Signal Transduction Mechanisms Research Articles

Unraveling the Molecular Mechanism of S-Nitrosation Mediated by N-Acetylmicroperoxidase-11.

Conversion of NO to stable S-nitrosothiols is perceived as a biologically important strategy of NO storage and a signal transduction mechanism. Transition-metal ions and metalloproteins are competent electron acceptors that may promote the formation of S-nitrosothiols from NO. We selected N-acetylmicroperoxidase (AcMP-11), a model of protein heme centers, to study NO incorporation to three biologically relevant thiols (glutathione, cysteine, and N-acetylcysteine). The efficient formation of S-nitrosothiols under anaerobic conditions was confirmed with spectrofluorimetric and electrochemical assays. AcMP-11-assisted incorporation of NO to thiols occurs via an intermediate characterized as an N-coordinated S-nitrosothiol, (AcMP-11)Fe2+(N(O)SR), which is efficiently converted to (AcMP-11)Fe2+(NO) in the presence of NO excess. Two possible mechanisms of S-nitrosothiol formation at the heme-iron were considered: a nucleophilic attack on (AcMP-11)Fe2+(NO+) by a thiolate and a reaction of (AcMP-11)Fe3+(RS) with NO. Kinetic studies, performed under anaerobic conditions, revealed that the reversible formation of (AcMP-11)Fe2+(N(O)SR) occurs in a reaction of RS- with (AcMP-11)Fe2+(NO+) and excluded the second mechanism, indicating that the formation of (AcMP-11)Fe3+(RS) is a dead-end equilibrium. Theoretical calculations revealed that N-coordination of RSNO to iron, forming (AcMP-11)Fe2+(N(O)SR), shortens the S-N bond and increases the complex stability compared to S-coordination. Our work unravels the molecular mechanism of heme-iron-assisted interconversion of NO and low-molecular-weight thiols to S-nitrosothiols and recognizes the reversible NO binding in the form of a heme-Fe2+(N(O)SR) motif as an important biological strategy of NO storage.

Anti-inflammatory potential of selective small compounds by targeting TNF-α & NF-kB signaling: a comprehensive molecular docking and simulation study

Tumor necrosis factor alpha (TNF-α) is the major cause of inflammation in autoimmune diseases like rheumatoid arthritis (RA). It’s mechanisms of signal transduction through nuclear factor kappa B (NF-kB) pathway via small molecules such as metabolite crosstalk are still elusive. In this study, we have targeted TNF-α and NF-kB through metabolites of RA, to inhibit TNF-α activity and deter NF-kB signaling pathways, thereby mitigating the disease severity of RA. TNF-α and NF-kB structure was obtained from PDB database and metabolites of RA were selected from literature survey. In-silico studies were carried out by molecular docking using AutoDock Vina software and further, known TNF-α and NF-kB inhibitors were compared and revealed metabolite’s capacity to targets the respective proteins. Most suitable metabolite was then validated by MD simulation to verify its efficiency against TNF-α. Total 56 known differential metabolites of RA were docked with TNF-α and NF-kB compared to their corresponding inhibitor compounds. Four metabolites such as Chenodeoxycholic acid, 2-Hydroxyestrone, 2-Hydroxyestradiol (2-OHE2), and 16-Hydroxyestradiol were identified as a common TNF-α inhibitor’s having binding energies ranging from −8.3 to −8.6 kcal/mol, followed by docking with NF-kB. Further, 2-OHE2 was selected because of having binding energy −8.5 kcal/mol, found to inhibit inflammation and the effectiveness was validated by root mean square fluctuation, radius of gyration and molecular mechanics with generalized born and surface area solvation against TNF-α. Thus 2-OHE2, an estrogen metabolite was identified as the potential inhibitor, attenuated inflammatory activation and can be utilized as a therapeutic target to disseminate severity of RA.

Mesenchymal stem/stromal cells-derived exosomes for osteoporosis treatment

Osteoporosis is a systemic bone disease, which leads to decreased bone mass and an increased risk of fragility fractures. Currently, there are many anti-resorption drugs and osteosynthesis drugs, which are effective in the treatment of osteoporosis, but their usage is limited due to their contraindications and side effects. In regenerative medicine, the unique repair ability of mesenchymal stem cells (MSCs) has been favored by researchers. The exosomes secreted by MSCs have signal transduction and molecular delivery mechanisms, which may have therapeutic effects. In this review, we describe the regulatory effects of MSCs-derived exosomes on osteoclasts, osteoblasts, and bone immunity. We aim to summarize the preclinical studies of exosome therapy in osteoporosis. Furthermore, we speculate that exosome therapy can be a future direction to improve bone health.

Discovery of SNP Molecular Markers and Candidate Genes Associated with Sacbrood Virus Resistance in Apis cerana cerana Larvae by Whole-Genome Resequencing.

Sacbrood virus (SBV) is a significant problem that impedes brood development in both eastern and western honeybees. Whole-genome sequencing has become an important tool in researching population genetic variations. Numerous studies have been conducted using multiple techniques to suppress SBV infection in honeybees, but the genetic markers and molecular mechanisms underlying SBV resistance have not been identified. To explore single nucleotide polymorphisms (SNPs), insertions, deletions (Indels), and genes at the DNA level related to SBV resistance, we conducted whole-genome resequencing on 90 Apis cerana cerana larvae raised in vitro and challenged with SBV. After filtering, a total of 337.47 gigabytes of clean data and 31,000,613 high-quality SNP loci were detected in three populations. We used ten databases to annotate 9359 predicted genes. By combining population differentiation index (FST) and nucleotide polymorphisms (π), we examined genome variants between resistant (R) and susceptible (S) larvae, focusing on site integrity (INT < 0.5) and minor allele frequency (MAF < 0.05). A selective sweep analysis with the top 1% and top 5% was used to identify significant regions. Two SNPs on the 15th chromosome with GenBank KZ288474.1_322717 (Guanine > Cytosine) and KZ288479.1_95621 (Cytosine > Thiamine) were found to be significantly associated with SBV resistance based on their associated allele frequencies after SNP validation. Each SNP was authenticated in 926 and 1022 samples, respectively. The enrichment and functional annotation pathways from significantly predicted genes to SBV resistance revealed immune response processes, signal transduction mechanisms, endocytosis, peroxisomes, phagosomes, and regulation of autophagy, which may be significant in SBV resistance. This study presents novel and useful SNP molecular markers that can be utilized as assisted molecular markers to select honeybees resistant to SBV for breeding and that can be used as a biocontrol technique to protect honeybees from SBV.

Transmembrane signaling by a synthetic receptor in artificial cells

Signal transduction across biological membranes is among the most important evolutionary achievements. Herein, for the design of artificial cells, we engineer fully synthetic receptors with the capacity of transmembrane signaling, using tools of chemistry. Our receptors exhibit similarity with their natural counterparts in having an exofacial ligand for signal capture, being membrane anchored, and featuring a releasable messenger molecule that performs enzyme activation as a downstream signaling event. The main difference from natural receptors is the mechanism of signal transduction, which is achieved using a self-immolative linker. The receptor scaffold is modular and can readily be re-designed to respond to diverse activation signals including biological or chemical stimuli. We demonstrate an artificial signaling cascade that achieves transmembrane enzyme activation, a hallmark of natural signaling receptors. Results of this work are relevant for engineering responsive artificial cells and interfacing them and/or biological counterparts in co-cultures.

The Role of Histone Modifications in Heat Signal Transduction in Plants.

Global warming and the more frequent occurrence of extremly high temperatures seriously affect crop yields. Heat stress (HS) has become a major environmental factor threatening food security worldwide. Understanding how plants sense and respond to HS is of clear interest to plant scientists and crop breeders. However, it is not trivial to elucidate the underlying signaling cascade, as specific cellular responses (ranging from detrimental to systemic effects) must be disentangled. Plants respond and adapt to high temperatures in many ways. In this review, recent progress in understanding heat signal transduction and the role of histone modifications in regulating the expression of genes involved in HS responses are discussed. The outstanding issues that are crucial for understanding the interactions between plants and HS are also discussed. The study of heat signal transduction mechanisms in plants is essential to facilitate the cultivation of heat-resistant crop varieties.

The role of cytokines in the development of systemic inflammation in chronic obstructive pulmonary disease and obesity

Patients with chronic obstructive pulmonary disease (COPD) are characterized by a variety of comorbid conditions, including both somatic (arterial hypertension, atherosclerosis, coronary heart disease, bronchial asthma, malignant neoplasms, diabetes mellitus, obesity, etc.) and mental (depressive disorders, suicide attempts). Against the background of various chronic diseases of the respiratory system, endocrine, metabolic disorders, the risks of exacerbations of COPD increase. The leading unifying mechanism of these conditions is systemic subclinical inflammation. Its excessive activity leads to the loss of the physiological functions of inflammation, which is accompanied by an imbalance in the endocrine system and the release of high concentrations of hormones and neurotransmitters. The result of this response is the uncoupling of cytokine mechanisms, which leads to an imbalance in the system of pro- and anti-inflammatory cytokines. The article describes the role of the pro-inflammatory chemokine IL-8 (interleukin 8), which is responsible for the migration of neutrophils to the site of inflammation. This is how the neutrophilic type of inflammation is formed. IL-4 and IL-10 are considered, which occupy a leading position in the formation of CD4+ type of immunoreactivity, which is observed in bronchial asthma. Attention is focused on the significance of IL-6, because it is an integral component of local and systemic inflammation. An increase in its concentration and, as a result, a potential risk of damage to the respiratory epithelium is the remodeling of the bronchial tree, resulting in a decrease in the elasticity of the epithelium of the respiratory tract. This mechanism leads to the formation of pulmonary emphysema and further potentiation of pathophysiological processes in patients with COPD. Since IL-6 is a cytokine with anti-inflammatory properties, its molecular activity is achieved by interacting with a special receptor complex consisting of two subunits: IL-6R and gp130. The former mediates IL-6 binding, while the latter triggers the JAK/STAT or MAPK signaling cascade pathways. The result of the reaction of IL-6 with the effector cell directly depends on the type of signaling. The paper describes three mechanisms of signal transduction into the target cell: classical, cluster, and transsignaling. Thus, by studying the role of cytokines in the systemic inflammatory response, we have shown the cross-talk between adipose tissue and the lungs in obesity, highlighting the main inflammatory mediators, which may indicate new therapeutic targets for preventing pulmonary dysfunction.

Commensal Bacillus siamensis LF4 induces antimicrobial peptides expression via TLRs and NLRs signaling pathways in intestinal epithelial cells of Lateolabrax maculatus

Antimicrobial peptides (AMPs) play an important role in modulating intestinal microbiota, and our previous study showed that autochthonous Baccilus siamensis LF4 could shape the intestinal microbiota of spotted seabass (Lateolabrax maculatus). In the present study, a spotted seabass intestinal epithelial cells (IECs) model was used to investigate whether autochthonous B. siamensis LF4 could modulate the expression of AMPs in IECs. And then, the IECs were treated with active, heat-inactivated LF4 and its supernatant to illustrate their AMPs inducing effects and the possible signal transduction mechanisms. The results showed that after 3 h of incubation with 108 CFU/mL B. siamensis LF4, lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), glutamic propylic transaminase (GPT) activities in supernatant decreased significantly and obtained minimum values, while supernatant alkaline phosphatase (AKP) activity, β-defensin protein level and IECs Na+/K+-ATPase activity, AMPs (β-defensin, hepcidin-1, NK-lysin, piscidin-5) genes expression increased significantly and obtained maximum values (P < 0.05). Further study demonstrated that the active, heat-inactivated LF4 and its supernatant treatments could effectively decrease the LDH, GOT, and GPT activities in IECs supernatant, increase AKP activity and β-defensin (except LF4 supernatant treatment) protein level in IECs supernatant and Na+/K+-ATPase and AMPs genes expression in IECs. Treatment with active and heat-inactivated B. siamensis LF4 resulted in significantly up-regulated the expressions of TLR1, TLR2, TLR3, TLR5, NOD1, NOD2, TIRAP, MyD88, IRAK1, IRAK4, TRAF6, TAB1, TAB2, ERK, JNK, p38, AP-1, IKKα, IKKβ and NF-κB genes. Treatment with B. siamensis LF4 supernatant also resulted in up-regulated these genes, but not the genes (ERK, JNK, p38, and AP-1) in MAPKs pathway. In summary, active, heat-inactivated and supernatant of B. siamensis LF4 can efficiently induce AMPs expression through activating the TLRs/NLRs-MyD88-dependent signaling, active and heat-inactivated LF4 activated both the downstream MAPKs and NF-κB pathways, while LF4 supernatant only activated NF-κB pathway.

Diurnal emission of herbivore-induced (Z)-3-hexenyl acetate and allo-ocimene activates sweet potato defense responses to sweet potato weevils

The sweet potato weevil (Cylas formicarius (Fab.) (Coleoptera: Brentidae)) is a pest that feeds on sweet potato (Ipomoea batatas (L.) Lam. (Solanales: Convolvulaceae)), causing substantial economic losses annually. However, no safe and effective methods have been found to protect sweet potato from this pest. Herbivore-induced plant volatiles (HIPVs) promote various defensive bioactivities, but their formation and the defense mechanisms in sweet potato have not been investigated. To identify the defensive HIPVs in sweet potato, the release dynamics of volatiles was monitored. The biosynthetic pathways and regulatory factors of the candidate HIPVs were revealed via stable isotope tracing and analyses at the transcriptional and metabolic levels. Finally, the anti-insect activities and the defense mechanisms of the gaseous candidates were evaluated. The production of (Z)-3-hexenyl acetate (z3HAC) and allo-ocimene was induced by sweet potato weevil feeding, with a distinct circadian rhythm. Ipomoea batatas ocimene synthase (IbOS) is first reported here as a key gene in allo-ocimene synthesis. Insect-induced wounding promoted the production of the substrate, (Z)-3-hexenol, and upregulated the expression of IbOS, which resulted in higher contents of z3HAC and allo-ocimene, respectively. Gaseous z3HAC and allo-ocimene primed nearby plants to defend themselves against sweet potato weevils. These results provide important data regarding the formation, regulation, and signal transduction mechanisms of defensive volatiles in sweet potato, with potential implications for improving sweet potato weevil management strategies.

Quorum-quenching potential of recombinant PvdQ-engineered bacteria for biofilm formation.

Quorum sensing (QS) is a core mechanism for bacteria to regulate biofilm formation, and therefore, QS inhibition or quorum quenching (QQ) is used as an effective and economically feasible strategy against biofilms. In this study, the PvdQ gene encoding AHL acylase was introduced into Escherichia coli (DE3), and a PvdQ-engineered bacterium with highly efficient QQ activity was obtained and used to inhibit biofilm formation. Gene sequencing and western blot analysis showed that the recombinant pET-PvdQ strain was successfully constructed. The color reaction of Agrobacterium tumefaciens A136 indicated that PvdQ engineering bacteria had shown strong AHL signal molecule quenching activity and significantly inhibited the adhesion (motility) of Pseudomonas aeruginosa and biofilm formation of activated sludge bacteria in Membrane Bio-Reactor (MBR; inhibition rate 51-85%, p < 0.05). In addition, qRT-PCR testing revealed that recombinant PvdQ acylase significantly reduced the transcription level of QS biofilm formation-related genes (cdrA, pqsA, and lasR; p < 0.05). In this study, QQ genetically engineered bacteria enhanced by genetic engineering could effectively inhibit the QS signal transduction mechanism and have the potential to control biofilm formation of pathogenic bacteria in the aquaculture environment, providing an environmentally friendly and alternative antibiotic strategy to suppress biofilm contamination.

Decoupling Transmission and Transduction for Improved Durability of Highly Stretchable, Soft Strain Sensing: Applications in Human Health Monitoring.

This work presents a modular approach to the development of strain sensors for large deformations. The proposed method separates the extension and signal transduction mechanisms using a soft, elastomeric transmission and a high-sensitivity microelectromechanical system (MEMS) transducer. By separating the transmission and transduction, they can be optimized independently for application-specific mechanical and electrical performance. This work investigates the potential of this approach for human health monitoring as an implantable cardiac strain sensor for measuring global longitudinal strain (GLS). The durability of the sensor was evaluated by conducting cyclic loading tests over one million cycles, and the results showed negligible drift. To account for hysteresis and frequency-dependent effects, a lumped-parameter model was developed to represent the viscoelastic behavior of the sensor. Multiple model orders were considered and compared using validation and test data sets that mimic physiologically relevant dynamics. Results support the choice of a second-order model, which reduces error by 73% compared to a linear calibration. In addition, we evaluated the suitability of this sensor for the proposed application by demonstrating its ability to operate on compliant, curved surfaces. The effects of friction and boundary conditions are also empirically assessed and discussed.

Analysis of Microbial Community Characteristics and Function Prediction of MBBR with Magnetic Biocarriers at Low Temperature

In order to clarify effect of magnetic biocarriers on the performance of MBBR at low temperatures, the microbial diversity, community structure, functional characteristics, and nitrogen metabolism of biofilm in the reaction system were investigated. The results indicated that MBBR with magnetic biocarriers had a better pollutant removal efficiency, with the average removal rates of NH4+-N and TN being 16.2% and 12.1% higher than those in the control group (commercial biocarriers), respectively. Illumina high-throughput sequencing analysis showed that higher diversity and richness of the bacterial community was established in the biofilm of magnetic biocarriers. There were obvious differences in microbial community structure of biofilm between the two biocarrier duos to bacterial magnetic susceptibility. The relative abundances of nitrifying bacteria (e.g., Nitrosomonas and Nitrospira) and denitrifying bacteria (e.g., Sphaerotilus and Zoogloea) were increased in the magnetic biocarriers. Functional prediction analysis with PICRUSt2 showed that the microorganism of magnetic biocarriers had a better total gene function expression level, which was significantly more increased than commercial biocarriers in gene-representing signal transduction mechanisms and intracellular trafficking, secretion, and vesicular transport. Furthermore, most of the abundances of nitrogen metabolism genes were raised in the biofilm of magnetic biocarriers (e.g., genes amo and hao, were responsible for nitrification, and genes nap and nor, which were responsible for denitrification). Magnetic biocarriers increased biofilm potential for denitrification at low temperatures. Our results explained the difference in performance between the two reactors from microbiology and provided the theoretical basis for magnetic biocarrier-enhanced performances of MBBR at low temperatures.

Signaling mechanisms in red blood cells: A view through the protein phosphorylation and deformability.

Intracellular signaling mechanisms in red blood cells (RBCs) involve various protein kinases and phosphatases and enable rapid adaptive responses to hypoxia, metabolic requirements, oxidative stress, or shear stress by regulating the physiological properties of the cell. Protein phosphorylation is a ubiquitous mechanism for intracellular signal transduction, volume regulation, and cytoskeletal organization in RBCs. Spectrin-based cytoskeleton connects integral membrane proteins, band 3 and glycophorin C to junctional proteins, ankyrin and Protein 4.1. Phosphorylation leads to a conformational change in the protein structure, weakening the interactions between proteins in the cytoskeletal network that confers a more flexible nature for the RBC membrane. The structural organization of the membrane and the cytoskeleton determines RBC deformability that allows cells to change their ability to deform under shear stress to pass through narrow capillaries. The shear stress sensing mechanisms and oxygenation-deoxygenation transitions regulate cell volume and mechanical properties of the membrane through the activation of ion transporters and specific phosphorylation events mediated by signal transduction. In this review, we summarize the roles of Protein kinase C, cAMP-Protein kinase A, cGMP-nitric oxide, RhoGTPase, and MAP/ERK pathways in the modulation of RBC deformability in both healthy and disease states. We emphasize that targeting signaling elements may be a therapeutic strategy for the treatment of hemoglobinopathies or channelopathies. We expect the present review will provide additional insights into RBC responses to shear stress and hypoxia via signaling mechanisms and shed light on the current and novel treatment options for pathophysiological conditions.

Genetic and transcriptome analysis of a cotton leaf variegation mutant

In eukaryotic photosynthetic organisms, chloroplast is not only a site for photosynthesis, but it also have a vital role in signal transduction mechanisms. Plants exhibit various colors in nature with various mutants induced by EMS, whose traits are regulated by developmental and environmental factors, making them ideal for studying the regulation of chloroplast development. In this study, the cotton leaf variegated mutant (VAR) induced by EMS was used for this experiment. Genetic analysis revealed that VAR phenotype was a dominant mutation and by performing freehand section inspection, it was noticed that the vascular bundles of VAR were smaller. Chloroplast ultrastructure showed that the stacking of grana thylakoid was thinner and the starch granules were increased significantly in VAR comparedto wild type (WT). Transcriptome analysis found that the KEGG was enriched in photosynthesis pathway, and GO was abundant in zinc ion transmembrane transport, electron transporter and cation binding terms. In addition, GhFTSH5 expression in VAR was significantly higher than WT and the promoter sequence of GhFTSH5 had differences. The results showed that the VAR plant had altered GhFTSH5 expression and disrupted chloroplast structure, which in turn affects plant photosynthesis. More importantly, this study lays a foundation for further analyzing molecular mechanism of cotton variegated phenotypes.

UNC93B1 facilitates TLR18-mediated NF-κB signal activation in Schizothorax prenanti

Toll-like receptor 18 (TLR18), a non-mammalian TLR, has been believed to play an important role in anti-bacterial immunity of teleost fishes. UNC93B1 is a classical molecular chaperone that mediates TLRs transport from endoplasmic reticulum to the located membrane. However, TLR18-mediated signal transduction mechanism and the regulatory effect of UNC93B1 to TLR18 are still unclear in teleost fishes. In this study, the coding sequences of TLR18 and UNC93B1 were cloned from Schizothorax prenanti, named spTLR18 and spUNC93B1, respectively. The spTLR18 and spUNC93B1 are 2583 bp and 1878 bp in length, encode 860 and 625 amino acids, respectively. The spTLR18 widely expressed in various tissues with the highest expression level in liver. After stimulation of Aeromonas hydrophila, lipopolysaccharide (LPS) and Poly(I:C), the expression levels of spTLR18 were significantly increased in spleen and head kidney. The spTLR18 located in the cell membrane, while spUNC93B1 located in the cytoplasm. Luciferase and overexpression analysis showed that spTLR18 activated NF-κB and type I IFN signal pathways, and spTLR18-mediated NF-κB activation might depend on the adaptor molecule MyD88. Besides, spUNC93B1 positively regulates spTLR18-mediated NF-κB signal. Our study first uncovers TLR18-UNC93B1-mediated signal transduction mechanism, which contributes to the understanding of TLR signaling pathway in teleost fishes.

DNA-templated silver nanocluster-based cleavable probes for nucleic acid detection.

DNA or RNA probes that fluoresce upon enzymatic digestion, such as TaqMan probes and DNaseAlert, allow for not only nuclease activity detection but also nucleic acid amplification assays. However, relying on FRET as a signal transduction mechanism, these short oligonucleotide probes need to be dually labeled with a donor and a quencher, thus suffering from the issues of high cost and singly labeled impurities. Here we demonstrate a new class of probe, based on DNA-templated silver nanoclusters (DNA/AgNCs). These nanoclusters exhibit fascinating photophysical properties that depend on the arrangement of the nucleobases in the DNA ligand. Not using FRET, our probes not only turn on upon nuclease cleavage but display a significantly altered fluorescence color. Although a variety of DNA/AgNC probes exist to detect proteins and nucleic acids, no AgNC probes have been designed to detect nuclease activity (enzymes that cleave the bonds between the nucleotides of nucleic acids such as DNA). Due to their multicolor nature, our probes also enable ratiometric sensing for the highly quantitative detection of nuclease activities. Here we show that our silver cluster probes can replace DNaseAlert for the enzymatic activity assay of DNase I. The electrophoresis gel shows clear digestion of our silver cluster probes by DNase I and the digested products have a significantly enhanced fluorescence with 700% increase in intensity and different emission color (90 nm red-shift). In addition, our silver cluster probes can substitute the FRET probes in novel CRISPR-Cas assays (such as DETECTR and SHERLOCK) for nucleic acid detection. We demonstrate that we can selectively detect SARS-CoV-2 cDNA in combination with CRISPR-Cas12a. Our results not only demonstrate that DNA-templated silver nanocluster probes can be a versatile nuclease activity probe but also provide insights into the interactions between nucleases and metallo-DNA nanomaterials.

Mechanism of ligand binding signal transduction in a transcriptional riboswitch.

Riboswitches are noncoding RNA molecules that regulate gene expression in bacteria. The riboswitch's aptamer domain (AD) senses the cognate ligand and induces a structural change in the expression platform (EP) to signal on/off to the gene expression machinery. Despite extensive studies, the question of how the ligand binding signal is transmitted from the AD to EP resulting in the structural transitions in the EP remains unanswered. We studied the ligand binding signal transduction from AD to EP in the preQ1-sensing transcriptional riboswitch. In the absence of preQ1, both AD and EP individually form stem-loops with no structural overlap between the two stem-loops. The segment between the AD and EP stem loops is rich in adenine. In the presence of preQ1, AD forms a pseudoknot to encapsulate preQ1. We performed coarse-grained molecular dynamics simulations of full-length preQ1 riboswitch in the presence of Mg2+ and preQ1 ligand. In the absence of preQ1 and variation in Mg2+ concentration ([Mg2+]), we observed that AD populates only stem-loop and does not form the pseudoknot required to capture the ligand. Only in the presence of preQ1 AD attains the pseudoknot conformation by wrapping the A-rich region around the pre-existing stem-loop to facilitate ligand binding. The pseudoknot formation at AD allows EP to form the terminator helix and sequester the poly-U through strand invasion. The wrapping of the A-rich region around the AD stem-loop forces the stem-loop at EP to rupture and slide by 10 nucleotides towards 3'-end and form the terminator helix. The propensity of pseudoknot and terminator formation through strand invasion in the presence of preQ1 is enhanced with increment in [Mg2+]. We show that the competition between base-pair formation and helix sliding rate is crucial for the ligand binding signal transduction.

In silico analysis of biased signaling in the serotonin 2A receptor.

The 5-HT2A receptor (5HT2AR) is a widely expressed and highly studied subtype of the serotonin receptor family. Many FDA-approved antidepressants and antipsychotics elicit their pharmacological effects through antagonism of 5HT2AR, a class A G protein-coupled receptor (GPCR). However, these drugs suffer from undesired hallucinogenic effects similar to those produced by classic psychedelics such as LSD and psilocybin. The binding of an agonist to 5HT2AR can induce conformational changes in the receptor and activate intracellular signaling through the G protein- and β-arrestin-mediated pathways. Interestingly, like other GPCRs, the 5HT2A receptor has been shown to exhibit biased signaling in which ligands can preferentially activate one pathway over another. A recent structural and pharmacological study demonstrated that β-arrestin biased agonists, such as IHCH-7086, can exhibit antidepressant properties without hallucinogenic effects. Notably, the IHCH-7086-bound receptor revealed altered conformational changes in a set of phenylalanine residues in the orthosteric sites. However, the subsequent signal transduction mechanism and how other residues in the signaling network are altered remain poorly understood. In this study, using molecular dynamics simulations, we investigated the conformational dynamics of the receptor bound to an antagonist, lumateperone (PDB ID: 7WC8), and IHCH-7086 (PDB ID: 7WC9) in the presence or absence of Gq protein. Our results show that the β-arrestin-biased partial agonist produces significant changes at the intracellular receptor-Gq protein interface residue interactions that are unfavorable for the binding. Also, there is a significant difference in the interactions of IHCH-7086 at the orthosteric binding site. Lastly, our network analysis shows differences in communications between residues among the studied receptor-ligand complexes. The knowledge obtained from this study will be used to develop structure-based pharmacophore models to identify high-affinity biased agonists for the 5HT2A receptor as potential therapeutics to treat 5HT2A receptor-related CNS diseases.

Lipid dependence of the conformational coupling across the membrane bilayer of full-length epidermal growth factor receptor.

Membrane proteins regulate several vital cellular processes, establishing them as major drug targets. A main function of membrane proteins is to transduce extracellular signal across the membrane bilayer. One such protein is the epidermal growth factor receptor (EGFR), which plays a crucial role in cell signaling and is implicated in cancer. While the structures of the extracellular and intracellular regions of this protein have been well-elucidated individually, conformational coupling connecting these two regions during signal transduction is challenging to probe due to the mixture of hydrophobic and hydrophilic domains intrinsic to membrane proteins. Here, I will discuss the signal transduction mechanism across full-length EGFR. We combine two biochemical tools, cell-free protein expression and nanodiscs, to isolate full-length, functional EGFR in a near-physiological environment. Using a multidisciplinary approach involving single-molecule Förster resonance energy transfer, mutagenesis, and molecular dynamics simulations, we observe a compaction in the intracellular domain of EGFR upon extracellular ligand binding in a neutral lipid environment. The ligand-induced extracellular/intracellular conformational coupling is reversed in the presence of anionic lipids, robust around the physiological 15-30% anionic content, and disappears in the presence of cholesterol. Our findings show how the extracellular and intracellular domains are coupled to each other in this critical receptor and the impact of the lipid composition in this conformational coupling. Our results could be universal to other membrane receptors which share the same structural homology and perform other significant functions.

Leukotrienes in Innate Immunity: Still Underappreciated after All These Years?

Leukotrienes (LTs) are lipid mediators derived from the 5-lipoxygenase pathway of arachidonate metabolism. Though best known for their role in asthma, they have broad actions that touch on virtually every aspect of mammalian biology. In a Brief Review published in the journal in 2005, we presented the existing evidence supporting a role for LTs in host defense. In this updated Brief Review, we focus on selected advances since then. We detail new insights into mechanisms and regulation of LT biosynthesis; the protective roles of LTs in the host response to diverse classes of pathogens, with an emphasis on viruses, including SARS-CoV-2; the phagocyte signal transduction mechanisms by which LTs exert their antimicrobial actions; the capacity for overexuberant LT production to promote tissue damage; and roles of LTs in the noninfectious immune-relevant conditions neuroinflammation and cancer.