Scholarly Works, Biochemistry

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Evolution at Spike protein position 519 in SARS-CoV-2 facilitated adaptation to humans
Cereghino, C.; Michalak, K.; DiGiuseppe, S.; Yu, D.; Faraji, A.; Sharp, A.K.; Brown, Anne M.; Kang, L.; Weger-Lucarelli, James; Michalak, P. (Springer Nature, 2024)
As the COVID-19 pandemic enters its fourth year, the pursuit of identifying a progenitor virus to SARSCoV- 2 and understanding the mechanism of its emergence persists, albeit against the backdrop of intensified efforts to monitor the ongoing evolution of the virus and the influx of new mutations. Surprisingly, few residues hypothesized to be essential forSARS-CoV-2 emergence and adaptation to humans have been validated experimentally, despite the importance that these mutations could contribute to the development of effective antivirals. To remedy this,we searched for genomic regions in the SARS-CoV-2 genome that show evidence of past selection around residues unique to SARSCoV- 2 compared with closely related coronaviruses. In doing so, we identified a residue at position 519 in Spike within the receptor binding domain that holds a static histidine in human-derived SARSCoV- 2 sequences but an asparagine in SARS-related coronaviruses from bats and pangolins. In experimental validation, the SARS-CoV-2 Spike protein mutant carrying the putatively ancestral H519N substitution showed reduced replication in human lung cells, suggesting that the histidine residue contributes to viral fitness in the human host. Structural analyses revealed a potential role of Spike residue 519 in mediating conformational transitions necessary for Spike prior to binding with ACE2. Pseudotyped viruses bearing the putatively ancestral N519 also demonstrated significantly reduced infectivity in cells expressing the human ACE2 receptor compared to H519. ELISA data corroborated that H519 enhances Spike binding affinity to the human ACE2 receptor compared to the putatively ancestral N519. Collectively, these findings suggest that the evolutionary transition at position 519 of the Spike protein played a critical role inSARS-CoV-2 emergence and adaptation to the human host. Additionally, this residue presents as a potential drug target for designing small molecule inhibitors tailored to this site.
Indirectly acquired fear memories have distinct, sex-specific molecular signatures from directly acquired fear memories
Navabpour, Shaghayegh; Patrick, Morgan B.; Omar, Nour A.; Kincaid, Shannon E.; Bae, Yeeun; Abraham, Jennifer; McGrew, Jacobi; Musaus, Madeline; Ray, W. Keith; Helm, Richard F.; Jarome, Timothy J. (PLOS, 2024-12-23)
Post-traumatic stress disorder (PTSD) is a severe anxiety disorder that affects women more than men. About 30% of patients suffering from PTSD develop the disorder by witnessing a traumatic event happen to someone else. However, as the focus has remained on those directly experiencing the traumatic event, whether indirectly acquired fear memories that underlie PTSD have the same molecular signature as those that are directly acquired remains unknown. Here, using a rodent indirect fear learning paradigm where one rat (observer) watches another rat (demonstrator) associate an auditory cue with foot shock, we found that fear can be indirectly acquired by both males and females regardless of the sex or novelty (familiarity) of the demonstrator animal. However, behaviorally, indirectly acquired fear responses resemble those of pseudoconditioning, a behavioral response that is thought to not represent learning. Despite this, using unbiased proteomics, we found that indirectly acquired fear memories have distinct protein degradation profiles in the amygdala and anterior cingulate cortex (ACC) relative to directly acquired fear memories and pseudoconditioning, which further differed significantly by sex. Additionally, Egr2 and c-fos expression in the retrosplenial cortex of observer animals resembled that of demonstrator rats but was significantly different than that of pseudoconditioned rats. Together, these findings reveal that indirectly acquired fear memories have sex-specific molecular signatures that differ from those of directly acquired fear memories or pseudoconditioning. These data have important implications for understanding the neurobiology of indirectly acquired fear memories that may underlie bystander PTSD.
Evaluation of ebony as a potential selectable marker for genetic sexing in Aedes aegypti
Nikolouli, Katerina; Compton, Austin; Tu, Zhijian J.; Bourtzis, Kostas (2025-02-25)
Background: Aedes aegypti is expected to invade previously unoccupied areas, mainly due to the climate change, the increase in travel and trade activities and the continuous transformation of the rural environment into urban areas. The sterile insect technique (SIT), which relies on the mass production and release of sterile males, is an environmentally friendly approach that can be applied for population control of Ae. aegypti. SIT programs can be greatly benefited by a genetic sexing strain (GSS) and a reliable sex sorting system to minimize any accidental female release. Visually detectable or conditionally lethal selectable markers can be used for the development of new GSSs. In this study, we evaluated the suitability and competence of a mutant Ae. aegypti ebony strain for the development of a new GSS. The ebony gene is known to be involved in the pigmentation pathway of several dipteran insects, including Ae. aegypti. Methods: An ebony gene knockout was developed though CRISPR/Cas9 mutagenesis. G0 individuals with the desired phenotype were crossed, and progeny were screened in every generation. PCR and sequencing were performed using gDNA from a pulled leg to determine the mutant genotype. Quality control tests, including pupae and adult recovery rates, male sex ratio and fecundity, were applied to the ebony mutant line to determine whether the mutation confers any fitness cost. Results: An Ae. aegypti ebony knockout mutant carrying a 5-bp deletion was obtained, which presented darker head and siphon phenotypes at the larval stage. However, genetic analysis revealed that this ebony mutation results in incomplete penetrance and variable expressivity. The establishment of a pure ebony mutant line was not possible because of the fitness costs conferred by the mutation. Conclusions: In this study, the adequacy and suitability of the ebony gene as a selectable marker for the development of a GSS in Ae. aegypti were assessed. Despite its clear phenotype early in larval development, the homozygous mutant line presented phenotypic inconsistency and loss of fertility. These drawbacks clearly indicate that this particular mutation is not suitable for the development of a new GSS. Nonetheless, it cannot be excluded that a different mutation will lead to a different expression and penetrance profile and a viable homozygous mutant line.
Identification of small molecule inhibitors of the Chloracidobacterium thermophilum type IV pilus protein PilB by ensemble virtual screening
McDonald-Ramos, Jay S.; Hicklin, Ian K.; Yang, Zhaomin; Brown, Anne M. (Elsevier, 2024-08-16)
Antivirulence strategy has been explored as an alternative to traditional antibiotic development. The bacterial type IV pilus is a virulence factor involved in host invasion and colonization in many antibiotic resistant pathogens. The PilB ATPase hydrolyzes ATP to drive the assembly of the pilus filament from pilin subunits. We evaluated Chloracidobacterium thermophilum PilB (CtPilB) as a model for structure-based virtual screening by molecular docking and molecular dynamics (MD) simulations. A hexameric structure of CtPilB was generated through homology modeling based on an existing crystal structure of a PilB from Geobacter metallireducens. Four representative structures were obtained from molecular dynamics simulations to examine the conformational plasticity of PilB and improve docking analyses by ensemble docking. Structural analyses after 1 μs of simulation revealed conformational changes in individual PilB subunits are dependent on ligand presence. Further, ensemble virtual screening of a library of 4234 compounds retrieved from the ZINC15 database identified five promising PilB inhibitors. Molecular docking and binding analyses using the four representative structures from MD simulations revealed that top-ranked compounds interact with multiple Walker A residues, one Asp-box residue, and one arginine finger, indicating these are key residues in inhibitor binding within the ATP binding pocket. The use of multiple conformations in molecular screening can provide greater insight into compound flexibility within receptor sites and better inform future drug development for therapeutics targeting the type IV pilus assembly ATPase.
Spatial transcriptomic analysis reveals local effects of intratumoral fusobacterial infection on DNA damage and immune signaling in rectal cancer
Duggan, William P.; Kisakol, Batuhan; Woods, Ina; Azimi, Mohammedreza; Dussmann, Heiko; Fay, Joanna; O'Grady, Tony; Maguire, Barry; Reynolds, Ian S.; Salvucci, Manuela; Slade, Daniel J.; McNamara, Deborah A.; Burke, John P.; Prehn, Jochen H. M. (Taylor & Francis, 2024-05-06)
Mucinous colorectal cancer (CRC) is a common histological subtype of colorectal adenocarcinoma, associated with a poor response to chemoradiotherapy. The commensal facultative anaerobes fusobacteria, have been associated with poor prognosis specifically in mesenchymal CRC. Interestingly, fusobacterial infection is especially prevalent in mucinous CRC. The objective of this study was therefore to increase our understanding of beneficial and detrimental effects of fusobacterial infection, by contrasting host cell signaling and immune responses in areas of high vs. low infection, using mucinous rectal cancer as a clinically relevant example. We employed spatial transcriptomic profiling of 106 regions of interest from 8 mucinous rectal cancer samples to study gene expression in the epithelial and immune segments across regions of high versus low fusobacterial infection. Fusobacteria high regions were associated with increased oxidative stress, DNA damage, and P53 signaling. Meanwhile regions of low fusobacterial prevalence were characterized by elevated JAK-STAT, Il-17, Il-1, chemokine and TNF signaling. Immune masks within fusobacterial high regions were characterized by elevated proportions of cytotoxic (CD8+) T cells (p = 0.037), natural killer (NK) cells (p < 0.001), B-cells (p < 0.001), and gamma delta T cells (p = 0.003). Meanwhile, fusobacteria low regions were associated with significantly greater M2 macrophage (p < 0.001), fibroblast (p < 0.001), pericyte (p = 0.002), and endothelial (p < 0.001) counts.
Context-specific variation in life history traits and behavior of Aedes aegypti mosquitoes
Vinauger, Clément ; Chandrasegaran, Karthikeyan (Frontiers, 2024-09-25)
Aedes aegypti, the vector for dengue, chikungunya, yellow fever, and Zika, poses a growing global epidemiological risk. Despite extensive research on Ae. aegypti’s life history traits and behavior, critical knowledge gaps persist, particularly in integrating these findings across varied experimental contexts. The plasticity of Ae. aegypti’s traits throughout its life cycle allows dynamic responses to environmental changes, yet understanding these variations within heterogeneous study designs remains challenging. A critical aspect often overlooked is the impact of using lab-adapted lines of Ae. aegypti, which may have evolved under laboratory conditions, potentially altering their life history traits and behavioral responses compared to wild populations. Therefore, incorporating field-derived populations in experimental designs is essential to capture the natural variability and adaptability of Ae. aegypti. The relationship between larval growing conditions and adult traits and behavior is significantly influenced by the specific context in which mosquitoes are studied. Laboratory conditions may not replicate the ecological complexities faced by wild populations, leading to discrepancies in observed traits and behavior. These discrepancies highlight the need for ecologically relevant experimental conditions, allowing mosquito traits and behavior to reflect field distributions. One effective approach is semi-field studies involving field-collected mosquitoes housed for fewer generations in the lab under ecologically relevant conditions. This growing trend provides researchers with the desired control over experimental conditions while maintaining the genetic diversity of field populations. By focusing on variations in life history traits and behavioral plasticity within these varied contexts, this review highlights the intricate relationship between larval growing conditions and adult traits and behavior. It underscores the significance of transstadial effects and the necessity of adopting study designs and reporting practices that acknowledge plasticity in adult traits and behavior, considering variations due to larval rearing conditions. Embracing such approaches paves the way for a comprehensive understanding of contextual variations in mosquito life history traits and behavior. This integrated perspective enables the synthesis of research findings across laboratory, semi-field, and field-based investigations, which is crucial for devising targeted intervention strategies tailored to specific ecological contexts to combat the health threat posed by this formidable disease vector effectively.
Juvenile hormone induces phosphorylation of insulin/insulin-like growth factor signaling proteins in previtellogenic Aedes aegypti mosquitoes
Zhao, Wenhao; Liu, Pengcheng; Saunders, Thomas R.; Zhu, Jinsong (Wiley, 2024-12-11)
Juvenile hormone (JH) plays a pivotal role in regulating post-emergence development and metabolism in previtellogenic female Aedes aegypti mosquitoes. In contrast, yolk protein precursor production and egg maturation after a blood meal are regulated by the steroid hormone 20-hydroxyecdysone, the insulin-like growth factor (IGF)/insulin signaling (IIS) pathway, and the mammalian target of rapamycin (mTOR) pathway. The role of IIS/mTOR signaling in female adults prior to blood feeding has not been thoroughly investigated. In this study, we identified a significant increase in the phosphorylation of key effector proteins in the IIS/mTOR signaling pathway, including eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), ribosomal protein S6 kinase (S6K) and forkhead box protein O1 (FoxO1), in previtellogenic females. In vitro fat body culture experiments suggest that JH induces these phosphorylations through rapid nongenomic signaling mediated by the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mTOR network. RNA interference experiments demonstrated that activation of IIS/mTOR signaling in previtellogenic females modulate metabolic gene expression, promoting the accumulation of energy reserves (glycogen and triglycerides), which influence mosquito fecundity. Additionally, depletion of either the insulin receptor (InR) or the JH receptor Methoprene-tolerant (Met) in adult mosquitoes abolished the phosphorylation of these proteins, indicating that both receptors are involved in JH-induced membrane-initiated signal transduction. Although the precise mechanisms remain unclear, this study uncovers a novel function of the IIS/mTOR pathway in adult mosquitoes before blood feeding, as well as a new mode of JH action through its crosstalk with the IIS pathway.
Cholesterol is required for transcriptional repression by BASP1
Loats, Amy E.; Carrera, Samantha; Fleming, Anna F.; Roberts, Abigail R. E.; Sherrard, Alice; Toska, Eneda; Moorhouse, Alexander J.; Medler, Kathryn F.; Roberts, Stefan G. E. (National Academy of Sciences, 2021-07-15)
Lipids are present within the cell nucleus, where they engage with factors involved in gene regulation. Cholesterol associates with chromatin in vivo and stimulates nucleosome packing in vitro, but its effects on specific transcriptional responses are not clear. Here, we show that the lipidated Wilms tumor 1 (WT1) transcriptional corepressor, brain acid soluble protein 1 (BASP1), interacts with cholesterol in the cell nucleus through a conserved cholesterol interaction motif. We demonstrate that BASP1 directly recruits cholesterol to the promoter region of WT1 target genes. Mutation of BASP1 to ablate its interaction with cholesterol or the treatment of cells with drugs that block cholesterol biosynthesis inhibits the transcriptional repressor function of BASP1. We find that the BASP1–cholesterol interaction is required for BASP1-dependent chromatin remodeling and the direction of transcription programs that control cell differentiation. Our study uncovers a mechanism for gene-specific targeting of cholesterol where it is required to mediate transcriptional repression.
Balancing Group 1 Monoatomic Ion-Polar Compound Interactions in the Polarizable Drude Force Field: Application in Protein and Nucleic Acid Systems
Nan, Yiling; Baral, Prabin; Orr, Asuka A.; Michel, Haley M.; Lemkul, Justin A.; Mackerell, Alexander D. (American Chemical Society, 2024-12-03)
An accurate force field (FF) is the foundation of reliable results from molecular dynamics (MD) simulations. In our recently published work, we developed a protocol to generate atom pair-specific Lennard-Jones (known as NBFIX in CHARMM) and through-space Thole dipole screening (NBTHOLE) parameters in the context of the Drude polarizable FF based on readily accessible quantum mechanical (QM) data to fit condensed phase experimental thermodynamic benchmarks, including the osmotic pressure, diffusion coefficient, ionic conductivity, and solvation free energy, when available. In the present work, the developed protocol is applied to generate NBFIX and NBTHOLE parameters for interactions between monatomic ions (specifically Li+, Na+, K+, Rb+, Cs+, and Cl-) and common functional groups found in proteins and nucleic acids. The parameters generated for each ion-functional group pair were then applied to the corresponding functional groups within proteins or nucleic acids followed by MD simulations to analyze the distribution of ions around these biomolecules. The modified FF successfully addresses the issue of overbinding observed in a previous iteration of the Drude FF. Quantitatively, the model accurately reproduces the effective charge of proteins and demonstrates a level of charge neutralization for a double-helix B-DNA in good agreement with the counterion condensation theory. Additionally, simulations involving ion competition correlate well with experimental results, following the trend Li+ > Na+ ≈ K+ > Rb+. These results validate the refined model for group 1 ion-biomolecule interactions that will facilitate the application of the polarizable Drude FF in systems in which group 1 ions play an important role.
Introductory Tutorials for Simulating Protein Dynamics with GROMACS
Lemkul, Justin A. (American Chemical Society, 2024-09-21)
Atomistic molecular dynamics (MD) simulations have become an indispensable tool for investigating the structure, dynamics, and energetics of biomolecules. Continual optimization of software algorithms and hardware has enabled investigators to access biologically relevant time scales in feasible amounts of computing time. Given the widespread use and utility of MD simulations, there is considerable interest in learning essential skills in performing them. Here, we present a set of introductory tutorials for performing MD simulations of proteins in the popular, open-source GROMACS package. Three exercises are detailed, including simulating a single protein, setting up a protein complex, and performing umbrella sampling simulations to model the unfolding of a short polypeptide. Essential features and input settings are illustrated throughout. The purpose of these tutorials is to provide new users with a general understanding of foundational workflows, from which they can design their own simulations.
Pyroglutamylation modulates electronic properties and the conformational ensemble of the amyloid β-peptide
Davidson, Darcy S.; Lemkul, Justin A. (Wiley, 2024-03-04)
Alzheimer's disease (AD) is a neurodegenerative disorder that is characterized by the formation of extracellular amyloid-β (Aβ) plaques. The underlying cause of AD is unknown, however, post-translational modifications (PTMs) of Aβ have been found in AD patients and are thought to play a role in protein aggregation. One such PTM is pyroglutamylation, which can occur at two sites in Aβ, Glu3 and Glu11. This modification of Aβ involves the truncation and charge-neutralization of N-terminal glutamate, causing Aβ to become more hydrophobic and prone to aggregation. The molecular mechanism by which the introduction of pyroglutamate (pE) promotes aggregation has not been determined. To gain a greater understanding of the role that charge neutralization and truncation of the N-terminus plays on Aβ conformational sampling, we used the Drude polarizable force field (FF) to perform molecular dynamics simulations on AβpE3–42 and AβpE11–42 and comparing their properties to previous simulations of Aβ1–42. The Drude polarizable FF allows for a more accurate representation of electrostatic interactions, therefore providing novel insights into the role that charge plays in protein dynamics. Here, we report the parametrization of pE in the Drude polarizable FF and the effect of pyroglutamylation on Aβ. We found that AβpE3–42 and AβpE11–42 alter the permanent and induced dipoles of the peptide. Specifically, we found that AβpE3–42 and AβpE11–42 have modification-specific backbone and sidechain polarization response and perturbed solvation properties that shift the Aβ conformational ensemble.
Base pair dynamics, electrostatics, and thermodynamics at the LTR-III quadruplex:duplex junction
Michel, Haley M.; Lemkul, Justin A. (Cell Press, 2024-04-04)
G-quadruplexes (GQs) play key regulatory roles within the human genome and have also been identified to play similar roles in other eukaryotes, bacteria, archaea, and viruses. Human immunodeficiency virus 1, the etiological agent of acquired immunodeficiency syndrome, can form two GQs in its long terminal repeat (LTR) promoter region, each of which act to regulate viral gene expression in opposing manners. The major LTR GQ, called LTR-III, is a distinct hybrid GQ containing a 12-nucleotide duplex loop attached to the quadruplex motif. The resulting quadruplex:duplex junction (QDJ) has been hypothesized to serve as a selective drug targeting site. To better understand the dynamics of this QDJ, we performed conventional and enhanced-sampling molecular dynamics simulations using the Drude-2017 force field. We observed unbiased and reversible formation of additional base pairs in the QDJ, between Ade4:Thy14 and Gua3:Thy14. Both base pairs were electrostatically favored, but geometric constraints within the junction may drive the formation of, and preference for, the Ade4:Thy14 base pair. Finally, we demonstrated that the base pairs are separated only by small energy barriers that may enable transitions between both base-paired states. Together, these simulations provide new insights into the dynamics, electrostatics, and thermodynamics of the LTR-III QDJ.
Neuromodulating Alkaloids from Millipede Defensive Secretions
Menegatti, Carla; Wood, Jared S.; Banks, Paige; Knott, Kenneth; Briganti, Jonathan S.; Briganti, Anthony J.; McNally, Samuel V. G.; Marek, Paul E.; Brown, Anne M.; Jones, Tappey H.; Williamson, R. Thomas; Mevers, Emily (American Chemical Society, 2024)
Millipedes have long been known to produce structurally diverse chemical defenses, including hydrogen cyanide, terpenoid alkaloids, and oxidized aromatics. Although the hydrogen cyanide and oxidized aromatic producing millipedes have been well studied, less than 10% of the terpenoid alkaloid producers have been chemically investigated. Several previous studies have shown that alkaloids disorient predators, but their biochemical target is currently unknown. Herein, we investigated the defensive secretions of a colobognath millipede, Ischnocybe plicata, and elucidated the constitution, absolute configuration, and conformation of four new highly oxidized terpenoid alkaloids, termed ischnocybines, using a range of analytical techniques. The ischnocybines are actively secreted from the defensive glands and were shown to disorient ants, a likely common predator. Evaluation of the ischnocybines in a panel of neuroreceptors revealed that ischnocybine A possesses potent (Ki 13.6 nM) and selective (100-fold) binding affinity for sigma-1, an orphan neuroreceptor, over sigma-2. These molecules represent the most complex alkaloids to be discovered from millipedes and provide the first potential insights into a biochemical target responsible for their defensive properties.
Widespread exposure to SARS-CoV-2 in wildlife communities
Goldberg, Amanda R.; Langwig, Kate E.; Brown, Katherine L.; Marano, Jeffrey M.; Rai, Pallavi; King, Kelsie M.; Sharp, Amanda K.; Ceci, Alessandro; Kailing, Christopher D.; Kailing, Macy J.; Briggs, Russell; Urbano, Matthew G.; Roby, Clinton; Brown, Anne M.; Weger-Lucarelli, James; Finkielstein, Carla V.; Hoyt, Joseph R. (Springer, 2024-07-29)
Pervasive SARS-CoV-2 infections in humans have led to multiple transmission events to animals. While SARS-CoV-2 has a potential broad wildlife host range, most documented infections have been in captive animals and a single wildlife species, the white-tailed deer. The full extent of SARS-CoV-2 exposure among wildlife communities and the factors that influence wildlife transmission risk remain unknown. We sampled 23 species of wildlife for SARS-CoV-2 and examined the effects of urbanization and human use on seropositivity. Here, we document positive detections of SARS-CoV-2 RNA in six species, including the deer mouse, Virginia opossum, raccoon, groundhog, Eastern cottontail, and Eastern red bat between May 2022–September 2023 across Virginia and Washington, D.C., USA. In addition, we found that sites with high human activity had three times higher seroprevalence than low human-use areas. We obtained SARS-CoV-2 genomic sequences from nine individuals of six species which were assigned to seven Pango lineages of the Omicron variant. The close match to variants circulating in humans at the time suggests at least seven recent human-to-animal transmission events. Our data support that exposure to SARS-CoV-2 has been widespread in wildlife communities and suggests that areas with high human activity may serve as points of contact for cross-species transmission.
Effects of Copper on Legionella pneumophila Revealed via Viability Assays and Proteomics
Song, Yang; Mena-Aguilar, Didier; Brown, Connor L.; Rhoads, William J.; Helm, Richard F.; Pruden, Amy; Edwards, Marc A. (MDPI, 2024-07-03)
Cu is an antimicrobial that is commonly applied to premise (i.e., building) plumbing systems for Legionella control, but the precise mechanisms of inactivation are not well defined. Here, we applied a suite of viability assays and mass spectrometry-based proteomics to assess the mechanistic effects of Cu on L. pneumophila. Although a five- to six-log reduction in culturability was observed with 5 mg/L Cu²⁺ exposure, cell membrane integrity only indicated a <50% reduction. Whole-cell proteomic analysis revealed that AhpD, a protein related to oxidative stress, was elevated in Cu-exposed Legionella relative to culturable cells. Other proteins related to cell membrane synthesis and motility were also higher for the Cu-exposed cells relative to controls without Cu. While the proteins related to primary metabolism decreased for the Cu-exposed cells, no significant differences in the abundance of proteins related to virulence or infectivity were found, which was consistent with the ability of VBNC cells to cause infections. Whereas the cell-membrane integrity assay provided an upper-bound measurement of viability, an amoebae co-culture assay provided a lower-bound limit. The findings have important implications for assessing Legionella risk following its exposure to copper in engineered water systems.
Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase
Johnson, Sydney B.; Li, Hao; Valentino, Hannah; Sobrado, Pablo (American Chemical Society, 2024-05-23)
OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a “cautious” flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme−substrate and enzyme− intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.
The Essential Oils Compounds of Lippia multiflora Moldenke and Cymbopogon schoenanthus (L.) Spreng Repel and Affect the Survival of the Maize Pest Spodoptera frugiperda (Lepidoptera: Noctuidae) Larvae
Wangrawa, Dimitri Wendgida; Waongo, Antoine; Traore, Fousséni; Sanou, Drissa; Lahondere, Chloé; Sané, Abdoul Razac; Borovsky, Dov; Ouédraogo, Sylvain Nafita; Sanon, Antoine (Hindawi, 2024-04-01)
Plant-derived insecticides, such as essential oils, can be an effective alternative to replace synthetic chemical insecticides against Spodoptera frugiperda, which becomes increasingly resistant to synthetic products. This study aims to evaluate essential oils (EOs) effects on larval growth and development following feeding inhibition, growth regulation, and repellency of EOs of Lippia multiflora (Verbenaceae), Cymbopogon schoenanthus (Poaceae), and their combination. Topical application of EOs was used on S. frugiperda larvae for larvicidal effect or treating filter paper with the EOs to find repellency. The effect of EOs on food intake and larval growth was also evaluated. Several types of compounds have been identified in the EOs, mainly monoterpenes with the appearance of new compounds in the 1 : 1 combination. Bioassay results show that individuals and combinations of EOs significantly influenced S. frugiperda larval development. L. multiflora caused 100% mortality of L2 larvae within 24 hours at 3%. The Lm + Cs (1 : 1) EOs combination was the most effective with LC50 and LC90 of 1.02% and 1.92%, respectively. Lm + Cs (1/4 : 3/4) EOs caused the highest inhibition of food consumption, 0.0160 g consumed after food was treated with 2.2% concentration compared to food consumption of 0.0602 g for the control group at 24 hours. Lower food consumption caused the inhibition of larval growth and weight loss of 0.0005 g/day at the 2.2% EOs concentration. The highest repellency effect of the EOs was found in EOs of L. multiflora, exhibiting a repulsion of 83.33% of the larvae after 3 hours of exposure. This diversity in the biological actions of the EOs tested on S. frugiperda represents valuable options for contributing to integrated pest management and an alternative to synthetic chemical insecticides.
Peptidoglycan in osteoarthritis synovial tissue is associated with joint inflammation
Holub, Meaghan N.; Wahhab, Amanda; Rouse, Joseph R.; Danner, Rebecca; Hackner, Lauren G.; Duris, Christine B.; McClune, Mecaila E.; Dressler, Jules M.; Strle, Klemen; Jutras, Brandon L.; Edelstein, Adam I.; Lochhead, Robert B. (2024-03-27)
Objectives: Peptidoglycan (PG) is an arthritogenic bacterial cell wall component whose role in human osteoarthritis is poorly understood. The purpose of this study was to determine if PG is present in synovial tissue of osteoarthritis patients at the time of primary total knee arthroplasty (TKA), and if its presence is associated with inflammation and patient reported outcomes. Methods: Intraoperative synovial tissue and synovial fluid samples were obtained from 56 patients undergoing primary TKA, none of whom had history of infection. PG in synovial tissue was detected by immunohistochemistry (IHC) and immunofluorescence microscopy (IFM). Synovial tissue inflammation and fibrosis were assessed by histopathology and synovial fluid cytokine quantification. Primary human fibroblasts isolated from arthritis synovial tissue were stimulated with PG to determine inflammatory cytokine response. Results: A total of 33/56 (59%) of primary TKA synovial tissue samples were positive for PG by IHC, and PG staining colocalized with markers of synovial macrophages and fibroblasts by IFM. Synovial tissue inflammation and elevated IL-6 in synovial fluid positively correlated with PG positivity. Primary human fibroblasts stimulated with PG secreted high levels of IL-6, consistent with ex vivo findings. Interestingly, we observed a significant inverse correlation between PG and age at time of TKA, indicating younger age at time of TKA was associated with higher PG levels. Conclusion: Peptidoglycan is commonly found in synovial tissue from patients undergoing TKA. Our data indicate that PG may play an important role in inflammatory synovitis, particularly in patients who undergo TKA at a relatively younger age.
Sinorhizobium meliloti Chemoreceptor McpV Senses Short-Chain Carboxylates via Direct Binding
Compton, K. Karl; Hildreth, Sherry B.; Helm, Richard F.; Scharf, Birgit E. (2018-12)
Sinorhizobium meliloti is a soil-dwelling endosymbiont of alfalfa that has eight chemoreceptors to sense environmental stimuli during its free-living state. The functions of two receptors have been characterized, with McpU and McpX serving as general amino acid and quaternary ammonium compound sensors, respectively. Both receptors use a dual Cache (calcium channels and chemotaxis receptors) domain for ligand binding. We identified that the ligand-binding periplasmic region (PR) of McpV contains a single Cache domain. Homology modeling revealed that McpVPR is structurally similar to a sensor domain of a chemoreceptor with unknown function from Anaeromyxobacter dehalogenans, which crystallized with acetate in its binding pocket. We therefore assayed McpV for carboxylate binding and S. meliloti for carboxylate sensing. Differential scanning fluorimetry identified 10 potential ligands for McpVPR. Nine of these are monocarboxylates with chain lengths between two and four carbons. We selected seven compounds for capillary assay analysis, which established positive chemotaxis of the S. meliloti wild type, with concentrations of peak attraction at 1 mM for acetate, propionate, pyruvate, and glycolate, and at 100 mM for formate and acetoacetate. Deletion of mcpV or mutation of residues essential for ligand coordination abolished positive chemotaxis to carboxylates. Using microcalorimetry, we determined that dissociation constants of the seven ligands with McpVPR were in the micromolar range. An McpVPR variant with a mutation in the ligand coordination site displayed no binding to isobutyrate or propionate. Of all the carboxylates tested as attractants, only glycolate was detected in alfalfa seed exudates. This work examines the relevance of carboxylates and their sensor to the rhizobium-legume interaction.
More than Rotating Flagella: Lipopolysaccharide as a Secondary Receptor for Flagellotropic Phage 7-7-1
Gonzalez, Floricel; Helm, Richard F.; Broadway, Katherine M.; Scharf, Birgit E. (2018-10)
Bacteriophage 7-7-1, a member of the family Myoviridae, infects the soil bacterium Agrobacterium sp. strain H13-3. Infection requires attachment to actively rotating bacterial flagellar filaments, with flagellar number, length, and rotation speed being important determinants for infection efficiency. To identify the secondary receptor(s) on the cell surface, we isolated motile, phage-resistant Agrobacterium sp. H13-3 transposon mutants. Transposon insertion sites were pinpointed using arbitrary primed PCR and bioinformatics analyses. Three genes were recognized, whose corresponding proteins had the following computationally predicted functions: AGROH133_07337, a glycosyltransferase; AGROH133_13050, a UDP-glucose 4-epimerase; and AGROH133_08824, an integral cytoplasmic membrane protein. The first two gene products are part of the lipopolysaccharide (LPS) synthesis pathway, while the last is predicted to be a relatively small (13.4-kDa) cytosolic membrane protein with up to four transmembrane helices. The phenotypes of the transposon mutants were verified by complementation and site-directed mutagenesis. Additional characterization of motile, phage-resistant mutants is also described. Given these findings, we propose a model for Agrobacterium sp. H13-3 infection by bacteriophage 7-7-1 where the phage initially attaches to the flagellar filament and is propelled down toward the cell surface by clockwise flagellar rotation. The phage then attaches to and degrades the LPS to reach the outer membrane and ejects its DNA into the host using its syringe-like contractile tail. We hypothesize that the integral membrane protein plays an important role in events following viral DNA ejection or in LPS processing and/or deployment. The proposed two-step attachment mechanism may be conserved among other flagellotropic phages infecting Gram-negative bacteria.

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