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Journal of Molecular Biology Oct 2023ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance...
ABCG2 is an ATP-binding cassette transporter that exports a wide range of xenobiotic compounds and has been recognized as a contributing factor for multidrug resistance in cancer cells. Substrate and inhibitor interactions with ABCG2 have been extensively studied and small molecule inhibitors have been developed that prevent the export of anticancer drugs from tumor cells. Here, we explore the potential for inhibitors that target sites other than the substrate binding pocket of ABCG2. We developed novel nanobodies against ABCG2 and used functional analyses to select three inhibitory nanobodies (Nb8, Nb17 and Nb96) for structural studies by single particle cryo-electron microscopy. Our results showed that these nanobodies allosterically bind to different regions of the nucleotide binding domains. Two copies of Nb8 bind to the apex of the NBDs preventing them from fully closing. Nb17 binds near the two-fold axis of the transporter and interacts with both NBDs. Nb96 binds to the side of the NBD and immobilizes a region connected to key motifs involved in ATP binding and hydrolysis. All three nanobodies prevent the transporter from undergoing conformational changes required for substrate transport. These findings advance our understanding of the molecular basis of modulation of ABCG2 by external binders, which may contribute to the development of a new generation of inhibitors. Furthermore, this is the first example of modulation of human multidrug resistance transporters by nanobodies.
Topics: Humans; ATP Binding Cassette Transporter, Subfamily G, Member 2; ATP-Binding Cassette Transporters; Cryoelectron Microscopy; Hydrolysis; Membrane Transport Proteins; Neoplasm Proteins; Single-Domain Antibodies
PubMed: 37597690
DOI: 10.1016/j.jmb.2023.168234 -
Critical Care (London, England) Nov 2023Critically ill patients suffer from acute muscle wasting, which is associated with significant physical functional impairment. We describe data from nested muscle biopsy...
BACKGROUND
Critically ill patients suffer from acute muscle wasting, which is associated with significant physical functional impairment. We describe data from nested muscle biopsy studies from two trials of functional electrical stimulation (FES) that did not shown improvements in physical function.
METHODS
Primary cohort: single-centre randomized controlled trial. Additional healthy volunteer data from patients undergoing elective hip arthroplasty. Validation cohort: Four-centre randomized controlled trial.
INTERVENTION
FES cycling for 60-90min/day.
ANALYSES
Skeletal muscle mRNA expression of 223 genes underwent hierarchal clustering for targeted analysis and validation.
RESULTS
Positively enriched pathways between healthy volunteers and ICU participants were "stress response", "response to stimuli" and "protein metabolism", in keeping with published data. Positively enriched pathways between admission and day 7 ICU participants were "FOXO-mediated transcription" (admission = 0.48 ± 0.94, day 7 = - 0.47 ± 1.04 mean log fold change; P = 0.042), "Fatty acid metabolism" (admission = 0.50 ± 0.67, day 7 = 0.07 ± 1.65 mean log fold change; P = 0.042) and "Interleukin-1 processing" (admission = 0.88 ± 0.50, day 7 = 0.97 ± 0.76 mean log fold change; P = 0.054). Muscle mRNA expression of UCP3 (P = 0.030) and DGKD (P = 0.040) decreased in both cohorts with no between group differences. Changes in IL-18 were not observed in the validation cohort (P = 0.268). Targeted analyses related to intramuscular mitochondrial substrate oxidation, fatty acid oxidation and intramuscular inflammation showed PPARγ-C1α; (P < 0.001), SLC25A20 (P = 0.017) and UCP3 (P < 0.001) decreased between admission and day 7 in both arms. LPIN-1 (P < 0.001) and SPT1 (P = 0.044) decreased between admission and day 7. IL-18 (P = 0.011) and TNFRSF12A (P = 0.009) increased in both arms between admission and day 7. IL-1β (P = 0.007), its receptor IL-1R1 (P = 0.005) and IL-6R (P = 0.001) decreased in both arms between admission and day 7. No between group differences were seen in any of these (all p > 0.05).
CONCLUSIONS
Intramuscular inflammation and altered substrate utilization are persistent in skeletal muscle during first week of critical illness and are not improved by the application of Functional Electrical Stimulation-assisted exercise. Future trials of exercise to prevent muscle wasting and physical impairment are unlikely to be successful unless these processes are addressed by other means than exercise alone.
Topics: Humans; Critical Illness; Interleukin-18; Intensive Care Units; Muscular Atrophy; Electric Stimulation; Fatty Acids; RNA, Messenger; Membrane Transport Proteins
PubMed: 37932834
DOI: 10.1186/s13054-023-04664-7 -
Communications Biology Apr 2024Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other...
Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other inflammatory factors. We identified a previously uncharacterized peptide in the Klebsiella pneumoniae secretome, which enters Streptococcus pneumoniae via its AmiA-AliA/AliB permease. Subsequent downregulation of genes for amino acid biosynthesis and peptide uptake was associated with reduction of pneumococcal growth in defined medium and human cerebrospinal fluid, irregular cell shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was specific to S. pneumoniae and Streptococcus pseudopneumoniae with no effect on Streptococcus mitis, Haemophilus influenzae, Staphylococcus aureus or K. pneumoniae. Peptide sequence and length were crucial to growth suppression. The peptide reduced pneumococcal adherence to primary human airway epithelial cell cultures and colonization of rat nasopharynx, without toxicity. We identified a peptide with potential as a therapeutic for pneumococcal diseases suppressing growth of multiple clinical isolates, including antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis.
Topics: Rats; Animals; Humans; Streptococcus pneumoniae; Klebsiella pneumoniae; Membrane Transport Proteins; Nasopharynx; Pneumococcal Infections; Peptides
PubMed: 38589539
DOI: 10.1038/s42003-024-06113-9 -
Microbiology (Reading, England) Nov 2023The controlled entry and expulsion of small molecules across the bacterial cytoplasmic membrane is essential for efficient cell growth and cellular homeostasis. While... (Review)
Review
The controlled entry and expulsion of small molecules across the bacterial cytoplasmic membrane is essential for efficient cell growth and cellular homeostasis. While much is known about the transcriptional regulation of genes encoding transporters, less is understood about how transporter activity is modulated once the protein is functional in the membrane, a potentially more rapid and dynamic level of control. In this review, we bring together literature from the bacterial transport community exemplifying the extensive and diverse mechanisms that have evolved to rapidly modulate transporter function, predominantly by switching activity off. This includes small molecule feedback, inhibition by interaction with small peptides, regulation through binding larger signal transduction proteins and, finally, the emerging area of controlled proteolysis. Many of these examples have been discovered in the context of metal transport, which has to finely balance active accumulation of elements that are essential for growth but can also quickly become toxic if intracellular homeostasis is not tightly controlled. Consistent with this, these transporters appear to be regulated at multiple levels. Finally, we find common regulatory themes, most often through the fusion of additional regulatory domains to transporters, which suggest the potential for even more widespread regulation of transporter activity in biology.
Topics: Membrane Transport Proteins; Cell Membrane; Bacteria
PubMed: 37948297
DOI: 10.1099/mic.0.001412 -
Biochimica Et Biophysica Acta. General... Jan 2024In advanced and recurrent endometrial carcinoma (EC), the current state of immuno- or targeted therapy remains in the clinical research phase. Our study aimed to explore...
BACKGROUND
In advanced and recurrent endometrial carcinoma (EC), the current state of immuno- or targeted therapy remains in the clinical research phase. Our study aimed to explore the role of the ESCRT machinery in maintaining cell membrane integrity and reversing pyroptotic cell death.
METHODS
Immunohistochemistry, western blotting, and co-immunoprecipitation were performed to determine the expression and relationship between GSDMD, CHMP4B, and VPS4A. We employed techniques such as FITC Annexin V/propidium iodide staining, Ca fluorescence intensity, IL-1β enzyme-linked immunosorbent assay, and lactate dehydrogenase release assay to detect pyroptosis in endometrial cancer cells. Plasma membrane perforations and CHMP4B/VPS4A puncta were observed through electron and fluorescence confocal microscopy.
RESULTS
We showed that GSDMD, CHMP4B, and VPS4A were differentially expressed in the pyroptotic EC xenograft mouse model group, as well as high, moderate, and mild expression in EC cells treated with LPS and nigericin compared to endometrial epithelial cells. Co-IP confirmed the interaction between GSDMD, CHMP4B, and VPS4A. We found that GSDMD knockdown reduced PI-positive cells, Ca efflux, IL-1β, and LDH release, while CHMP4B and VPS4A depletion enhanced these indicators in HEC1A and AN3CA cells. Electron microscopy showed membrane perforations correspondingly decreased with inactivated GSDMD and increased or decreased after CHMP4B and VPS4A depletion or overexpression in EC cells. Fluorescence confocal microscopy detected CHMP4B protein puncta associated with VPS4A at the injured plasma membrane in GSDMD cells.
CONCLUSIONS
We preliminary evidenced that CHMP4B and VPS4A reverses GSDMD-mediated pyroptosis by facilitating cell membrane remodeling in endometrial carcinoma. Targeting CHMP4B related proteins may promote pyroptosis in endometrial tumors.
Topics: Female; Humans; Mice; Animals; Pyroptosis; Intracellular Signaling Peptides and Proteins; Cell Membrane; Disease Models, Animal; Endometrial Neoplasms; Endosomal Sorting Complexes Required for Transport; ATPases Associated with Diverse Cellular Activities; Vacuolar Proton-Translocating ATPases; Gasdermins; Phosphate-Binding Proteins
PubMed: 37931722
DOI: 10.1016/j.bbagen.2023.130497 -
Zhurnal Nevrologii I Psikhiatrii Imeni... 2024This review highlights literature data on potential genetic markers that potentially influence the development of postoperative cognitive dysfunction, such as and The... (Review)
Review
This review highlights literature data on potential genetic markers that potentially influence the development of postoperative cognitive dysfunction, such as and The main pathogenetic mechanisms triggered by these genes and leading to the development of cognitive impairment after anesthesia are described. The paper systematizes previously published works that provide evidence of the impact of specific genetic variants on the development of postoperative cognitive dysfunction.
Topics: Humans; Postoperative Cognitive Complications; Apolipoproteins E; Mitochondrial Precursor Protein Import Complex Proteins; Methyltransferases; Membrane Glycoproteins; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Membrane Transport Proteins; Genetic Markers; Reelin Protein; Cognitive Dysfunction; Genetic Predisposition to Disease; Receptors, Immunologic
PubMed: 38676676
DOI: 10.17116/jnevro202412404143 -
Identification of potential prognostic markers for lung adenocarcinoma using comprehensive analysis.Molecular Medicine Reports Aug 2023Lung adenocarcinoma (LUAD) is a common malignancy throughout the world with high levels of mortality and morbidity. In the present study, potential biomarkers and...
Lung adenocarcinoma (LUAD) is a common malignancy throughout the world with high levels of mortality and morbidity. In the present study, potential biomarkers and treatment targets for LUAD were investigated using data from The Cancer Genome Atlas. Overall, 4,485 differentially expressed genes (DEGs) were identified (1,857 upregulated and 2,628 downregulated) between tumor and adjacent control tissues. Functional analysis with Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Gene Set Variation Analysis and Gene Set Enrichment Analysis revealed significant enrichment of the DEGs in pathways related to system development, cell cycle and cell adhesion. Weighted gene co‑expression network analysis distinguished ten co‑expression modules on inclusion of the clinical profiles of patients with LUAD. Of these, the blue/turquoise modules showed peak association with tumor onset. Analysis of hub modules identified five hub genes, namely , , , and (also known as ). Survival analysis revealed associations between hub‑gene expression profiles and patient prognosis. Downregulation of in LUAD tumor tissues was confirmed using immunohistochemistry. Additional assays (Cell Counting Kit‑8, colony formation, scratch assay, cell cycle, Transwell invasion assay and cell adhesion assay) revealed that overexpression inhibited A549 cell growth, invasion and migration. The findings demonstrated that the hub genes could act as treatment targets or new biomarkers for LUAD.
Topics: Humans; Prognosis; Biomarkers, Tumor; Adenocarcinoma of Lung; Gene Expression Profiling; Lung Neoplasms; Serotonin Plasma Membrane Transport Proteins; Receptor-Like Protein Tyrosine Phosphatases, Class 3; Angiopoietin-like Proteins; Angiopoietin-Like Protein 7
PubMed: 37350390
DOI: 10.3892/mmr.2023.13036 -
Cells Sep 2023Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs...
Cell-penetrating peptides (CPPs) are short peptide sequences that have the ability to cross the cell membrane and deliver cargo. Although it is critical that CPPs accomplish this task with minimal off-target effects, such actions have in many cases not been robustly screened. We presently investigated whether the commonly used CPPs TAT and the polyarginines Arg and Arg exert off-target effects on cellular Ca homeostasis. In experiments employing myocytes and homogenates from the cardiac left ventricle or soleus muscle, we observed marked inhibition of Ca recycling into the sarcoplasmic reticulum (SR) following incubation with polyarginine CPPs. In both tissues, the rate of SR Ca leak remained unchanged, indicating that protracted Ca removal from the cytosol stemmed from inhibition of the SR Ca ATPase 2 (SERCA2). No such inhibition occurred following treatment with TAT, or in preparations from the SERCA1-expressing extensor digitorum longus muscle. Experiments in HEK cells overexpressing individual SERCA isoforms confirmed that polyarginine incubation specifically inhibited the activity of SERCA2a and 2b, but not SERCA1 or 3. The attenuation of SERCA2 activity was not dependent on the presence of phospholamban, and ELISA-based analyses rather revealed direct interaction between the polyarginines and the actuator domain of the protein. Surface plasmon resonance experiments confirmed strong binding within this region of SERCA2, and slow dissociation between the two species. Based on these observations, we urge caution when employing polyarginine CPPs. Indeed, as SERCA2 is expressed in diverse cell types, the wide-ranging consequences of SERCA2 binding and inhibition should be anticipated in both experimental and therapeutic settings.
Topics: Sarcoplasmic Reticulum Calcium-Transporting ATPases; Cell-Penetrating Peptides; Muscle, Skeletal; Protein Isoforms
PubMed: 37830576
DOI: 10.3390/cells12192358 -
The Journal of Biological Chemistry Feb 2024The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of...
The mammalian SID-1 transmembrane family members, SIDT1 and SIDT2, are multipass transmembrane proteins that mediate the cellular uptake and intracellular trafficking of nucleic acids, playing important roles in the immune response and tumorigenesis. Previous work has suggested that human SIDT1 and SIDT2 are N-glycosylated, but the precise site-specific N-glycosylation information and its functional contribution remain unclear. In this study, we use high-resolution liquid chromatography tandem mass spectrometry to comprehensively map the N-glycosites and quantify the N-glycosylation profiles of SIDT1 and SIDT2. Further molecular mechanistic probing elucidates the essential role of N-linked glycans in regulating cell surface expression, RNA binding, protein stability, and RNA uptake of SIDT1. Our results provide crucial information about the potential functional impact of N-glycosylation in the regulation of SIDT1-mediated RNA uptake and provide insights into the molecular mechanisms of this promising nucleic acid delivery system with potential implications for therapeutic applications.
Topics: Humans; Biological Transport; Glycosylation; Mammals; Membrane Proteins; Nucleotide Transport Proteins; RNA
PubMed: 38237680
DOI: 10.1016/j.jbc.2024.105654 -
JACC. Clinical Electrophysiology Dec 2023Atrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular...
BACKGROUND
Atrial fibrillation (AF), the most common cardiac arrhythmia, is widely associated with inflammation, vascular dysfunction, and elevated levels of the vascular leak-inducing cytokine, vascular endothelial growth factor (VEGF). Mechanisms underlying AF are poorly understood and current treatments only manage this progressive disease, rather than arresting the underlying pathology. The authors previously identified edema-induced disruption of sodium channel (NaV1.5)-rich intercalated disk nanodomains as a novel mechanism for AF initiation secondary to acute inflammation. Therefore, we hypothesized that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias.
OBJECTIVES
In this study the authors tested the hypothesis that protecting the vascular barrier can prevent vascular leak-induced atrial arrhythmias. They identified 2 molecular targets for vascular barrier protection, connexin43 (Cx43) hemichannels and pannexin-1 (Panx1) channels, which have been implicated in cytokine-induced vascular leak.
METHODS
The authors undertook in vivo electrocardiography, electron microscopy, and super-resolution light microscopy studies in mice acutely treated with a clinically relevant level of VEGF.
RESULTS
AF incidence was increased in untreated mice exposed to VEGF relative to vehicle control subjects. VEGF also increased the average number of AF episodes. VEGF shifted Na1.5 signal to longer distances from Cx43 gap junctions, measured by a distance transformation-based spatial analysis of 3-dimensional confocal images of intercalated disks. Similar effects were observed with Na1.5 localized near mechanical junctions composed of neural cadherin. Blocking connexin43 hemichannels (αCT11 peptide) or Panx1 channels (PxIL2P peptide) significantly reduced the duration of AF episodes compared with VEGF alone with no treatment. Concurrently, both peptide therapies preserved Na1.5 distance from gap junctions to control levels and reduced mechanical junction-adjacent intermembrane distance in these hearts. Notably, similar antiarrhythmic efficacy was also achieved with clinically-relevant small-molecule inhibitors of Cx43 and Panx1.
CONCLUSIONS
These results highlight vascular barrier protection as an antiarrhythmic strategy following inflammation-induced vascular leak.
Topics: Animals; Humans; Mice; Anti-Arrhythmia Agents; Atrial Fibrillation; Connexin 43; Connexins; Cytokines; Inflammation; Myocytes, Cardiac; Nanostructures; Nerve Tissue Proteins; Vascular Endothelial Growth Factor A
PubMed: 38032579
DOI: 10.1016/j.jacep.2023.10.013