Search Results (67)

Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation channel, and its activator, the alcohol breakdown product acetaldehyde, plays a key role in the pathomechanism of alcoholic liver disease (ALD). We hypothesized that TRPA1 is expressed in the liver, can be activated by alcohol breakdown products, and plays a role in ALD. We aimed (1) to confirm the presence of TRPA1 in liver samples from C57BL6/J mice by RNAscope in situ hybridization combined with immunostaining, (2) to prove that alcohol breakdown products may activate human TRPA1 by calcium-imaging, and (3) to investigate the role of TRPA1 in a chronic continuous 20% alcohol drinking model involving Trpa1 gene-deficient (KO) mice. The liver enzyme levels were evaluated; moreover, the steatosis, portal and interface inflammatory infiltrations were assessed in PAS–hematoxylin-stained sections. We detected Trpa1 expression in both hepatocytes and liver macrophages. We observed elevated liver enzyme levels in wild-type mice. Significant inflammatory infiltration and steatosis developed in both WT and KO mice in response to alcohol; however, no significant differences were found between the genotypes. We conclude that Trpa1 is expressed in hepatocytes and liver macrophages; however, the chronic alcohol-induced steatosis and inflammatory infiltration develop through a TRPA1-independent mechanism. Full article

Introduction: cigarette smoking is a major source of systemic oxidative stress and a well-established risk factor for cardiovascular disease. Heated tobacco products (HTPs) are increasingly promoted as reduced-risk alternatives, yet their cellular effects remain incompletely understood. Methods: this study compared the oxidative stress-mediated effects of conventional cigarette smoking and HTP use on human erythrocytes. Erythrocytes from healthy non-smokers, conventional smokers, and HTP users were analyzed using biochemical, functional, and cytological approaches to assess redox status, membrane and cytoskeletal organization, anion exchanger 1 (AE1) function, antioxidant response, and redox-sensitive signaling pathways. Results: conventional smokers exhibited higher intracellular reactive oxygen species (ROS) levels, thiol depletion, methemoglobin and hemichrome formation, whereas HTP users showed marked lipid peroxidation despite lower ROS availability. Both groups instead displayed altered expression and distribution of key membrane and cytoskeletal proteins, including glycophorin A, AE1, spectrin, ankyrin, and band 4.1, indicating impaired membrane–cytoskeleton interactions. Functional analyses revealed an accelerated AE1-mediated anion exchange in erythrocytes from conventional smokers, whereas cells from HTP users exhibited a reduced sulfate accumulation, indicating altered transport capacity. In both groups, G6PDH activity was significantly increased, and redox-sensitive signaling pathways involving ERK, AKT, and eNOS were activated, accompanied by sex-dependent alterations in estrogen receptor expression and distribution. Conclusions: collectively, these findings identify erythrocytes as sensitive biomarkers of tobacco-related systemic damage and indicate that smoking-induced erythrocyte dysfunction, including that associated with HTP use, may actively contribute to vascular impairment. This evidence challenges the assumption that heated tobacco products confer a substantially reduced cardiovascular risk compared with conventional cigarettes. Full article

The Transient Receptor Potential Ankyrin 1 (TRPA1) channel is a non-selective cation channel activated by a range of physical and chemical stimuli. While primarily studied in neuronal tissues, TRPA1 is also expressed in human keratinocytes, where its role remains poorly understood. Here, we investigated TRPA1 expression and function in keratinocytes and examined the effects of its activation on cellular proliferation, immune activation, and neuropeptide release under both basal and inflammatory stimuli. TRPA1 expression was detected in basal keratinocytes and was upregulated by pro-inflammatory cytokines. Stimulation with the TRPA1 agonist allyl isothiocyanate (AITC) induced a rapid calcium influx, confirming functional channel activity. AITC at 5 µM did not induce cytotoxicity but significantly reduced keratinocyte proliferation and caused cell cycle arrest. Under stimulation with TNF-α and IFN-γ, TRPA1 activation decreased the surface expression of HLA-DR and ICAM-1, and downregulated mRNA levels of CXCL10, CXCL8, CCL5, and CCL20, while IL-6 expression remained unchanged. Furthermore, AITC treatment reduced the secretion of Substance P, but not CGRP. These findings indicate that TRPA1 functions as a cytokine-inducible, immunomodulatory receptor in human keratinocytes, capable of attenuating proliferation and inflammatory activation without compromising cell viability, thereby suggesting a potential role in maintaining skin homeostasis and modulating cutaneous inflammation. Full article

Cathepsin B (CatB) is a lysosomal cysteine protease that plays a major role in various pathologies and is therefore considered a valuable therapeutic target. To address species-specific inhibitor challenges, we characterized the selective binding of designed ankyrin repeat protein (DARPin) 4m3 toward mouse cathepsin B (mCatB) over human CatB (hCatB). The mCatB–DARPin 4m3 complex was validated by size-exclusion chromatography (SEC), nano-differential scanning fluorimetry (nano-DSF), and surface plasmon resonance (SPR), revealing high affinity binding (K_D = 65.7 nM) and potent inhibition (Ki = 26.7 nM; mixed competitive/noncompetitive). DARPin 4m3 showed no binding/inhibition toward hCatB. The 1.67 Å crystal structure of the complex—the first for mCatB—identified key interaction residues (e.g., I65/Q66 in mCatB vs. S65/M66 in hCatB) conferring selectivity. Proteomic analysis of endogenous substrates using a support vector machine (SVM) revealed greater similarity between mCatB and hCatB cleavages (Area Under the Curve (AUC) = 0.733) than between mCatB and other human cathepsins (AUC = 0.939–0.965). Clustering and SVM methods offer broadly applicable tools for protease specificity profiling in drug discovery. This study demonstrates the utility of DARPins for species-selective targeting and highlights the importance of integrated structural and proteomic approaches for dissecting protein–protein interactions. Full article

Radionuclide molecular imaging of epidermal growth factor receptor (EGFR) expression might permit the selection of patients for EGFR-targeting therapies. Designed ankyrin repeat protein (DARPin) E01 with a high affinity to the ectodomain III of the EGFR is a possible EGFR imaging probe. The goal of this study was to evaluate the potential of radiolabeled DARPin E01 for in vivo imaging of EGFR. DARPin E01 containing the (HE)₃-tag was site-specifically labeled with a residualizing ^99mTc (using ^99mTc]Tc(CO)₃). Two methods providing non-residualizing ¹²³I labels, direct electrophilic radioiodination and indirect radioiodination using [¹²³I]I-para-iodobenzoate (PIB), were tested. [^99mTc]Tc-(HE)₃-E01 and [¹²³I]I-(HE)₃-E01-PIB preserved specific binding to EGFR-expressing cells and affinity in the single-digit nanomolar range. Direct labeling with ¹²³I resulted in a substantial loss of binding. In vitro cellular processing studies showed that both [^99mTc]Tc-(HE)₃-E01 and [¹²³I]I-(HE)₃-E01-PIB had rapid binding and relatively slow internalization. Evaluation of [^99mTc]Tc-(HE)₃-E01 biodistribution in normal CD1 mice showed that its hepatic uptake was non-saturable, suggesting that this tracer does not bind to murine EGFR. A side-by-side comparison of biodistribution and tumor targeting of [^99mTc]Tc-(HE)₃-E01 and [¹²³I]I-(HE)₃-E01-PIB was performed in Nu/j mice bearing EGFR-positive A-431 and EGFR-negative Ramos human cancer xenografts. Both radiolabeled DARPins demonstrated EGFR-specific tumor uptake. However, [¹²³I]I-(HE)₃-E01-PIB had appreciably lower uptake in normal organs compared to [^99mTc]Tc-(HE)₃-E01, which provided significantly (p < 0.05) higher tumor-to-organ ratios. Gamma-camera imaging confirmed that [¹²³I]I-(HE)₃-E01-PIB demonstrated a higher imaging contrast in preclinical models than [^99mTc]Tc-(HE)₃-E01. In conclusion, DARPin (HE)₃-E01 labeled using a non-residualizing [¹²³I]I-para-iodobenzoate (PIB) label is the preferred radiotracer for in vivo imaging of EGFR expression in cancer. 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

Designed ankyrin repeat protein (DARPin) Ec1, a small scaffold protein (18 kDa), binds with high affinity the epithelial cell adhesion molecule (EpCAM) that is overexpressed in several carcinomas. To enhance the targeted delivery of cytotoxic drugs using Ec1, we investigated the potential of fusing Ec1 with an albumin-binding domain (ABD) to improve its circulation time and decrease renal uptake. Two fusion proteins were created, Ec1-ABD, with the ABD at the C-terminus, and ABD-Ec1, with the ABD at the N-terminus. Both variants were labeled with ¹¹¹In. ABD-fused variants bound specifically to EpCAM-expressing cells with picomolar affinity. Adding human albumin reduced the affinity. This effect was more pronounced for Ec1-ABD; however, the affinity remained in the subnanomolar range. The position of the ABD did not influence the internalization rate of both variants by human cancer cells. In mouse models with human cancer xenografts, both variants demonstrated over 10-fold lower renal uptake compared to the Ec1. Tumor uptake of the ABD-fused variants was higher than the uptake of Ec1. ABD-Ec1 provided two-fold higher tumor uptake, indicating fusion with an ABD as a promising way to modulate the targeting properties of an Ec1-based construct. However, the effect of fusion depends on the order of the domains. Full article

Anal Squamous Cell Carcinoma (SCCA) is a rare Human Papillomavirus type 16 (HPV16)-associated carcinoma whose pathogenesis is still poorly understood. Recent studies based on biopsy and Next Generation Sequencing (NGS) approaches have linked the viral episomal status to aggressive SCCA phenotypes, suggesting a potential role of the 16E5 oncoprotein in tumor development. Our previous findings indicated that 16E5 induces Fibroblast Growth Factor Receptor 2 (FGFR2) isoform switching, aberrant mesenchymal FGFR2c expression, Epithelial Mesenchymal Transition (EMT), and cell invasion in various in vitro human keratinocyte models, as well as in the in vivo context of cervical Low-grade Squamous Intraepithelial Lesions (LSILs). To further explore the role of 16E5 in epithelial carcinogenesis, this study aims to investigate the molecular profile in HPV-related anal lesions. The results showed a significant positive correlation between 16E5 and FGFR2c, as well as 16E5 or FGFR2c and key EMT-related transcription factors, particularly in the group of HPV16 positive anal samples not containing without high grade lesions. Additionally, by coupling the molecular analysis with an interactome investigation, we hypothesized a potential functional interplay between the Ca²⁺ channel Transient Receptor Potential Ankyrin 1 (TRPA1) and FGFR2c, mediated by 16E5 during the establishment of the oncogenic signaling. These findings will help to elucidate the actual relevance of 16E5 in the early progression of anal lesions and contribute to determine its potential as target for future preventive approaches for HPV16-positive SCCA. Full article

Non-immunoglobulin-based scaffold proteins (SPs) represent one of the key therapeutic target-specific and high-affinity binders in modern medicine. Among their cellular targets are signaling receptors, in particular, receptor tyrosine kinases, whose dysfunction leads to the development of cancer and other serious diseases. Successful applications of SPs have been reported for HER receptor type 2 (HER2), a member of the human epidermal growth factor receptor family that regulates cell growth and differentiation. To extend the blood residence of SPs and prevent their high accumulation in the kidneys, these proteins are often fused with serum albumin. Promising results for HER2-binding activity were obtained for SP G3 from the DARPins (Designed Ankyrin Repeat Proteins) family fused with an albumin-binding domain (ABD). Interestingly, the detected HER2–G3 binding strongly depended on the position of the G3 module in the sequence of the constructs. Further improvement of these constructs for biomedical applications requires deciphering the molecular mechanism responsible for this effect. Here, we investigate the structural and dynamic aspects of ABD–G3 and G3–ABD chimeras using NMR spectroscopy and molecular modeling. Based on biophysical data, we come to the conclusion that extensive inter-domain contacts form in both constructs, although their binding interfaces and complex stability are somewhat different. Also, it is shown that the domain linker plays an important role—it limits the accessibility of the detected protein–protein binding sites, depending on the order of the domains in the chimeric molecules. These results create a solid structural basis for the rational design of new effective SP constructs targeting the signaling receptors in cells. Full article

Preeclampsia is a complex pregnancy-related hypertensive disorder which poses significant risks for both maternal and fetal health. Preeclampsia affects 5–8% of pregnancies in the United States, causing a significant public health and economic burden. Despite extensive research, the etiology and pathogenesis of preeclampsia remain elusive, but have been correlated with maternal conditions such as obesity. In recent decades, the incidence of preeclampsia increased along with the prevalence of obesity among women of reproductive age. Maternal obesity has been shown to negatively affect pregnancy in almost all aspects. However, the precise mechanisms by which obesity influences preeclampsia are unclear. Ankyrin repeat and SOCS Box Containing protein 4 (ASB4) is an E3 ubiquitin ligase that can promote the degradation of a wide range of target proteins. ASB4-null mice display a full spectrum of preeclampsia-like phenotypes during pregnancy including hypertension, proteinuria, and decreased litter size. Furthermore, maternal obesity induced by a high-fat diet aggravates preeclampsia-like phenotypes in pregnant mice lacking ASB4. Variants in the ASB4 gene have been associated with obesity in humans, and a functional connection between the ASB4 gene and obesity has been established in mice. This review discusses the connections between preeclampsia, obesity, and ASB4. Full article

The ammonia/ammonium (NH₃/NH₄⁺, AM) concentration in human erythrocytes (RBCs) is significantly higher than in plasma. Two main possible mechanisms for AM transport, including simple and facilitated diffusion, are described; however, the driving force for AM transport is not yet fully characterized. Since the erythroid ammonium channel RhAG forms a structural unit with anion exchanger 1 (eAE1) within the ankyrin core complex, we hypothesized the involvement of eAE1 in AM transport. To evaluate the functional interaction between eAE1 and RhAG, we used a unique feature of RBCs to swell and lyse in isotonic NH₄⁺ buffer. The kinetics of cell swelling and lysis were analyzed by flow cytometry and an original laser diffraction method, adapted for accurate volume sensing. The eAE1 role was revealed according to (i) the changes in cell swelling and lysis kinetics, and (ii) changes in intracellular pH, triggered by eAE1 inhibition or the modulation of eAE1 main ligand concentrations (Cl⁻ and HCO₃⁻). Additionally, the AM import kinetics was analyzed enzymatically and colorimetrically. In NH₄⁺ buffer, RBCs concentration-dependently swelled and lysed when [NH₄⁺] exceeded 100 mM. Cell swelling and hemolysis were tightly regulated by chloride concentration. The complete substitution of chloride with glutamate prevented NH₄⁺-induced cell swelling and hemolysis, and the restoration of [Cl⁻] dose-dependently amplified the rates of RBC swelling and lysis and the percentage of hemolyzed cells. Similarly, eAE1 inhibition impeded cell swelling and completely prevented hemolysis. Accordingly, eAE1 inhibition, or a lack of chloride anions in the buffer, significantly decreased NH₄⁺ import. Our data indicate that the eAE1-mediated chloride gradient is required for AM transport. Taken together, our data reveal a new player in AM transport in RBCs. Full article

HBV infection is challenging to cure due to the persistence of viral covalently closed circular viral DNA (cccDNA). The dedicator of cytokinesis 11 (DOCK11) is recognized as a guanine nucleotide exchange factor (GEF) for CDC42 that has been reported to be required for HBV persistence. DOCK11 is expressed in both the cytoplasm and nucleus of human hepatocytes and is functionally associated with retrograde trafficking proteins Arf-GAP with GTPase domain, ankyrin repeat, and pleckstrin homology domain-containing protein 2 (AGAP2), and ADP-ribosylation factor 1 (ARF1), together with the HBV capsid, in the trans-Golgi network (TGN). This opens an alternative retrograde trafficking route for HBV from early endosomes (EEs) to the TGN and then to the endoplasmic reticulum (ER), thereby avoiding lysosomal degradation. DOCK11 also facilitates the association of cccDNA with H3K4me3 and RNA Pol II for activating cccDNA transcription. In addition, DOCK11 plays a crucial role in the host DNA repair system, being essential for cccDNA synthesis. This function can be inhibited by 10M-D42AN, a novel DOCK11-binding peptide, leading to the suppression of HBV replication both in vitro and in vivo. Treatment with a combination of 10M-D42AN and entecavir may represent a promising therapeutic strategy for patients with chronic hepatitis B (CHB). Consequently, DOCK11 may be seen as a potential candidate molecule in the development of molecularly targeted drugs against CHB. Full article

Intellectual disability (ID), which affects around 2% to 3% of the population, accounts for 0.63% of the overall prevalence of neurodevelopmental disorders (NDD). ID is characterized by limitations in a person’s intellectual and adaptive functioning, and is caused by pathogenic variants in more than 1000 genes. Here, we report a rare missense variant (c.350T>C; p.(Leu117Ser)) in HACE1 segregating with NDD syndrome with clinical features including ID, epilepsy, spasticity, global developmental delay, and psychomotor impairment in two siblings of a consanguineous Pakistani kindred. HACE1 encodes a HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1), which is involved in protein ubiquitination, localization, and cell division. HACE1 is also predicted to interact with several proteins that have been previously implicated in the ID phenotype in humans. The p.(Leu117Ser) variant replaces an evolutionarily conserved residue of HACE1 and is predicted to be deleterious by various in silico algorithms. Previously, eleven protein truncating variants of HACE1 have been reported in individuals with NDD. However, to our knowledge, p.(Leu117Ser) is the second missense variant in HACE1 found in an individual with NDD. Full article

Designed ankyrin repeat protein (DARPin) G3 is an engineered scaffold protein. This small (14.5 kDa) targeting protein binds with high affinity to human epidermal growth factor receptor 2 (HER2). HER2 is overexpressed in several cancers. The use of the DARPin G3 for radionuclide therapy is complicated by its high renal reabsorption after clearance via the glomeruli. We tested the hypothesis that a fusion of the DARPin G3 with an albumin-binding domain (ABD) would prevent rapid renal excretion and high renal reabsorption resulting in better tumour targeting. Two fusion proteins were produced, one with the ABD at the C-terminus (G3-ABD) and another at the N-terminus (ABD-G3). Both variants were labelled with ¹⁷⁷Lu. The binding properties of the novel constructs were evaluated in vitro and their biodistribution was compared in mice with implanted human HER2-expressing tumours. Fusion with the ABD increased the retention time of both constructs in blood compared with the non-ABD-fused control. The effect of fusion with the ABD depended strongly on the order of the domains in the constructs, resulting in appreciably better targeting properties of [¹⁷⁷Lu]Lu-G3-ABD. Our data suggest that the order of domains is critical for the design of targeting constructs based on scaffold proteins. Full article

Osteosarcoma is a highly malignant, painful cancer with poor treatment opportunities and a bad prognosis. Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are non-selective cation channels that have been of great interest in cancer, as their expression is increased in some malignancies. In our study we aim to characterize the expression and functionality of the TRPA1 and TRPV1 channels in human and mouse osteosarcoma tissues and in a mouse cell line. TRPA1/Trpa1 and TRPV1/Trpv1 mRNA expressions were demonstrated by PCR gel electrophoresis and RNAscope in situ hybridization. The function of these channels was confirmed by their radioactive ₄₅Ca²⁺ uptake in response to the TRPA1 agonist, Allyl-isothiocyanate (AITC), and TRPV1 agonist, capsaicin, in K7M2 cells. An ATP-based K2M7 cell viability luminescence assay was used to determine cell viability after AITC or capsaicin treatments. Both TRPA1/Trpa1 and TRPV1/Trpv1 were expressed similarly in human and mouse osteosarcoma tissues, while Trpa1 transcripts were more abundantly present in K7M2 cells. TRPA1 activation with 200 µM AITC induced a significant ₄₅Ca²⁺ influx into K7M2 cells, and the antagonist attenuated this effect. In accordance with the lower Trpv1 expression, capsaicin induced a moderate ₄₅Ca²⁺ uptake, which did not reach the level of statistical significance. Both AITC and capsaicin significantly reduced K7M2 cell viability, demonstrating EC₅₀ values of 22 µM and 74 µM. The viability-decreasing effect of AITC was significantly but only partially antagonized by HC-030031, but the action of capsaicin was not affected by the TRPV1 antagonist capsazepine. We provide here the first data on the functional expression of the TRPA1 and TRPV1 ion channels in osteosarcoma, suggesting novel diagnostic and/or therapeutic perspectives. Full article