Search Results (624)

Previous studies revealed that low expression of Selenoprotein S (SELS) could enhance osteogenic differentiation, but the underlying mechanisms remain unclear. In this study, we aimed to elucidate the role of SELS and its transcription-factor-based regulatory mechanism during osteogenic differentiation. In comparison with 12-week-old mice, which represent the stage of stable osteogenic differentiation, 3-week-old mice, representing the active ossification stage, showed significantly higher levels of SELS in the mandible. Transcriptomic analysis revealed that SELS is primarily associated with extracellular matrix organization and collagen biosynthesis during mandibular development. In bone marrow mesenchymal stem cells (BMSCs) with SELS knockdown, SP7 levels were elevated after 7 days of osteogenic induction in vitro. Consistently, immunohistochemical and immunofluorescence staining confirmed increased SP7 expression in the mandibles of 7-week-old Sels knockout mice. Dual-luciferase reporter assays and chromatin immunoprecipitation (ChIP) analysis demonstrated that SP7 directly binds to the heat shock protein 47 (HSP47) promoter and negatively regulates its transcription. Consequently, upregulation of SP7 following SELS knockdown led to downregulation of HSP47 and concurrent upregulation of the SP7 downstream targets, collagen type I alpha 1 chain (COL1A1) and Secreted protein acidic and rich in cysteine (SPARC). SELS expression is upregulated during active osteogenesis. Low expression of SELS regulates osteogenic differentiation in a bidirectional and fine-tuned manner through the SP7–HSP47/COL1A1/SPARC axis. Full article

The growing population and increasing concerns about food security and sustainability demand innovative solutions to minimize food waste and transform by-products into functional ingredients valuable to the food sector. Brewery by-products, including brewer’s spent grain (BSG) and brewer’s spent yeast (BSY), are underutilized resources despite their high protein contents and potential as sustainable food ingredients. This study aimed to transform BSG and BSY into protein hydrolysates (BSGH and BSYH, respectively) through enzymatic hydrolysis and thus add value to these brewery industry by-products to be used in the food industry. These protein hydrolysates were incorporated into non-alcoholic malt beverages at three different concentrations, and their effects on the physicochemical properties, including color, kinematic viscosity, turbidity, foaming capacity and foam stability, of the non-alcoholic malt beverages were evaluated. Both BSGH and BSYH exhibited higher water solubility (WS) and lower water binding capacity (WBC) values when compared to their native non-hydrolyzed forms, enhancing their suitability as ideal ingredients for protein supplementation of a wide range of food and beverage products. The production of peptides of varying sizes underscored the effectiveness of enzymatic hydrolysis which resulted in an increase in cysteine and methionine levels in BSYH but a decrease in BSGH. The addition of BSGH and BSYH increased the kinematic viscosity and turbidity but reduced the lightness values in color of the non-alcoholic malt beverages. When the properties of the protein hydrolysates were compared, BSYH was more effective than BSGH in forming foams and maintaining their stability for longer periods. These findings highlight the potential of brewery by-products, after enzymatic hydrolysis, as protein-rich ingredients that can support more sustainable food systems and contribute to the nutritional enhancement of various low-protein food and beverage products. Full article

Almost every cell of a multicellular organism is in contact with the extracellular matrix (ECM), which provides the shape and mechanic stability of tissue, organs and the entire body. At the molecular level, cells contact the ECM via integrins. Integrins are transmembrane cell adhesion molecules that connect the ECM to the cytoskeleton, which they bind with their extracellular and intracellular domains. Cysteine residues are abundant in both integrin subunits α and β. If pairwise oxidized into disulfide bridges, they stabilize the folding and molecular structure of the integrin. However, despite the oxidative environment of the extracellular space, not all pairs of cysteines in the extracellular integrin domains are permanently engaged in disulfide bridges. Rather, the reversible and temporary linkage of cystine bridges of these cysteine pairs by oxidation or their reductive cleavage can cause major conformational changes within the integrin, thereby changing ligand binding affinity and altering cellular functions such as adhesion and migration. During recent years, several oxidoreductases and thiol isomerases have been characterized which target such allosteric disulfide bridges. This outlines much better, albeit not comprehensively, the role that such thiol switches play in the redox regulation of integrins. The platelet integrin αIIbβ3 is the best examined example so far. Mostly referring to this integrin, this review will provide insights into the thiol switch-based redox regulation of integrins and the known effects of their allosteric disulfide bridges on conformational changes and cell functions, as well as on the machinery of redox-modifying enzymes that contribute to the redox regulation of cell contacts with the ECM. Full article

Green synthesis of gold nanoparticles (AuNPs) provides a significantly eco-friendly and low-impact counterpart to conventional chemical methods. In the present study, we synthesized gold nanoparticles using Schinus molle (P-AuNPs) aqueous extract as a reducing and stabilizing agent. The obtained nanoparticles were then stabilized by another biocompatible agent, the chiral amino acids L-cysteine (L-Cys-AuNPs) and D-cysteine (D-Cys-AuNPs), to estimate the potential of the surface modification for enhancing AuNPs surface chemistry and antimicrobial action. The synthesized gold nanoparticles were confirmed by UV-Vis spectroscopy, FTIR, XRD, and circular dichroism to validate their formation, crystalline structure, surface properties, and chirality. Physicochemical characterization confirmed the formation of crystalline AuNPs with size and morphology modulated by chiral functionalization. TEM and DLS analyses showed that L-cysteine-functionalized AuNPs were smaller and more uniform, while FTIR and circular dichroism spectroscopy confirmed surface binding and the induction of optical activity, respectively. L-Cys-AuNPs exhibited the highest antimicrobial efficacy against a broad spectrum of microorganisms, including Escherichia coli, Salmonella enterica, Listeria monocytogenes, Staphylococcus aureus, Staphylococcus epidermidis, and, notably, Candida albicans. L-Cys-AuNPs showed the lowest MIC and MBC values, highlighting the synergistic effect of chirality on biological performance. These findings suggest that L-cysteine surface engineering significantly enhances the therapeutic potential of AuNPs, particularly in combating drug-resistant fungal pathogens such as C. albicans. This research paves the way for the development of next-generation antimicrobial agents, reinforcing the relevance of green nanotechnology in the field of materials science and nanotechnology. Full article

The apoplast is often the first point of contact between plant cells and invading pathogens, serving as an important site for defense signaling. Pokeweed antiviral protein (PAP), a ribosome-inactivating protein from Phytolacca americana (pokeweed), is localized to the apoplast and is hypothesized to accompany a pathogen to the cytosol, where it would inactivate host ribosomes to prevent pathogen spread. However, it is not known whether PAP interacts with other proteins in the apoplast. In this study, we identified Phytolacca americana cysteine protease 1 (PaCP1), an extracellular cysteine protease, as a novel PAP interactor. Sequence and structural analyses classified PaCP1 as a member of the C1A subfamily of papain-like cysteine proteases. Immunoprecipitation, mass spectrometry, and yeast two-hybrid analysis showed that PAP specifically binds the mature, active form of PaCP1. Curiously, PaCP1 cleaves PAP at its N- and C-termini, generating peptides that enhance MAPK phosphorylation in pokeweed leaves, indicating their potential role in stress signaling. PaCP1 processing of PAP to generate bioactive peptides diversifies the function of a ribosome-inactivating protein beyond its canonical inhibition of translation. Our findings present a novel extracellular role for PAP and advance our understanding of how protein interactions in the apoplast contribute to plant immune responses. Full article

Biothiols, including cysteine (Cys), homocysteine (Hcy), and glutathione (GSH), are crucial for physiological regulation and their imbalance poses severe health risks. Herein, we developed a pH-responsive liquid crystal (LC)-based sensing platform for detection of biothiols by doping 4-n-pentylbiphenyl-4-carboxylic acid (PBA) into 4-n-pentyl-4-cyanobiphenyl (5CB). Urease catalyzed urea hydrolysis to produce OH⁻, triggering the deprotonation of PBA, thereby inducing a vertical alignment of LC molecules at the interface corresponding to dark optical appearances. Heavy metal ions (e.g., Hg²⁺) could inhibit urease activity, under which condition LC presents bright optical images and LC molecules maintain a state of tilted arrangement. However, biothiols competitively bind to Hg²⁺, the activity of urease is maintained which enables the occurrence of urea hydrolysis. This case triggers LC molecules to align in a vertical orientation, resulting in bright optical images. This pH-driven reorientation of LCs provides a visual readout (bright-to-dark transition) correlated with biothiol concentration. The detection limits of Cys/Hcy and GSH for the PBA-doped LC platform are 0.1 μM and 0.5 μM, respectively. Overall, this study provides a simple, label-free and low-cost strategy that has a broad application prospect for the detection of biothiols. Full article

Many vertebrates have evolved resistance to snake venom as a result of coevolutionary chemical arms races. In Australian skinks (family Scincidae), who often encounter venomous elapid snakes, the frequency, diversity, and molecular basis of venom resistance have been unexplored. This study investigated the evolution of neurotoxin resistance in Australian skinks, focusing on mutations in the muscle nicotinic acetylcholine receptor (nAChR) α1 subunit’s orthosteric site that prevent pathophysiological binding by α-neurotoxins. We sampled a broad taxonomic range of Australian skinks and sequenced the nAChR α1 subunit gene. Key resistance-conferring mutations at the toxin-binding site (N-glycosylation motifs, proline substitutions, arginine insertions, changes in the electrochemical state of the receptor, and novel cysteines) were identified and mapped onto the skink organismal phylogeny. Comparisons with other venom-resistant taxa (amphibians, mammals, and reptiles) were performed, and structural modelling and binding assays were used to evaluate the impact of these mutations. Multiple independent origins of α-neurotoxin resistance were found across diverse skink lineages. Thirteen lineages evolved at least one resistance motif and twelve additional motifs evolved within these lineages, for a total of twenty-five times of α-neurotoxic venoms resistance. These changes sterically or electrostatically inhibit neurotoxin binding. Convergent mutations at the orthosteric site include the introduction of N-linked glycosylation sites previously known from animals as diverse as cobras and mongooses. However, an arginine (R) substitution at position 187 was also shown to have evolved on multiple occasions in Australian skinks, a modification previously shown to be responsible for the Honey Badger’s iconic resistance to cobra venom. Functional testing confirmed this mode of resistance in skinks. Our findings reveal that venom resistance has evolved extensively and convergently in Australian skinks through repeated molecular adaptations of the nAChR in response to the enormous selection pressure exerted by elapid snakes subsequent to their arrival and continent-wide dispersal in Australia. These toxicological findings highlight a remarkable example of convergent evolution across vertebrates and provide insight into the adaptive significance of toxin resistance in snake–lizard ecological interactions. Full article

The ZC4H2 gene is the site of congenital mutations linked to neurodevelopmental and musculoskeletal pathologies collectively termed ZARD (ZC4H2-Associated Rare Disorders). ZC4H2 consists of a coiled coil and a single novel zinc finger with four cysteines and two histidines, from which the protein obtains its name. Alpha Fold 3 confidently predicts a structure for the zinc finger but also for similarly sized random sequences, providing equivocal information on its folding status. We show using synthetic peptide fragments that the zinc finger of ZC4H2 is genuine and folds upon binding a zinc ion with picomolar affinity. NMR pH titration of histidines and UV–Vis of a cobalt complex of the peptide indicate its four cysteines coordinate zinc, while two histidines do not participate in binding. The experimental NMR structure of the zinc finger has a novel structural motif similar to RANBP2 zinc fingers, in which two orthogonal hairpins each contribute two cysteines to coordinate zinc. Most of the nine ZARD mutations that occur in the ZC4H2 zinc finger are likely to perturb this structure. While the ZC4H2 zinc finger shares the folding motif and cysteine-ligand spacing of the RANBP2 family, it is missing key substrate-binding residues. Unlike the NZF branch of the RANBP2 family, the ZC4H2 zinc finger does not bind ubiquitin. Since the ZC4H2 zinc finger occurs in a single copy, it is also unlikely to bind DNA. Based on sequence homology to the VAB-23 protein, the ZC4H2 zinc finger may bind RNA of a currently undetermined sequence or have alternative functions. Full article

This review explores the dual role of reactive oxygen species (ROS) and free radicals in the pathogenesis of endometriosis, aiming to deepen our understanding of these processes through a systematic literature review. To assess the induction and involvement of ROS in endometriosis, we conducted a comprehensive literature review using Cochrane Libraries, EMBASE, Google Scholar, PubMed, and SCOPUS databases. Of 30 qualifying papers ultimately reviewed, 28 reported a significant contribution of ROS to the pathogenesis of endometriosis, while two found no association. The presence of ROS in endometriosis is associated with infertility, irregular menstrual cycles, painful menstruation, and chronic pelvic discomfort. Among individual ROS types studied, hydrogen peroxide was most frequently investigated, followed by lipid peroxides and superoxide radicals. Notable polymorphisms associated with ROS in endometriosis include those for AT-rich interactive domain 1A (ARID1A) and quinone oxidoreductase 1 (NQO1) isoforms. Key enzymes for ROS scavenging and detoxification include superoxide dismutase, glutathione, and glutathione peroxidase. Effective inhibitors of ROS related to endometriosis are vitamins C and E, astaxanthin, fatty acid-binding protein 4, cerium oxide nanoparticles (nanoceria), osteopontin, sphingosine 1-phosphate, N-acetyl-L-cysteine, catalase, and a high-antioxidant diet. Elevated levels of ROS and free radicals are involved in the pathogenesis of endometriosis, suggesting that targeting these molecules could offer potential therapeutic strategies. Full article

Crustins are a family of cysteine-rich antimicrobial peptides (AMPs), predominantly found in crustaceans, and play important roles in innate immunity. However, among the many reported crustins, few studies have explored their immunomodulatory functions. In this study, we investigated the immune function of a type I crustin (LvCrustinIa-2) in Litopenaeus vannamei, with particular emphasis on comparing the roles of its different domains. LvCrustinIa-2 possesses cationic patchy surface and amphipathic structure, and its expression was significantly induced in hemocytes after pathogen challenge. Both the recombinant LvCrustinIa-2 (rLvCrustinIa-2) and its whey acidic protein (WAP) domain (rLvCrustinIa-2-WAP) exhibited significant inhibitory activities against the tested Gram-positive bacteria. They also showed binding affinity not only for Gram-positive bacteria but also for Gram-negative bacteria. Furthermore, rLvCrustinIa-2 induced membrane leakage and structure damage in the target bacteria. Notably, chemotaxis assays revealed that rLvCrustinIa-2 and the synthetic cysteine-rich region (LvCrustinIa-2-CR) significantly enhanced the chemotactic activity of shrimp hemocytes in vitro. Knockdown of LvCrustinIa-2 triggered significant transcriptional activation of genes involved in calcium transport, inflammation, redox regulation, and NF-κB pathway. Taken together, these findings elucidate the distinct roles of the cysteine-rich region and WAP domain in type Ia crustin and provide the first evidence of a crustacean AMP with chemotactic and immunomodulatory activities. Full article

The Steinernema carpocapsae nematode is known to release several excretory/secretory products (ESPs) in its venom upon contact and during the parasitic infection process of insect hosts. A recurrent family of proteins found in this nematode’s venom is the CAP (cysteine-rich secretory protein/antigen 5/pathogenesis-related 1) protein, but the functional role of these proteins remains unknown. To elucidate the biological function, this study focused on characterising the secreted protein, first identified in the venom of the nematode’s parasitic stage, and the sequence retrieved from transcriptomic analysis. The structural comparisons of the Sc-CAP protein model, as determined by AlphaFold2, revealed related structures from other parasitic nematodes of vertebrates. Some of these closely related proteins are reported to have sterol-binding ability. The Sc-CAP recombinant protein was successfully produced in Escherichia coli in conjunction with a chaperone protein. The results showed that the Sc-CAP protein binds to cholesterol, and docking analyses of sterols on the protein revealed potential molecular interactions. Immunoassays performed in Galleria mellonella larvae revealed that this venom protein has an inhibitory effect against phenoloxidase and the antimicrobial response of insects. This suggests that the venom protein has an immunomodulatory function against insects, emphasising its importance during the parasite–host interaction. Full article

An electrochemical immunosensor for the quantification of prostate-specific antigens (PSAs) using silver nanocrystals (AgNCs) is reported. The silver nanocrystals were synthesized using a conventional citrate reduction protocol. The silver nanocrystals were characterized using scanning electron microscopy (SEM) and field effect scanning electron microscopy (FESEM), X-ray diffraction (XRD), high-resolution transmission electron microscopy (HRTEM), Fourier-transform infrared spectroscopy (FTIR), UV-Vis spectroscopy, and small-angle X-ray scattering (SAXS). The proposed immunosensor was fabricated on a glassy carbon electrode (GCE), sequentially, by drop-coating AgNCs, the electro-deposition of EDC-NHS, the immobilization of anti-PSA antibody (Ab), and dropping of bovine serum albumin (BSA) to prevent non-specific binding sites. Each stage of the fabrication process was characterized by cyclic voltammetry (CV). Using square wave voltammetry (SWV), the proposed immunosensor displayed high sensitivity in detecting PSA over a concentration range of 1 to 10 ng/mL with a detection limit of 1.14 ng/mL and R² of 0.99%. The immunosensor was selective in the presence of interfering substances like glucose, urea, L-cysteine, and alpha-methylacyl-CoA racemase (AMACR) and it showed good stability and repeatability. These results compare favourably with some previously reported results on similar or related technologies for PSA detection. Full article

The ReAV enzyme from Rhizobium etli, a representative of Class 3 L-asparaginases, is sequentially and structurally different from other known L-asparaginases. This distinctiveness makes ReAV a candidate for novel antileukemic therapies. ReAV is a homodimeric protein, with each subunit containing a highly specific zinc-binding site created by two cysteines, a lysine, and a water molecule. Two Ser-Lys tandems (Ser48-Lys51, Ser80-Lys263) are located in the close proximity of the metal binding site, with Ser48 hypothesized to be the catalytic nucleophile. To further investigate the catalytic process of ReAV, site-directed mutagenesis was employed to introduce alanine substitutions at residues from the Ser-Lys tandems and at Arg47, located near the Ser48-Lys51 tandem. These mutational studies, along with enzymatic assays and X-ray structure determinations, demonstrated that substitution of each of these highly conserved residues abolished the catalytic activity, confirming their essential role in enzyme mechanism. Full article

The tomato leaf miner, Tuta absoluta (Lepidoptera: Gelechiidae), is a destructive pest of Solanaceae crops worldwide. Its olfactory system plays an important role in locating mating partners and recognizing host plants. Understanding its olfactory recognition mechanism, particularly the function of odorant-binding proteins (OBPs), may reveal potential targets for pest management. In this study, we characterized two antenna-specific OBPs, TabsOBP45 and TabsOBP46, which were identified from the T. absoluta genome. Sequence analysis revealed that both TabsOBPs belong to the classic OBP subfamily, which is characterized by the presence of six conserved cysteine residues and an N-terminal signal peptide. Both TabsOBPs showed predominant antennal expression in quantitative real-time PCR (qRT-PCR) assays, suggesting their key roles in olfactory perception. Fluorescence competitive binding assays with a total of 63 tested volatiles revealed that 13 compounds exhibited strong binding affinities (Ki < 22 µM) to TabsOBP45, with the highest binding affinity to β-ionone, β-caryophyllene, terpinolene, and cinnamaldehyde. Nine compounds showed strong binding affinities to TabsOBP46, with the strongest binding to 4-anisaldehyde, 4-methoxybenzaldehyde, cinnamaldehyde, and β-ionone. Molecular docking analysis revealed the key residues involved in β-ionone binding: TabsOBP45 interacted with ILE8, ALA9, PHE12, TRP37, ILE92, PHE94, THR115, and PHE118, while TabsOBP46 interacted with ILE8, PHE12, PHE36, TRP37, ILE92, LEU94, PHE118, and VAL134. These results provide new insights into the olfactory mechanism of T. absoluta and potential molecular targets for the development of olfactory-based pest control strategies. Full article

TRPA1 (Transient Receptor Potential Ankyrin 1), a cation channel predominantly expressed in sensory neurons, plays a critical role in detecting noxious stimuli and mediating pain signal transmission. As a key player in nociceptive signaling pathways, TRPA1 has emerged as a promising therapeutic target for the development of novel analgesics. Crotalphine (CRP), a 14-amino acid peptide, has been demonstrated to specifically activate TRPA1 and elicit potent analgesic effects. Previous cryo-EM (cryo-electron microscopy) studies have elucidated the structural mechanisms of TRPA1 activation by small-molecule agonists, such as iodoacetamide (IA), through covalent modification of N-terminal cysteine residues. However, the molecular interactions between TRPA1 and peptide ligands, including crotalphine, remain unclear. Here, we present the cryo-EM structure of ligand-free human TRPA1 consistent with the literature, as well as TRPA1 complexed with crotalphine, with resolutions of 3.1 Å and 3.8 Å, respectively. Through a combination of single-particle cryo-EM studies, patch-clamp electrophysiology, and microscale thermophoresis (MST), we have identified the cysteine residue at position 621 (Cys621) within the TRPA1 ion channel as the primary binding site for crotalphine. Upon binding to the reactive pocket containing C621, crotalphine induces rotational and translational movements of the transmembrane domain. This allosteric modulation coordinately dilates both the upper and lower gates, facilitating ion permeation. Full article