCTD5 engages the E3 ligase ARIH1 to ubiquitylate Gßγ in an E3-E3 superassembly, and extension of the structure to include full-length CUL3 with RBX1 and an ARIH1~ubiquitin conjugate reveals that some conformational states position the ARIH1~ubiquitin thioester bond to within 10 Å of lysine-23 of Gß and likely represent priming complexes. Most previously described CRL/substrate structures have consisted of monovalent complexes and have involved flexible peptide substrates. The structure of the KCTD5/CUL3NTD/Gßγ complex shows that the oligomerization of a substrate receptor can generate a polyvalent E3 ligase complex and that the internal dynamics of the substrate receptor can position a structured target for ubiquitylation in a CRL3 complex.

The HisRS-like domain of GCN2 is a pseudoenzyme that can bind uncharged tRNA.

GCN2 is a stress response kinase that phosphorylates the translation initiation factor eIF2α to inhibit general protein synthesis when activated by uncharged tRNA and stalled ribosomes. The presence of a HisRS-like domain in GCN2, normally associated with tRNA aminoacylation, led to the hypothesis that eIF2α kinase activity is regulated by the direct binding of this domain to uncharged tRNA. Here we solved the structure of the HisRS-like domain in the context of full-length GCN2 by cryoEM. Structure and function analysis shows the HisRS-like domain of GCN2 has lost histidine and ATP binding but retains tRNA binding abilities. Hydrogen deuterium exchange mass spectrometry, site-directed mutagenesis and computational docking experiments support a tRNA binding model that is partially shifted from that employed by bona fide HisRS enzymes. These results demonstrate that the HisRS-like domain of GCN2 is a pseudoenzyme and advance our understanding of GCN2 regulation and function.

Direct structural analysis of a single acyl carrier protein domain in fatty acid synthase from the fungus Saccharomyces cerevisiae.

Acyl carrier protein (ACP) is the work horse of polyketide (PKS) and fatty acid synthases (FAS) and acts as a substrate shuttling domain in these mega enzymes. In fungi, FAS forms a 2.6 MDa symmetric assembly with six identical copies of FAS1 and FAS2 polypeptides. However, ACP spatial distribution is not restricted by symmetry owing to the long and flexible loops that tether the shuttling domain to its corresponding FAS2 polypeptide. This symmetry breaking has hampered experimental investigation of substrate shuttling route in fungal FAS. Here, we develop a protein engineering and expression method to isolate asymmetric fungal FAS proteins containing odd numbers of ACP domains. Electron cryomicroscopy (cryoEM) observation of the engineered complex reveals a non-uniform distribution of the substrate shuttling domain relative to its corresponding FAS2 polypeptide at 2.9 Å resolution. This work lays the methodological foundation for experimental study of ACP shuttling route in fungi.

Expression of concern: Cobalt metal-organic framework-based ZIF-67 for the trace determination of herbicide molinate by ion mobility spectrometry: investigation of different morphologies.

Expression of concern for 'Cobalt metal-organic framework-based ZIF-67 for the trace determination of herbicide molinate by ion mobility spectrometry: investigation of different morphologies' by Mehdi Davoodi et al., RSC Adv., 2021, 11, 2643-2655, DOI: https://doi.org/10.1039/D0RA09298C.

Expression of concern: Synthesis and characterization of a new ZIF-67@MgAl2O4 nanocomposite and its adsorption behaviour.

Expression of concern for 'Synthesis and characterization of a new ZIF-67@MgAl2O4 nanocomposite and its adsorption behaviour' by Mehdi Davoodi et al., RSC Adv., 2021, 11, 13245-13255, https://doi.org/10.1039/D1RA01056E.

Atomic model for core modifying region of human fatty acid synthase in complex with Denifanstat.

Fatty acid synthase (FASN) catalyzes the de novo synthesis of palmitate, a 16-carbon chain fatty acid that is the primary precursor of lipid metabolism and an important intracellular signaling molecule. FASN is an attractive drug target in diabetes, cancer, fatty liver diseases, and viral infections. Here, we develop an engineered full-length human FASN (hFASN) that enables isolation of the condensing and modifying regions of the protein post-translation. The engineered protein enables electron cryo-microscopy (cryoEM) structure determination of the core modifying region of hFASN to 2.7 Å resolution. Examination of the dehydratase dimer within this region reveals that unlike its close homolog, porcine FASN, the catalytic cavity is close-ended and is accessible only through one opening in the vicinity of the active site. The core modifying region exhibits two major global conformational variabilities that describe long-range bending and twisting motions of the complex in solution. Finally, we solved the structure of this region bound to an anti-cancer drug, Denifanstat (i.e., TVB-2640), demonstrating the utility of our approach as a platform for structure guided design of future hFASN small molecule inhibitors.

Identification of triazenyl indoles as inhibitors of fungal fatty acid biosynthesis with broad-spectrum activity.

Rising drug resistance among pathogenic fungi, paired with a limited antifungal arsenal, poses an increasing threat to human health. To identify antifungal compounds, we screened the RIKEN natural product depository against representative isolates of four major human fungal pathogens. This screen identified NPD6433, a triazenyl indole with broad-spectrum activity against all screening strains, as well as the filamentous mold Aspergillus fumigatus. Mechanistic studies indicated that NPD6433 targets the enoyl reductase domain of fatty acid synthase 1 (Fas1), covalently inhibiting its flavin mononucleotide-dependent NADPH-oxidation activity and arresting essential fatty acid biosynthesis. Robust Fas1 inhibition kills Candida albicans, while sublethal inhibition impairs diverse virulence traits. At well-tolerated exposures, NPD6433 extended the lifespan of nematodes infected with azole-resistant C. albicans. Overall, identification of NPD6433 provides a tool with which to explore lipid homeostasis as a therapeutic target in pathogenic fungi and reveals a mechanism by which Fas1 function can be inhibited.

Structure of the immunoregulatory sialidase NEU1.

Sialic acids linked to glycoproteins and glycolipids are important mediators of cell and protein recognition events. These sugar residues are removed by neuraminidases (sialidases). Neuraminidase-1 (sialidase-1 or NEU1) is a ubiquitously expressed mammalian sialidase located in lysosomes and on the cell membrane. Because of its modulation of multiple signaling processes, it is a potential therapeutic target for cancers and immune disorders. Genetic defects in NEU1 or in its protective protein cathepsin A (PPCA, CTSA) cause the lysosomal storage diseases sialidosis and galactosialidosis. To further our understanding of this enzyme's function at the molecular level, we determined the three-dimensional structure of murine NEU1. The enzyme oligomerizes through two self-association interfaces and displays a wide substrate-binding cavity. A catalytic loop adopts an inactive conformation. We propose a mechanism of activation involving a conformational change in this loop upon binding to its protective protein. These findings may facilitate the development of selective inhibitor and agonist therapies.

Silica aerogel modified electrospun polyacrylonitrile as a sorbent for thin-film microextraction of chlorpyrifos from real samples coupled with corona discharge ion mobility spectrometry detection.

In this paper, modified polyacrylonitrile/silica aerogel fibers were prepared and used as an adsorbent for thin-film microextraction of chlorpyrifos. The extracted analyte was analyzed by corona discharge ion mobility spectrometry. The electrospinning method was applied for the preparation of polyacrylonitrile fibers. The alkaline hydrolyzation technique was used to modify the electrospun film surface. Silica aerogel was synthesized on the surface of modified electrospun polyacrylonitrile fibers by the in situ growth technique. To access a high extraction yield, effective synthesis and extraction parameters such as NaOH concentration, reaction temperature and time, thin-film pretreatment, gelation time, solution pH, ionic strength, and extraction time were studied. The linearity range and the limit of detection of the method were 1-100 µg L-1 and 0.3 µg L-1, respectively. The precision of the method was 4 and 12% for the concentration levels of 5 and 60 µg L-1, respectively. Chlorpyrifos was successfully determined by the method in well water, river water, agricultural wastewater, and tangerine samples.

The Trisubstituted Isoxazole MMV688766 Exerts Broad-Spectrum Activity against Drug-Resistant Fungal Pathogens through Inhibition of Lipid Homeostasis.

Candida species are among the most prevalent causes of systemic fungal infection, posing a growing threat to public health. While Candida albicans is the most common etiological agent of systemic candidiasis, the frequency of infections caused by non-albicans Candida species is rising. Among these is Candida auris, which has emerged as a particular concern. Since its initial discovery in 2009, it has been identified worldwide and exhibits resistance to all three principal antifungal classes. Here, we endeavored to identify compounds with novel bioactivity against C. auris from the Medicines for Malaria Venture's Pathogen Box library. Of the five hits identified, the trisubstituted isoxazole MMV688766 emerged as the only compound displaying potent fungicidal activity against C. auris, as well as other evolutionarily divergent fungal pathogens. Chemogenomic profiling, as well as subsequent metabolomic and phenotypic analyses, revealed that MMV688766 disrupts cellular lipid homeostasis, driving a decrease in levels of early sphingolipid intermediates and fatty acids and a concomitant increase in lysophospholipids. Experimental evolution to further probe MMV688766's mode of action in the model fungus Saccharomyces cerevisiae revealed that loss of function of the transcriptional regulator HAL9 confers resistance to MMV688766, in part through the upregulation of the lipid-binding chaperone HSP12, a response that appears to assist in tolerating MMV688766-induced stress. The novel mode of action we have uncovered for MMV688766 against drug-resistant fungal pathogens highlights the broad utility of targeting lipid homeostasis to disrupt fungal growth and how screening structurally-diverse chemical libraries can provide new insights into resistance-conferring stress responses of fungi. IMPORTANCE As widespread antimicrobial resistance threatens to propel the world into a postantibiotic era, there is a pressing need to identify mechanistically distinct antimicrobial agents. This is of particular concern when considering the limited arsenal of drugs available to treat fungal infections, coupled with the emergence of highly drug-resistant fungal pathogens, including Candida auris. In this work, we demonstrate that existing libraries of drug-like chemical matter can be rich resources for antifungal molecular scaffolds. We discovered that the small molecule MMV688766, from the Pathogen Box library, displays previously undescribed broad-spectrum fungicidal activity through perturbation of lipid homeostasis. Characterization of the mode of action of MMV688766 provided new insight into the protective mechanisms fungi use to cope with the disruption of lipid homeostasis. Our findings highlight that elucidating the genetic circuitry required to survive in the presence of cellular stress offers powerful insights into the biological pathways that govern this important phenotype.

Corona discharge ionization ion mobility spectrometry for ultra-trace determination of methamphetamine extracted from urine and plasma samples by dispersive liquid-liquid microextraction.

In this work, dispersive liquid-liquid microextraction (DLLME) based on high-density extraction solvent was applied as a simple, fast and sensitive method for extraction and preconcentration of methamphetamine from human plasma and urine samples. The efficiency of positive corona discharge ionization ion mobility spectrometry was investigated for direct analysis of the extracted analyte. Effective parameters on the extraction efficiency, such as type and volume of the extraction and disperser solvents, centrifugation time, and sample solution pH were optimized. Trichloromethane and isopropanol were selected as the extracting and disperser solvents, respectively. Under the optimized conditions, the linear dynamic range (R2 = 0.9969) was found to be 0.5-18 µg/L, and 0.15 µg/L was calculated as the limit of detection. The relative standard deviations of intra- and inter-day were obtained 4 and 10%, respectively, and finally, in the analysis of human plasma and urine samples, the extraction recovery was obtained 104%.

SARS-CoV-2 impairs interferon production via NSP2-induced repression of mRNA translation.

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a mechanism by which the SARS-CoV-2 virus coopts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-ß. We reveal that the SARS-CoV-2 encoded nonstructural protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-ß production, which inhibits SARS-CoV-2 replication. Our findings reveal a target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

Investigation of different alcoholic modifiers for the separation and determination of two isomers of dinitrotoluene (2,4 and 2,6) by ion mobility spectrometry.

Some alcoholic modifier gases were applied to separate isomer peaks in ion mobility spectrometry (IMS). Different mechanisms have been investigated on the separation, such as collision cross-section and analyte-modifier cluster formation. In this regard, some parameters that affected the cluster formation, such as dipole moment, electron affinity, the position of functional groups, and the modifier structure, were evaluated. On the other hand, some effective experimental parameters, including cell temperature and the flow rates of the drift and modifier gases, were also optimized. The combination of dispersive liquid-liquid microextraction with thin-film evaporation (DLLME-TFE) was used as a sample preparation method for the extraction of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) isomers (as the target analytes). Isobutanol was selected as the alcoholic modifier to separate the ion molecular peaks of these isomers. The limit of detection and the limit of quantification obtained were 15 and 50 µg L-1 , and the linear dynamic range (50-700 µg L-1 ) with coefficient of determination of 0.9941 and 0.9914 were obtained for 2,4-DNT and 2,6-DNT, respectively. The intra- and inter-day relative standard deviations were obtained between 3% and 5%. For validation of the method, determination of the isomers was accomplished for a red wastewater field sample, resulting in relative recovery values of about 96%.

High efficient solid-phase microextraction based on a covalent organic framework for determination of trifluralin and chlorpyrifos in water and food samples by GC-CD-IMS.

Novel porous covalent organic framework (COF) based on condensation reaction between cyanuric chloride, 4,4'-ethylendianiline, and 3,4,9,10-perylenetetracarboxylic dianhydride was synthesized via sealed tube condition. The results COF was used as a new adsorbent for solid-phase microextraction (SPME) for extracting trifluralin and chlorpyrifos from vegetables, fruit samples, and wastewater. Gas chromatograph with a corona discharge-ion mobility spectrometer as the detector was also used for analyzing the target analytes. Some parameters that affected the extraction, such as stirring rate, time and temperature of extraction and pH were investigated, exhaustively. The detection limits were 0.13, and 0.15 µg/L and the linear ranges of 0.45-20 and 0.50-25 µg/L with a linearity coefficient of 0.9965 and 0.9987 were also obtained for trifluralin and chlorpyrifos, respectively. The method was applied successfully to analyze some real samples of cucumber, carrot, grape, and agriculture wastewater, and the results showed a relative recovery in the range of 87% to 110%.

Structural Characterization of Endogenous Tuberous Sclerosis Protein Complex Revealed Potential Polymeric Assembly.

Tuberous sclerosis protein complex (pTSC) nucleates a proteinaceous signaling hub that integrates information about the internal and external energy status of the cell in the regulation of growth and energy consumption. Biochemical and cryo-electron microscopy studies of recombinant pTSC have revealed its structure and stoichiometry and hinted at the possibility that the complex may form large oligomers. Here, we have partially purified endogenous pTSC from fasted mammalian brains of rat and pig by leveraging a recombinant antigen binding fragment (Fab) specific for the TSC2 subunit of pTSC. We demonstrate Fab-dependent purification of pTSC from membrane-solubilized fractions of the brain homogenates. Negative stain electron microscopy of the samples purified from pig brain demonstrates rod-shaped protein particles with a width of 10 nm, a variable length as small as 40 nm, and a high degree of conformational flexibility. Larger filaments are evident with a similar 10 nm width and a ≤1 µm length in linear and weblike organizations prepared from pig brain. Immunogold labeling experiments demonstrate linear aggregates of pTSC purified from mammalian brains. These observations suggest polymerization of endogenous pTSC into filamentous superstructures.

Self-rotating stir mesh screen sorptive extraction for analyzing chlorpyrifos by ion mobility spectrometry.

A mesh screen was electrochemically coated with polypyrrole and used as a sorptive extractor device, for the first time. This configuration acts in such a way that it is self-rotating in the presence of a magnetic force and can be used for extraction and concentration of analytes. Actually, applying a mesh screen instead of a bar or plate in sorptive extraction provided a more effective contact area between the sorptive materials and sample solution, resulting in higher sorption efficiency. The device performance was assessed by using chlorpyrifos pesticide as a model analyte. A thermal desorption unit was coupled to an ion mobility spectrometer and applied for evaporating the extracted analyte. Different parameters affecting the extraction efficiency during the electro-polymerization and the extraction process, including the time of electrodeposition, the concentration of pyrrole, oxalic acid and salt, temperature and time of extraction, and the stirring rate of the extractor device were investigated and optimized, simultaneously. The detection and quantification limits of the method were calculated to be 0.035 and 0.1 µg L-1, respectively. The linear dynamic range obtained was from 0.1 to 20 µg L-1, with a determination coefficient of 0.9984. The intra-day and inter-day-relative standard deviations (RSD, n = 3) were lower than 3% and 8%, respectively. Under the optimal conditions, the absolute recovery and the enrichment factor were found to be 97% and 5820, respectively. Finally, the relative recoveries of the proposed method were calculated to be in the range of 86-111% for spiked water, wastewater, and apple samples. The results obtained from the method were validated by EPA method 622.

Structure of the murine lysosomal multienzyme complex core.

The enzymes ß-galactosidase (GLB1) and neuraminidase 1 (NEU1; sialidase 1) participate in the degradation of glycoproteins and glycolipids in the lysosome. To remain active and stable, they associate with PPCA [protective protein cathepsin A (CTSA)] into a high-molecular weight lysosomal multienzyme complex (LMC), of which several forms exist. Genetic defects in these three proteins cause the lysosomal storage diseases GM1-gangliosidosis/mucopolysaccharidosis IV type B, sialidosis, and galactosialidosis, respectively. To better understand the interactions between these enzymes, we determined the three-dimensional structure of the murine LMC core. This 0.8-MDa complex is composed of three GLB1 dimers and three CTSA dimers, adopting a triangular architecture maintained through six copies of a unique GLB1-CTSA polar interface. Mutations in this contact surface that occur in GM1-gangliosidosis prevent formation of the LMC in vitro. These findings may facilitate development of therapies for lysosomal storage disorders.

Centrifuge-free dispersive liquid-liquid microextraction coupled with thin-film microextraction for the preconcentration of molinate in real samples by ion mobility spectrometry.

A tandem microextraction method, centrifuge free dispersive liquid-liquid microextraction and thin-film microextraction (DLLME-TFME), was used for analyzing molinate in environmental samples by ion mobility spectrometry (IMS). Considering the IMS as a competitive detection system, coupling these two popular sample preparation methods reduces the effect of solvent interference and improves the sensitivity of the technique. Trichloromethane and methanol were used as the extraction, and dispersive solvents for the DLLME method and electrospun polyacrylonitrile/copper-benzene-1,4-dicarboxylic acid fibers were used as a sorbent in the TFME method. Some effective experimental variables influencing the extraction efficiency of an analyte such as type and volume of dispersive and extraction solvents, solution pH, ionic strength, sonication time, and extraction time were studied. The linear dynamic range of 0.5-50 µg L-1 and the limit of detection of 0.1 µg L-1 were obtained under optimized conditions. The relative standard deviations for intra-and inter-day analysis were calculated less than 10%. The present method was used for the determination of molinate in different real samples such as agricultural wastewater, well water, river water, and apple, and the recovery was obtained between 82% and 113%, for the spiked samples.

Covalent triazine-based framework-grafted functionalized fibrous silica sphere as a solid-phase microextraction coating for simultaneous determination of fenthion and chlorpyrifos by ion mobility spectrometry.

A novel covalent triazine-based framework (CTF)-grafted phenyl-functionalized fibrous silica nanosphere, KCC-1 (named as RS-2) was synthesized via a simple and effective Friedel-Crafts approach. The microporous CTF with fluorene backbone was coupled and grown uniformly on the surface of phenyl-functionalized KCC-1 to prepare a hybrid extended porous framework. The prepared materials were characterized, and FE-SEM and TEM images revealed a flower-like structure for RS-2. The synthesized RS-2 showed excellent thermal stability, so the weight loss was about 30% at 800 °C. RS-2 was applied as a new coating in the solid-phase microextraction procedure to extract chlorpyrifos and fenthion pesticides from water, wastewater, and fruit samples, before determining by corona discharge-ion mobility spectrometry. Some experimental factors affecting the extraction yield of the analytes, including ionic strength, stirring rate, sample pH, extraction temperature, and extraction time, were investigated. Under optimum conditions, the linear dynamic ranges were 0.1-10 µg L-1 and 1.0-70 µg L-1, and the limits of detection were 0.05 and 0.55 µg L-1 for chlorpyrifos and fenthion, respectively. The proposed method showed recovery values in the range 86-117% with a precision of 3.0-7.1% for real samples. Covalent triazine-based framework (CTF)-grafted phenyl-functionalized fibrous silica nanosphere (named as RS-2) was synthesized. RS-2 was applied as a sorbent for solid-phase microextraction (SPME) of chlorpyrifos and fenthion from fruit and water samples followed by corona discharge ionization ion mobility spectrometry (CD-IMS).

Correction: Synthesis and characterization of a new ZIF-67@MgAl2O4 nanocomposite and its adsorption behaviour.

[This corrects the article DOI: 10.1039/D1RA01056E.].