-
Prehemolytic impact of phenothiazine drugs on the attachment of spectrin network in red blood cells.Folia Medica Oct 2023Chlorpromazine, thioridazine, and trifluoperazine are phenothiazine drugs that cause colloid-osmotic hemolysis of human erythrocytes by unknown mechanism. To clarify...
Chlorpromazine, thioridazine, and trifluoperazine are phenothiazine drugs that cause colloid-osmotic hemolysis of human erythrocytes by unknown mechanism. To clarify this mechanism, the impact of these drugs on the βsp (1.4 MHz) and γ1sp (9 MHz) dielectric relaxations was investigated. Each relaxation was shown to reduce its strength on the severing specific bridge that connects the spectrin network with the lipid membrane. For βsp relaxation, this is the spectrin-actin-glycophorin C bridge while for γ1sp relaxation this is the spectrin-ankyrin-band 3 bridge.
Topics: Humans; Spectrin; Erythrocyte Membrane; Erythrocytes; Glycophorins; Phenothiazines
PubMed: 38351761
DOI: 10.3897/folmed.65.e97410 -
Pathology, Research and Practice Mar 2024Confirmatory diagnosis of celiac disease (CD) include histopathology of duodenal biopsy and tissue trans-glutaminase-IgA. Identification of tissue-specific histological...
Confirmatory diagnosis of celiac disease (CD) include histopathology of duodenal biopsy and tissue trans-glutaminase-IgA. Identification of tissue-specific histological markers is warranted to improve the diagnosis. A genetic study in CD identified the association of ankyrin-G that connects E-cadherin with β2-spectrin in epithelial cells of the duodenal tissue. We attempted to investigate the differential expression of ankyrin-G, E-cadherin and β2-spectrin in duodenal biopsy of CD subjects compared to non-CD controls. Duodenal tissue was collected from 83 study participants, of which 50 were CD, and 33 were non-CD controls. Whole RNA was isolated from 32 CD and 23 non-CD controls from available tissues, and differential mRNA expression was measured using real-time PCR. Tissue sections from 18 CD cases and 10 non-CD controls were immunostained using monoclonal antibodies. Tissue immunohistochemistry were evaluated for differential expression and pattern of expression. RT-PCR revealed significantly reduced expression of ankyrin-G (fold change=0.63; p=0.03) and E-cadherin (fold change=0.50; p=0.02) among CD subjects compared to non-CD controls. Tissue immunohistochemistry confirmed the reduced expression of ankyrin-G and E-cadherin in CD. Differential expression is grossly limited within the outer columnar epithelial cell layer. Expression fold change of E-cadherin was seen to partially correlate with the serum tTG level (r=0.4; p=0.04). In CD, reduced expression of two key cytoskeletal proteins (ankyrin-G and E-cadherin) in duodenum mucosa was observed, which indicates its implication in disease biology and could be tested as a tissue-specific biomarker for CD. Functional studies may unravel the specific contribution of these proteins in CD pathophysiology.
Topics: Humans; Celiac Disease; Ankyrins; Spectrin; Transglutaminases; Duodenum; Biopsy; Intestinal Mucosa; Cadherins
PubMed: 38324966
DOI: 10.1016/j.prp.2024.155164 -
Nucleosides, Nucleotides & Nucleic Acids Feb 2024Hereditary spherocytosis (HS) is the most common hereditary hemolytic disorder induced by red blood cell (RBC) membrane defect. This study was undertaken to determine...
Hereditary spherocytosis (HS) is the most common hereditary hemolytic disorder induced by red blood cell (RBC) membrane defect. This study was undertaken to determine mutations in genes associated with RBC membrane defect in patients with HS such as α-spectrin gene (SPTA1), β-spectrin gene (SPTB), ankyrin gene (ANK1), band 3 anion transport gene (SLC4A1) and erythrocyte membrane protein band 4.1 gene (EPB41). Blood samples were collected from 23 unrelated patients with HS. Patients were diagnosed according to the guidelines from the British Society for Hematology. All hematological examinations for the determination of RBC abnormalities and osmotic fragility tests were conducted. Genomic DNA were extracted from peripheral blood cells and coding exons of known genes for hereditary spherocytosis were enriched using Roche/KAPA sequence capture technology and sequenced on an Illumina system next-generation sequencing (NGS). The data showed that most of the HS patients confirmed splenomegaly and showed elevated reticulocytes and abnormal bilirubin values. NGS analysis identified the heterozygous variant c.5501G > A in the exon 39 of SPTA1 gene, resulted in a Trp1834*, which leads to a premature stop codon and subsequent mRNA degradation (nonsense- mediated decay) or truncation in α spectrin. Moreover, our data also revealed conventional mutations in genes SPTB, ANK, SLC4A1 and EBP41 in severe patients of HS. In short, this is the first report that determined a novel mutation c.5501G > A in SPTA1 gene in the Saudi population. To the best of our knowledge, this variant c.5501G > A has not been described in global literature so far. This novel mutation in SPTA1 gene is unique in the Saudi population.
PubMed: 38319988
DOI: 10.1080/15257770.2024.2310703 -
ELife Feb 2024Barrier functions of proliferative epithelia are constantly challenged by mechanical and chemical constraints. How epithelia respond to and cope with disturbances of...
Barrier functions of proliferative epithelia are constantly challenged by mechanical and chemical constraints. How epithelia respond to and cope with disturbances of barrier functions to allow tissue integrity maintenance is poorly characterised. Cellular junctions play an important role in this process and intracellular traffic contribute to their homeostasis. Here, we reveal that, in pupal , alteration of the bi- or tricellular septate junctions (SJs) triggers a mechanism with two prominent outcomes. On one hand, there is an increase in the levels of E-cadherin, F-actin, and non-muscle myosin II in the plane of adherens junctions. On the other hand, β-integrin/Vinculin-positive cell contacts are reinforced along the lateral and basal membranes. We found that the weakening of SJ integrity, caused by the depletion of bi- or tricellular SJ components, alters ESCRT-III/Vps32/Shrub distribution, reduces degradation and instead favours recycling of SJ components, an effect that extends to other recycled transmembrane protein cargoes including Crumbs, its effector β-Heavy Spectrin Karst, and β-integrin. We propose a mechanism by which epithelial cells, upon sensing alterations of the SJ, reroute the function of Shrub to adjust the balance of degradation/recycling of junctional cargoes and thereby compensate for barrier junction defects to maintain epithelial integrity.
Topics: Animals; Drosophila; Drosophila melanogaster; Drosophila Proteins; Epithelial Cells; Intercellular Junctions; Integrins
PubMed: 38305711
DOI: 10.7554/eLife.91246 -
Cellular and Molecular Biology... Dec 2023Colorectal cancer (CRC) is one of the most common and lethal malignancies. According to our analysis in the GEPIA database, SPTBN2 was found to be significantly elevated...
Colorectal cancer (CRC) is one of the most common and lethal malignancies. According to our analysis in the GEPIA database, SPTBN2 was found to be significantly elevated in COAD patients.Western blot also verified this result, and SPTBN2 was highly expressed in two types of colorectal cancer cells, Caco2 and HCT-8. Therefore, we knocked down SPTBN2 to investigate its function in colorectal cancer, and the results of CCK-8 and Transwell assays showed that SPTBN2 deletion inhibited the proliferation, migration and invasion of CRC cells. In addition, we found that SPTBN2 may be a target of miR-214-3p through the staebase database. miR-214-3p inhibitors promote CRC cell proliferation, migration and invasion. And inhibition of SPTBN2 partially reversed the effect of miR-214-3p in CRC. Taken together, we demonstrated that SPTBN2 acts as an important target of miR-214-3p in CRC. Our study lays the foundation for the mechanism of CRC.
Topics: Humans; Caco-2 Cells; Cell Line, Tumor; Cell Movement; Cell Proliferation; Colorectal Neoplasms; MicroRNAs; Spectrin
PubMed: 38279463
DOI: 10.14715/cmb/2023.69.14.20 -
Biomolecules Dec 2023Piezo1 is a mechanosensitive ion channel required for various biological processes, but its regulation remains poorly understood. Here, we used erythrocytes to address...
Piezo1 is a mechanosensitive ion channel required for various biological processes, but its regulation remains poorly understood. Here, we used erythrocytes to address this question since they display Piezo1 clusters, a strong and dynamic cytoskeleton and three types of submicrometric lipid domains, respectively enriched in cholesterol, GM1 ganglioside/cholesterol and sphingomyelin/cholesterol. We revealed that Piezo1 clusters were present in both the rim and the dimple erythrocyte regions. Upon Piezo1 chemical activation by Yoda1, the Piezo1 cluster proportion mainly increased in the dimple area. This increase was accompanied by Ca influx and a rise in echinocytes, in GM1/cholesterol-enriched domains in the dimple and in cholesterol-enriched domains in the rim. Conversely, the effects of Piezo1 activation were abrogated upon membrane cholesterol depletion. Furthermore, upon Piezo1-independent Ca influx, the above changes were not observed. In healthy donors with a high echinocyte proportion, Ca influx, lipid domains and Piezo1 fluorescence were high even at resting state, whereas the cytoskeleton membrane occupancy was lower. Accordingly, upon decreases in cytoskeleton membrane occupancy and stiffness in erythrocytes from patients with hereditary spherocytosis, Piezo1 fluorescence was increased. Altogether, we showed that Piezo1 was differentially controlled by lipid domains and the cytoskeleton and was favored by the stomatocyte-discocyte-echinocyte transformation.
Topics: Humans; Cholesterol; Cytoskeleton; Erythrocytes; G(M1) Ganglioside; Microtubules; Lipid Bilayers; Ion Channels
PubMed: 38254651
DOI: 10.3390/biom14010051 -
Redox Biology Apr 2024The function of SLC7A11 in the process of ferroptosis is well-established, as it regulates the synthesis of glutathione (GSH), thereby influencing tumor development...
The function of SLC7A11 in the process of ferroptosis is well-established, as it regulates the synthesis of glutathione (GSH), thereby influencing tumor development along with drug resistance in non-small cell lung cancer (NSCLC). However, the determinants governing SLC7A11's membrane trafficking and localization remain unknown. Our study identified SPTBN2 as a ferroptosis suppressor, enhancing NSCLC cells resistance to ferroptosis inducers. Mechanistically, SPTBN2, through its CH domain, interacted with SLC7A11 and connected it with the motor protein Arp1, thus facilitating the membrane localization of SLC7A11 - a prerequisite for its role as System Xc, which mediates cystine uptake and GSH synthesis. Consequently, SPTBN2 suppressed ferroptosis through preserving the functional activity of System Xc on the membrane. Moreover, Inhibiting SPTBN2 increased the sensitivity of NSCLC cells to cisplatin through ferroptosis induction, both in vitro and in vivo. Using Abrine as a potential SPTBN2 inhibitor, its efficacy in promoting ferroptosis and sensitizing NSCLC cells to cisplatin was validated. Collectively, SPTBN2 is a potential therapeutic target for addressing ferroptosis dysfunction and cisplatin resistance in NSCLC.
Topics: Humans; Amino Acid Transport System y+; Biological Transport; Carcinoma, Non-Small-Cell Lung; Cisplatin; Ferroptosis; Glutathione; Lung Neoplasms; Spectrin
PubMed: 38241838
DOI: 10.1016/j.redox.2024.103039 -
Life Sciences Feb 2024Cancer metastasis significantly contributes to mortality in lung cancer patients. Calmodulin-regulated spectrin-associated protein family member 2 (CAMSAP2) plays a...
AIMS
Cancer metastasis significantly contributes to mortality in lung cancer patients. Calmodulin-regulated spectrin-associated protein family member 2 (CAMSAP2) plays a significant role in cancer cell migration; however, its role in lung cancer metastasis and the underlying mechanism remain largely unknown. The present study aimed to investigate the impact of CAMSAP2 on lung cancer.
MAIN METHODS
The clinical relevance of CAMSAP2 in lung cancer patients was assessed using public database. RNA interference experiments were conducted to investigate role of CAMSAP2 in cell migration through transwell and wound healing assays. Molecular mechanisms were explored by identifying the possible interacting partners and pathways using the BioGRID and KEGG pathway analyses. The impact of CAMSAP2 on Ras protein activator-like 2 (RASAL2)-mediated lung cancer metastasis was investigated through biochemical assays. Additionally, in vivo experimentation using a murine tail vein metastasis model was performed to comprehend CAMSAP2's influence on metastasis.
KEY FINDINGS
A high expression level of CAMSAP2 was associated with poor overall survival in lung cancer patients and it positively correlated with cell migration in non-small cell lung cancer (NSCLC) cell lines. Knockdown of CAMSAP2 inhibited lung cancer cell motility in vitro and metastasis in vivo. Proteomic and biochemical analyses revealed the interaction between CAMSAP2 and RASAL2, which facilitates the degradation of RASAL2 through the ubiquitin-proteasome system. These degradation processes resulted in the activation of the extracellular signal-regulated kinase (ERK) signaling pathway, thereby promoting lung cancer metastasis. Collectively, the results of this study suggest that CAMSAP2 is a crucial regulator of cancer cell migration and metastasis and a promising therapeutic target for lung cancer.
Topics: Humans; Mice; Animals; Lung Neoplasms; Carcinoma, Non-Small-Cell Lung; Spectrin; Proteomics; Cell Movement; Family; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Microtubule-Associated Proteins; GTPase-Activating Proteins
PubMed: 38159595
DOI: 10.1016/j.lfs.2023.122391 -
Nature Communications Dec 2023Force transmission at integrin-based adhesions is important for cell migration and mechanosensing. Talin is an essential focal adhesion (FA) protein that links F-actin...
Force transmission at integrin-based adhesions is important for cell migration and mechanosensing. Talin is an essential focal adhesion (FA) protein that links F-actin to integrins. F-actin constantly moves on FAs, yet how Talin simultaneously maintains the connection to F-actin and transmits forces to integrins remains unclear. Here we show a critical role of dynamic Talin unfolding in force transmission. Using single-molecule speckle microscopy, we found that the majority of Talin are bound only to either F-actin or the substrate, whereas 4.1% of Talin is linked to both structures via elastic transient clutch. By reconstituting Talin knockdown cells with Talin chimeric mutants, in which the Talin rod subdomains are replaced with the stretchable β-spectrin repeats, we show that the stretchable property is critical for force transmission. Simulations suggest that unfolding of the Talin rod subdomains increases in the linkage duration and work at FAs. This study elucidates a force transmission mechanism, in which stochastic molecular stretching bridges two cellular structures moving at different speeds.
Topics: Actins; Talin; Actin Cytoskeleton; Integrins; Focal Adhesions
PubMed: 38123541
DOI: 10.1038/s41467-023-44018-z -
Frontiers in Genetics 2023Hereditary spherocytosis (HS) is a congenital haemolytic anaemia attributed to dysregulation or abnormal quantities of erythrocyte membrane proteins. Currently, the...
Hereditary spherocytosis (HS) is a congenital haemolytic anaemia attributed to dysregulation or abnormal quantities of erythrocyte membrane proteins. Currently, the most common erythrocytic gene, spectrin β (), variants are located in exons and give rise to mRNA defects. However, the genetic characteristics and pathogenic mechanisms of intronic variants are not completely understood. This study aimed to analyse a rare intronic inversion variant in the gene associated with HS, and explore the impact of the variant on mRNA splicing. The clinical manifestations of the patient were summarised and analysed for spherocytosis phenotype diagnosis. The pathogenic variant was identified in the proband using targeted next-generation and Sanger sequencing. RNA sequencing was performed to analyse whether gene splicing and expression were affected. Targeted next-generation sequencing identified a novel disease-associated intronic inversion variant of the gene in the proband. The inversion variant was located between intron 19 and 20, and contained the entire exon 20 and partial sequences of adjacent introns. Sanger sequencing confirmed that the intronic inversion variant only appeared in the genome of the proband, not in his parents. RNA sequencing revealed that the variant could result in the skipping of exon 20 and reduced expression of mRNA. This study identifies a rare intronic inversion variant in the gene associated with hereditary spherocytosis. The pathogenic variant can lead to exon 20 skipping and decreased gene expression. This finding has not been previously reported in any literature. This study can expand the intronic variant spectrum of the gene, deepen our understanding of HS pathogenesis, and contribute to the genetic diagnosis and clinical management of patients.
PubMed: 38111681
DOI: 10.3389/fgene.2023.1309040