RÉSUMÉ
Hearing, the ability to sense sounds, and the processing of auditory information are important for perception of the world. Mice lacking expression of neuroplastin (Np), a type-1 transmembrane glycoprotein, display deafness, multiple cognitive deficiencies, and reduced expression of plasma membrane calcium (Ca2+) ATPases (PMCAs) in cochlear hair cells and brain neurons. In this study, we transferred the deafness causing missense mutations pitch (C315S) and audio-1 (I122N) into human Np (hNp) constructs and investigated their effects at the molecular and cellular levels. Computational molecular dynamics show that loss of the disulfide bridge in hNppitch causes structural destabilization of immunoglobulin-like domain (Ig) III and that the novel asparagine in hNpaudio-1 results in steric constraints and an additional N-glycosylation site in IgII. Additional N-glycosylation of hNpaudio-1 was confirmed by PNGaseF treatment. In comparison to hNpWT, transfection of hNppitch and hNpaudio-1 into HEK293T cells resulted in normal mRNA levels but reduced the Np protein levels and their cell surface expression due to proteasomal/lysosomal degradation. Furthermore, hNppitch and hNpaudio-1 failed to promote exogenous PMCA levels in HEK293T cells. In hippocampal neurons, expression of additional hNppitch or hNpaudio-1 was less efficient than hNpWT to elevate endogenous PMCA levels and to accelerate the restoration of basal Ca2+ levels after electrically evoked Ca2+ transients. We propose that mutations leading to pathological Np variants, as exemplified here by the deafness causing Np mutants, can affect Np-dependent Ca2+ regulatory mechanisms and may potentially cause intellectual and cognitive deficits in humans.
Sujet(s)
Encéphale , Calcium , Surdité , Glycoprotéines membranaires , Mutation faux-sens , Neurones , Plasma Membrane Calcium-Transporting ATPases , Humains , Surdité/métabolisme , Surdité/génétique , Surdité/anatomopathologie , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Plasma Membrane Calcium-Transporting ATPases/génétique , Neurones/métabolisme , Cellules HEK293 , Glycoprotéines membranaires/métabolisme , Glycoprotéines membranaires/génétique , Calcium/métabolisme , Animaux , Encéphale/métabolisme , Encéphale/anatomopathologie , Membrane cellulaire/métabolisme , Souris , GlycosylationRÉSUMÉ
Our research aimed to elucidate the mechanism by which aurintricarboxylic acid (ATA) inhibits plasma membrane Ca2+-ATPase (PMCA), a crucial enzyme responsible for calcium transport. Given the pivotal role of PMCA in cellular calcium homeostasis, understanding how it is inhibited by ATA holds significant implications for potentially regulating physiopathological cellular processes in which this pump is involved. Our experimental findings revealed that ATA employs multiple modes of action to inhibit PMCA activity, which are influenced by ATP but also by the presence of calcium and magnesium ions. Specifically, magnesium appears to enhance this inhibitory effect. Our experimental and in-silico results suggest that, unlike those reported in other proteins, ATA complexed with magnesium (ATA·Mg) is the molecule that inhibits PMCA. In summary, our study presents a novel perspective and establishes a solid foundation for future research efforts aimed at the development of new pharmacological molecules both for PMCA and other proteins.
Sujet(s)
Acide aurintricarboxylique , Calcium , Magnésium , Plasma Membrane Calcium-Transporting ATPases , Magnésium/métabolisme , Magnésium/pharmacologie , Acide aurintricarboxylique/pharmacologie , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Plasma Membrane Calcium-Transporting ATPases/antagonistes et inhibiteurs , Calcium/métabolisme , Adénosine triphosphate/métabolisme , Membrane cellulaire/métabolisme , Membrane cellulaire/effets des médicaments et des substances chimiques , Animaux , HumainsRÉSUMÉ
In electrochemotherapy, permeabilization of the cell membrane by electric pulses increases the anti-tumour effect of chemotherapeutics. In calcium electroporation, chemotherapy is replaced by calcium chloride with obvious benefits. This study explores the effect and underlying mechanisms of calcium electroporation on basal cell carcinomas using either high- or low-frequency electroporation. Low-risk primary basal cell carcinomas were treated in local anaesthesia with intratumoral calcium chloride followed by electroporation with high (167 kHz) or low (5 kHz) frequencies. Non-complete responders were retreated after 3 months. The primary endpoint was tumour response 3 months after last calcium electroporation. Plasma membrane calcium ATPase was examined in various cell lines as plasma membrane calcium ATPase levels have been associated with calcium electroporation efficacy. Twenty-two out of 25 included patients complete the study and 7 of these (32%) achieved complete response at 3 months with no difference in efficacy between high- and low-frequency pulses. High-frequency calcium electroporation was significantly less painful (p=0.03). Plasma membrane calcium ATPase was increased 16-32-fold in basal cell carcinoma cell lines compared with 4 other cancer cell lines. Calcium electroporation for low-risk basal cell carcinomas does not fulfil the requirements of a new dermatological basal cell carcinoma treatment but may be useful as adjuvant treatment to surgery in more advanced basal cell carcinomas. The elevated PMCA levels in basal cell carcinomas may contribute to low efficacy.
Sujet(s)
Carcinome basocellulaire , Électrochimiothérapie , Tumeurs cutanées , Humains , Carcinome basocellulaire/anatomopathologie , Carcinome basocellulaire/thérapie , Tumeurs cutanées/anatomopathologie , Tumeurs cutanées/thérapie , Mâle , Femelle , Adulte d'âge moyen , Sujet âgé , Résultat thérapeutique , Électrochimiothérapie/méthodes , Lignée cellulaire tumorale , Chlorure de calcium/administration et posologie , Sujet âgé de 80 ans ou plus , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Facteurs temps , ÉlectroporationRÉSUMÉ
High serum estrogen concentrations are associated with asthma development and severity, suggesting a link between estradiol and airway hyperresponsiveness (AHR). 17ß-estradiol (E2) has non-genomic effects via Ca2+ regulatory mechanisms; however, its effect on the plasma membrane Ca2+-ATPases (PMCA1 and 4) and sarcoplasmic reticulum Ca2+-ATPase (SERCA) is unknown. Hence, in the present study, we aim to demonstrate if E2 favors AHR by increasing intracellular Ca2+ concentrations in guinea pig airway smooth muscle (ASM) through a mechanism involving Ca2+-ATPases. In guinea pig ASM, Ca2+ microfluorometry, muscle contraction, and Western blot were evaluated. Then, we performed molecular docking analysis between the estrogens and Ca2+ ATPases. In tracheal rings, E2 produced AHR to carbachol. In guinea pig myocytes, acute exposure to physiological levels of E2 modified the transient Ca2+ peak induced by caffeine to a Ca2+ plateau. The incubation with PMCA inhibitors (lanthanum and carboxyeosin, CE) partially reversed the E2-induced sustained plateau in the caffeine response. In contrast, cyclopiazonic acid (SERCA inhibitor), U-0126 (an inhibitor of ERK 1/2), and choline chloride did not modify the Ca2+ plateau produced by E2. The mitochondrial uniporter activity and the capacitative Ca2+ entry were unaffected by E2. In guinea pig ASM, Western blot analysis demonstrated PMCA1 and PMCA4 expression. The results from the docking modeling demonstrate that E2 binds to both plasma membrane ATPases. In guinea pig tracheal smooth muscle, inhibiting the PMCA with CE, induced hyperresponsiveness to carbachol. 17ß-estradiol produces hyperresponsiveness by inhibiting the PMCA in the ASM and could be one of the mechanisms responsible for the increase in asthmatic crisis in women.
Sujet(s)
Calcium , Oestradiol , Simulation de docking moléculaire , Plasma Membrane Calcium-Transporting ATPases , Animaux , Cochons d'Inde , Oestradiol/pharmacologie , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Calcium/métabolisme , Muscles lisses/effets des médicaments et des substances chimiques , Muscles lisses/métabolisme , Sarcoplasmic Reticulum Calcium-Transporting ATPases/métabolisme , Mâle , Trachée/effets des médicaments et des substances chimiques , Trachée/métabolisme , Contraction musculaire/effets des médicaments et des substances chimiques , Hypersensibilité respiratoire/induit chimiquement , Hypersensibilité respiratoire/métabolisme , Membrane cellulaire/effets des médicaments et des substances chimiques , Membrane cellulaire/métabolisme , Carbachol/pharmacologie , Myocytes du muscle lisse/effets des médicaments et des substances chimiques , Myocytes du muscle lisse/métabolismeRÉSUMÉ
Macropinocytosis (MPC) is a large-scale endocytosis pathway that involves actin-dependent membrane ruffle formation and subsequent ruffle closure to generate macropinosomes for the uptake of fluid-phase cargos. MPC is categorized into two types: constitutive and stimuli-induced. Constitutive MPC in macrophages relies on extracellular Ca2+ sensing by a calcium-sensing receptor. However, the link between stimuli-induced MPC and Ca2+ remains unclear. Here, we find that both intracellular and extracellular Ca2+ are required for epidermal growth factor (EGF)-induced MPC in A431 human epidermoid carcinoma cells. Through investigation of mammalian homologs of coelomocyte uptake defective (CUP) genes, we identify ATP2B4, encoding for a Ca2+ pump called the plasma membrane calcium ATPase 4 (PMCA4), as a Ca2+-related regulator of EGF-induced MPC. Knockout (KO) of ATP2B4, as well as depletion of extracellular/intracellular Ca2+, inhibited ruffle closure and macropinosome formation, without affecting ruffle formation. We demonstrate the importance of PMCA4 activity itself, independent of interactions with other proteins via its C-terminus known as a PDZ domain-binding motif. Additionally, we show that ATP2B4-KO reduces EGF-stimulated Ca2+ oscillation during MPC. Our findings suggest that EGF-induced MPC requires ATP2B4-dependent Ca2+ dynamics.
Sujet(s)
Calcium , Facteur de croissance épidermique , Pinocytose , Plasma Membrane Calcium-Transporting ATPases , Humains , Facteur de croissance épidermique/métabolisme , Facteur de croissance épidermique/pharmacologie , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Plasma Membrane Calcium-Transporting ATPases/génétique , Calcium/métabolisme , Lignée cellulaire tumoraleRÉSUMÉ
The plasma membrane Ca2+-ATPase (PMCA) is crucial for the fine tuning of intracellular calcium levels in eukaryotic cells. In this study, we show the presence of CARC sequences in all human and rat PMCA isoforms and we performed further analysis by molecular dynamics simulations. This analysis focuses on PMCA1, containing three CARC motifs, and PMCA4, with four CARC domains. In PMCA1, two CARC motifs reside within transmembrane domains, while the third is situated at the intracellular interface. The simulations depict more stable RMSD values and lower RMSF fluctuations in the presence of cholesterol, emphasizing its potential stabilizing effect. In PMCA4, a distinct dynamic was found. Notably, the total energy differences between simulations with cholesterol and phospholipids are pronounced in PMCA4 compared to PMCA1. RMSD values for PMCA4 indicate a more energetically favorable conformation in the presence of cholesterol, suggesting a robust interaction between CARCs and this lipid in the membranes. Furthermore, RMSF analysis for CARCs in both PMCA isoforms exhibit lower values in the presence of cholesterol compared to POPC alone. The analysis of H-bond occupancy and total energy values strongly suggests the potential interaction of CARCs with cholesterol. Given the crucial role of PMCAs in physiological calcium regulation and their involvement in diverse pathological processes, this study underscores the significance of CARC motifs and their interaction with cholesterol in elucidating PMCA function. These insights into the energetic preferences associated with CARC-cholesterol interactions offer valuable implications for understanding PMCA function in maintaining calcium homeostasis and addressing potential associated pathologies.
Sujet(s)
Cholestérol , Plasma Membrane Calcium-Transporting ATPases , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Plasma Membrane Calcium-Transporting ATPases/composition chimique , Cholestérol/métabolisme , Humains , Animaux , Rats , Simulation de dynamique moléculaire , Motifs d'acides aminés , Membrane cellulaire/métabolismeRÉSUMÉ
BACKGROUND: LncRNA ATP2B1-AS1 (ATP2B1-AS1) is involved in the occurrence and development of various diseases, while the relationship between lung adenocarcinoma (LUAD) and ATP2B1-AS1 is unclear. This study was to investigate the expression of ATP2B1-AS1 in LUAD and its influence on survival and prognosis of patients. METHODS: LUAD tissue samples from patients participating in this study were collected, and the expression levels of ATP2B1-AS1 and miR-141-3p in LUAD sampleswere detected by real-time quantitative polymerase chain reaction (RT-qPCR). The effect of ATP2B1-AS1 on the growth of A549 cells was investigated through cell counting kit-8 (CCK-8) and transwell experiments. Besides, the prognostic value of ATP2B1-AS1 in LUAD was assessed via Kaplan-Meier curve and multivariate Cox regression. RESULTS: ATP2B1-AS1 was downregulated in LUAD tissues and cells, whereas miR-141-3p was upregulated. After pcDNA3.1-ATP2B1-AS1 was transfected into A549 cells, the proliferation ability of A549 cells was decreased, and the migration level and invasion of A549 cells were also inhibited. High expression of ATP2B1-AS1 sponge miR-141-3p exerted prognostic value. CONCLUSIONS: ATP2B1-AS1 sponge miR-141-3p alleviated the progression of LUAD, and ATP2B1-AS1 may be deemed as a prognostic marker for LUAD.
Sujet(s)
Adénocarcinome , Tumeurs du poumon , microARN , ARN long non codant , Humains , microARN/génétique , microARN/métabolisme , ARN long non codant/génétique , Lignée cellulaire tumorale , Mouvement cellulaire/génétique , Prolifération cellulaire/génétique , Tumeurs du poumon/anatomopathologie , Poumon/anatomopathologie , Régulation de l'expression des gènes tumoraux , Plasma Membrane Calcium-Transporting ATPases/génétique , Plasma Membrane Calcium-Transporting ATPases/métabolismeRÉSUMÉ
ATP2B1 encodes plasma membrane calcium-transporting-ATPase1 and plays an essential role in maintaining intracellular calcium homeostasis that regulates diverse signaling pathways. Heterozygous de novo missense and truncating ATP2B1 variants are associated with a neurodevelopmental phenotype of variable expressivity. We describe a proband with distinctive craniofacial gestalt, Pierre-Robin sequence, neurodevelopmental and growth deficit, periventricular heterotopia, brachymesophalangy, cutaneous syndactyly, and persistent hypocalcemia from primary hypoparathyroidism. Proband-parent trio exome sequencing identified compound heterozygous ATP2B1 variants: a maternally inherited splice-site (c.3060+2 T > G) and paternally inherited missense c.2938 G > T; p.(Val980Leu). Reverse-transcription-PCR on the proband's fibroblast-derived mRNA showed aberrantly spliced ATP2B1 transcripts targeted for nonsense-mediated decay. All correctly-spliced ATP2B1 mRNA encoding p.(Val980Leu) functionally causes decreased cellular Ca2+ extrusion. Immunoblotting showed reduced fibroblast ATP2B1. We conclude that biallelic ATP2B1 variants are the likely cause of the proband's phenotype, strengthening the association of ATP2B1 as a neurodevelopmental gene and expanding the phenotypic characterization of a biallelic loss-of-function genotype.
Sujet(s)
Calcium , Hypoparathyroïdie , Humains , Calcium/métabolisme , Phénotype , Génotype , ARN messager , Hypoparathyroïdie/génétique , Plasma Membrane Calcium-Transporting ATPases/génétique , Plasma Membrane Calcium-Transporting ATPases/métabolismeRÉSUMÉ
Alpha-synuclein (aSN) is a membrane-associated and intrinsically disordered protein, well known for pathological aggregation in neurodegeneration. However, the physiological function of aSN is disputed. Pull-down experiments have pointed to plasma membrane Ca2+ -ATPase (PMCA) as a potential interaction partner. From proximity ligation assays, we find that aSN and PMCA colocalize at neuronal synapses, and we show that calcium expulsion is activated by aSN and PMCA. We further show that soluble, monomeric aSN activates PMCA at par with calmodulin, but independent of the autoinhibitory domain of PMCA, and highly dependent on acidic phospholipids and membrane-anchoring properties of aSN. On PMCA, the key site is mapped to the acidic lipid-binding site, located within a disordered PMCA-specific loop connecting the cytosolic A domain and transmembrane segment 3. Our studies point toward a novel physiological role of monomeric aSN as a stimulator of calcium clearance in neurons through activation of PMCA.
Sujet(s)
Calcium , alpha-Synucléine , Calcium/métabolisme , alpha-Synucléine/génétique , alpha-Synucléine/métabolisme , Plasma Membrane Calcium-Transporting ATPases/génétique , Plasma Membrane Calcium-Transporting ATPases/composition chimique , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Membrane cellulaire/métabolisme , Adenosine triphosphatases/métabolisme , Sites de fixationRÉSUMÉ
ABSTRACT: GLI1 gene alterations (rearrangement or amplification) have been found in several bone and soft tissue tumors including pericytic tumors, gastric plexiform fibromyxoma, gastroblastoma, and a various group of epithelioid tumors with regional recurrence or distant metastasis. In this article, we describe a case of primary cutaneous epithelioid mesenchymal tumor harboring hitherto not reported ATP2B4::GLI1 gene fusion. A 42-year-old man presented with a growing firm lesion on the left postauricular scalp. Microscopically, the shave biopsy specimen revealed a dermal-based nodular proliferation of relatively monotonous epithelioid cells with round to ovoid nuclei and pale eosinophilic cytoplasm, accompanied by prominent stromal vasculature. Significant cytologic atypia, necrosis, and mitotic activity were absent. The tumor cells were partially positive for CD34 and S-100 protein, but were negative for other markers, including SOX-10, keratins, and myogenic markers. An ATP2B4::GLI1 gene fusion was identified by next-generation sequencing. Array CGH was also performed, but it did not show relevant chromosomal copy number changes. Awareness of this rare cutaneous tumor, and thus, reporting of additional cases is necessary for further delineating its full clinicopathologic spectrum.
Sujet(s)
Tumeurs cutanées , Tumeurs des tissus mous , Mâle , Humains , Adulte , Protéine à doigt de zinc GLI1/génétique , Protéine à doigt de zinc GLI1/métabolisme , Tumeurs cutanées/anatomopathologie , Tumeurs des tissus mous/génétique , Tumeurs des tissus mous/anatomopathologie , Fusion de gènes , Protéines S100/génétique , Marqueurs biologiques tumoraux/génétique , Plasma Membrane Calcium-Transporting ATPases/génétique , Plasma Membrane Calcium-Transporting ATPases/métabolismeRÉSUMÉ
Acarbose (ACA), a well-studied and effective inhibitor of α-amylase and α-glucosidase, is a postprandial-acting antidiabetic medicine. The membrane of the erythrocyte is an excellent tool for analyzing different physiological and biochemical activities since it experiences a range of metabolic alterations throughout aging. It is uncertain if ACA modulates erythrocyte membrane activities in an age-dependent manner. As a result, the current study was conducted to explore the influence of ACA on age-dependent deteriorated functions of transporters/exchangers, disrupted levels of various biomarkers such as lipid hydroperoxides (LHs), protein carbonyl (PCO), sialic acid (SA), total thiol (-SH), and erythrocyte membrane osmotic fragility. In addition to a concurrent increase in Na+/H+ exchanger activity and concentration of LH, PCO, and osmotic fragility, we also detected a considerable decrease in membrane-linked activities of Ca2+-ATPase (PMCA) and Na+/K+-ATPase (NKA), as well as concentrations of SA and -SH in old-aged rats. The aging-induced impairment of the activities of membrane-bound ATPases and the changed levels of redox biomarkers were shown to be effectively restored by ACA treatment.
Sujet(s)
Acarbose , Vieillissement , Membrane érythrocytaire , Inhibiteurs des glycoside hydrolases , Plasma Membrane Calcium-Transporting ATPases , Sodium-Potassium-Exchanging ATPase , Acarbose/pharmacologie , Inhibiteurs des glycoside hydrolases/pharmacologie , Membrane érythrocytaire/composition chimique , Membrane érythrocytaire/effets des médicaments et des substances chimiques , Membrane érythrocytaire/enzymologie , Vieillissement/effets des médicaments et des substances chimiques , Vieillissement/métabolisme , Peroxydes lipidiques/analyse , Acides sialiques/analyse , Carbonylation des protéines/effets des médicaments et des substances chimiques , Thiols/analyse , Fragilité osmotique/effets des médicaments et des substances chimiques , Animaux , Rats , Mâle , Rat Wistar , Plasma Membrane Calcium-Transporting ATPases/analyse , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Sodium-Potassium-Exchanging ATPase/analyse , Sodium-Potassium-Exchanging ATPase/métabolisme , Oxydoréduction/effets des médicaments et des substances chimiques , Marqueurs biologiques/analyse , Marqueurs biologiques/métabolismeRÉSUMÉ
BACKGROUND: Sepsis is a life-threatening disease with dominant mortality. Its early diagnosis and treatment can improve prognosis and reduce mortality. Long noncoding RNAs (lncRNAs) ATPase plasma membrane Ca2+ transporting 1 antisense RNA 1 (ATP2B1-AS1) is dysregulated and is involved in the progression of various diseases. Nevertheless, the role of ATP2B1-AS1 in sepsis remains unclear. METHODS: A human monocytic cell line, THP-1 cells, was stimulated to induce a model of sepsis in vitro. The levels of ATP2B1-AS1, miR-23a-3p, and TLR4 were assessed by real-time quantitative polymerase chain reaction. The role of ATP2B1-AS1 in cell apoptosis and inflammation was explored by flow cytometry, Western blot analysis and enzyme-linked immunosorbent serologic assay. The binding sites between ATP2B1-AS1 and miR-23a-3p, and between miR-23a-3p and TLR4 were predicted by BiBiServ and the Encyclopedia of RNA Interactomes (ENCORI) online sites, respectively, and confirmed by the luciferase assay. RESULTS: The level of ATP2B1-AS1 was increased in lipopolysaccharide (LPS)-treated THP-1 cells. LPS increased apoptosis ratio, relative protein expressions of pro-apoptotic factors, and relative messenger RNA (mRNA) level and concentrations of pro-inflammatory cytokines, but decreased the relative expression of anti-apoptosis protein and relative mRNA level and concentrations of anti-inflammatory factor. All these alterations were reversed with transfection of shATP2B1-AS1 into THP-1 cells. Moreover, ATP2B1-AS1 directly bound miR-23a-3p and negatively modulated the level of miR-23a-3p. Meanwhile, TLR4 was directly targeted by miR-23a-3p, and negatively and positively modulated by miR-23a-3p and ATP2B1-AS1, respectively. CONCLUSION: ATP2B1-AS1 aggravated apoptosis and inflammation by modulating miR-23a-3p/TLR4 axis in LPS-treated THP-1 cells.
Sujet(s)
microARN , Sepsie , Humains , microARN/génétique , microARN/métabolisme , Récepteur de type Toll-4/génétique , Récepteur de type Toll-4/métabolisme , Lipopolysaccharides , Inflammation/métabolisme , Prolifération cellulaire/génétique , Plasma Membrane Calcium-Transporting ATPases/métabolismeRÉSUMÉ
The hippocampus is an important part of the limbic system in the human brain that has essential roles in spatial navigation and cognitive functions. It is still unknown how gene expression changes in single-cell in different spatial locations of the hippocampus of Parkinson's disease. The purpose of this study was to analyze the gene expression features of single cells in different spatial locations of mouse hippocampus, and to explore the effects of gene expression regulation on learning and memory mechanisms. Here, we obtained 74 single-cell samples from different spatial locations in a mouse hippocampus through microdissection technology, and used single-cell RNA-sequencing and spatial transcriptome sequencing to visualize and quantify the single-cell transcriptome features of tissue sections. The results of differential expression analysis showed that the expression of Sv2b, Neurod6, Grp and Stk32b genes in a hippocampus single cell at different locations was significantly different, and the marker genes of CA1, CA3 and DG subregions were identified. The results of gene function enrichment analysis showed that the up-regulated differentially expressed genes Tubb2a, Eno1, Atp2b1, Plk2, Map4, Pex5l, Fibcd1 and Pdzd2 were mainly involved in neuron to neuron synapse, vesicle-mediated transport in synapse, calcium signaling pathway and neurodegenerative disease pathways, thus affecting learning and memory function. It revealed the transcriptome profile and heterogeneity of spatially located cells in the hippocampus of PD for the first time, and demonstrated that the impaired learning and memory ability of PD was affected by the synergistic effect of CA1 and CA3 subregions neuron genes. These results are crucial for understanding the pathological mechanism of the Parkinson's disease and making precise treatment plans.
Sujet(s)
Maladies neurodégénératives , Maladie de Parkinson , Souris , Humains , Animaux , Maladie de Parkinson/métabolisme , Maladies neurodégénératives/métabolisme , Hippocampe/métabolisme , Analyse de profil d'expression de gènes , Transcriptome , Plasma Membrane Calcium-Transporting ATPases/métabolismeRÉSUMÉ
BACKGROUND: Polymorphisms in ATP2B4 coding for PMCA4b, the primary regulator of erythrocyte calcium concentration, have been shown by GWAS and cross-sectional studies to protect against severe malaria but the mechanism remains unknown. METHODS: Using a recall-by-genotype design, we investigated the impact of a common haplotype variant in ATP2B4 using in vitro assays that model erythrocyte stage malaria pathogenesis. Ninety-six donors representing homozygote (carriers of the minor allele, C/C), heterozygote (T/C) and wildtype (T/T) carriers of the tagging SNP rs1541252 were selected from a cohort of over 12,000 participants in the Keneba Biobank. RESULTS: Red blood cells (RBCs) from homozygotes showed reduced PMCA4b protein expression (mean fluorescence intensities (MFI = 2428 ± 124, 3544 ± 159 and 4261 ± 283], for homozygotes, heterozygotes and wildtypes respectively, p < 0.0001) and slower rates of calcium expulsion (calcium t½ ± SD = 4.7 ± 0.5, 1.8 ± 0.3 and 1.9 ± 0.4 min, p < 0.0001). Growth of a Plasmodium falciparum laboratory strain (FCR3) and two Gambian field isolates was decreased in RBCs from homozygotes compared to heterozygotes and wildtypes (p < 0.01). Genotype group did not affect parasite adhesion in vitro or var-gene expression in malaria-infected RBCs. Parasite growth was inhibited by a known inhibitor of PMCA4b, aurintricarboxylic acid (IC50 = 122uM CI: 110-134) confirming its sensitivity to calcium channel blockade. CONCLUSION: The data support the hypothesis that this ATP2B4 genotype, common in The Gambia and other malaria-endemic areas, protects against severe malaria through the suppression of parasitaemia during an infection. Reduction in parasite density plays a pivotal role in disease outcome by minimizing all aspects of malaria pathogenesis. Follow up studies are needed to further elucidate the mechanism of protection and to determine if this ATP2B4 genotype carries a fitness cost or increases susceptibility to other human disease.
Sujet(s)
Paludisme à Plasmodium falciparum , Plasma Membrane Calcium-Transporting ATPases , Adulte , Humains , Calcium/métabolisme , Études transversales , Érythrocytes/parasitologie , Gambie , Paludisme à Plasmodium falciparum/génétique , Plasma Membrane Calcium-Transporting ATPases/génétique , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Plasmodium falciparum , Polymorphisme de nucléotide simpleRÉSUMÉ
Disruption of calcium (Ca2+) homeostasis is emerging as a prevalent feature of aging and aging-associated neurodegenerative diseases, including Alzheimer's disease (AD), the most common type of tauopathy. This disease is characterized by the combined presence of extracellular neuritic plaques composed by amyloid ß-peptides (Aß) and neurofibrillary tangles of tau. The association of calcium dyshomeostasis with Aß has been extensively studied, however its link with tau has been less investigated. Thus, this review will concentrate on the functional link between tau and the plasma membrane Ca2+ pump (PMCA) and other membrane proteins involved in the regulation of intracellular calcium and/or its association with neurodegeneration.
Sujet(s)
Maladie d'Alzheimer , Peptides bêta-amyloïdes , Humains , Peptides bêta-amyloïdes/métabolisme , Calcium/métabolisme , Adenosine triphosphatases/métabolisme , Protéines tau/métabolisme , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Maladie d'Alzheimer/métabolisme , Membrane cellulaire/métabolisme , Enchevêtrements neurofibrillaires/métabolismeRÉSUMÉ
Calcium concentration must be finely tuned in all eukaryotic cells to ensure the correct performance of its signalling function. Neuronal activity is exquisitely dependent on the control of Ca2+ homeostasis: its alterations ultimately play a pivotal role in the origin and progression of many neurodegenerative processes. A complex toolkit of Ca2+ pumps and exchangers maintains the fluctuation of cytosolic Ca2+ concentration within the appropriate threshold. Two ubiquitous (isoforms 1 and 4) and two neuronally enriched (isoforms 2 and 3) of the plasma membrane Ca2+ATPase (PMCA pump) selectively regulate cytosolic Ca2+ transients by shaping the sub-plasma membrane (PM) microdomains. In humans, genetic mutations in ATP2B1, ATP2B2 and ATP2B3 gene have been linked with hearing loss, cerebellar ataxia and global neurodevelopmental delay: all of them were found to impair pump activity. Here we report three additional mutations in ATP2B3 gene corresponding to E1081Q, R1133Q and R696H amino acids substitution, respectively. Among them, the novel missense mutation (E1081Q) immediately upstream the C-terminal calmodulin-binding domain (CaM-BD) of the PMCA3 protein was present in two patients originating from two distinct families. Our biochemical and molecular studies on PMCA3 E1081Q mutant have revealed a splicing variant-dependent effect of the mutation in shaping the sub-PM [Ca2+]. The E1081Q substitution in the full-length b variant abolished the capacity of the pump to reduce [Ca2+] in the sub-PM microdomain (in line with the previously described ataxia-related PMCA mutations negatively affecting Ca2+ pumping activity), while, surprisingly, its introduction in the truncated a variant selectively increased Ca2+ extrusion activity in the sub-PM Ca2+ microdomains. These results highlight the importance to set a precise threshold of [Ca2+] by fine-tuning the sub-PM microdomains and the different contribution of the PMCA splice variants in this regulation.
Sujet(s)
Ataxie cérébelleuse , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Acides aminés , Ataxie/génétique , Ataxie/métabolisme , Calcium/métabolisme , Calmoduline/génétique , Membrane cellulaire/métabolisme , Ataxie cérébelleuse/génétique , Ataxie cérébelleuse/métabolisme , Humains , Mutation/génétique , Plasma Membrane Calcium-Transporting ATPases/composition chimique , Plasma Membrane Calcium-Transporting ATPases/génétique , Isoformes de protéines/génétique , Isoformes de protéines/métabolismeRÉSUMÉ
Ca2+ is a ubiquitous second messenger, which allows eukaryotic cells to respond to external stimuli. The use of genetically encoded Ca2+ indicators allows real-time monitoring of cytosolic Ca2+ levels to study such responses. Here we explored the possibility of using the ratiometric Ca2+ indicator GEM-GECO for monitoring cytosolic Ca2+ concentration ([Ca2+]cyt) in the yeast Ogataea parapolymorpha. High-level production of GEM-GECO led to a severe growth defect in cells lacking the vacuolar Ca2+ ATPase Pmc1, which is involved in [Ca2+]cyt control, and prompted a phenotype resembling that of Pmc1 deficiency, in a strain with wild-type PMC1. This was likely due to the presence of the calmodulin domain in GEM-GECO. In contrast to previous studies of genetically-encoded calcium indicators in neuronal cells, our results suggest that physiological effects of GEM-GECO expression in yeast cells are due not to Ca2+ depletion, but to excessive Ca2+ signaling. Despite these drawbacks, study of fluorescence in individual cells revealed switching of GEM-GECO from the Ca2+-free to Ca2+-bound state minutes after external addition of CaCl2. This was followed by gradual return of GEM-GECO to a Ca2+-free-state that was impaired in the pmc1-Δ mutant. These results demonstrate GEM-GECO usability for [Ca2+]cyt monitoring in budding yeast.
Sujet(s)
Protéines de Saccharomyces cerevisiae , Saccharomycetales , Calcium/métabolisme , Calcium-Transporting ATPases/métabolisme , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Saccharomyces cerevisiae/métabolisme , Protéines de Saccharomyces cerevisiae/génétique , Protéines de Saccharomyces cerevisiae/métabolisme , Saccharomycetales/génétique , Saccharomycetales/métabolismeRÉSUMÉ
Plasma membrane calcium ATPase 1 (PMCA1, Atp2b1) is emerging as a key contributor to cardiac physiology, involved in calcium handling and myocardial signalling. In addition, genome wide association studies have associated PMCA1 in several areas of cardiovascular disease including hypertension and myocardial infarction. Here, we investigated the role of PMCA1 in basal cardiac function and heart rhythm stability. Cardiac structure, heart rhythm and arrhythmia susceptibility were assessed in a cardiomyocyte-specific PMCA1 deletion (PMCA1CKO) mouse model. PMCA1CKO mice developed abnormal heart rhythms related to ventricular repolarisation dysfunction and displayed an increased susceptibility to ventricular arrhythmias. We further assessed the levels of cardiac ion channels using qPCR and found a downregulation of the voltage-dependent potassium channels, Kv4.2, with a corresponding reduction in the transient outward potassium current which underlies ventricular repolarisation in the murine heart. The changes in heart rhythm were found to occur in the absence of any structural cardiomyopathy. To further assess the molecular changes occurring in PMCA1CKO hearts, we performed proteomic analysis. Functional characterisation of differentially expressed proteins suggested changes in pathways related to metabolism, protein-binding, and pathways associated cardiac function including ß-adrenergic signalling. Together, these data suggest an important role for PMCA1 in basal cardiac function in relation to heart rhythm control, with reduced cardiac PMCA1 expression resulting in an increased risk of arrhythmia development.
Sujet(s)
Plasma Membrane Calcium-Transporting ATPases , Dysfonction ventriculaire , Animaux , Souris , Troubles du rythme cardiaque/métabolisme , Calcium/métabolisme , Étude d'association pangénomique , Myocytes cardiaques/métabolisme , Plasma Membrane Calcium-Transporting ATPases/génétique , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Protéomique , Dysfonction ventriculaire/métabolismeRÉSUMÉ
The plasma membrane calcium ATPase (PMCA) extrudes calcium from the cytosol to the extracellular space to terminate calcium-dependent signaling. Although the distribution of PMCA is crucial for its function, the molecular mechanisms that regulate the localization of PMCA isoforms are not well understood. PLEKHA7 is implicated by genetic studies in hypertension and the regulation of calcium handling. PLEKHA7 recruits the small adapter protein PDZD11 to adherens junctions, and together they control the trafficking and localization of plasma membrane associated proteins, including the Menkes copper ATPase. Since PDZD11 binds to the C-terminal domain of b-isoforms of PMCA, PDZD11 and its interactor PLEKHA7 could control the localization and activity of PMCA. Here, we test this hypothesis using cultured cell model systems. We show using immunofluorescence microscopy and a surface biotinylation assay that KO of either PLEKHA7 or PDZD11 in mouse kidney collecting duct epithelial cells results in increased accumulation of endogenous PMCA at lateral cell-cell contacts and PDZ-dependent ectopic apical localization of exogenous PMCA4x/b isoform. In HeLa cells, coexpression of PDZD11 reduces membrane accumulation of overexpressed PMCA4x/b, and analysis of cytosolic calcium transients shows that PDZD11 counteracts calcium extrusion activity of overexpressed PMCA4x/b, but not PMCA4x/a, which lacks the PDZ-binding motif. Moreover, KO of PDZD11 in either endothelial (bEnd.3) or epithelial (mouse kidney collecting duct) cells increases the rate of calcium extrusion. Collectively, these results suggest that the PLEKHA7-PDZD11 complex modulates calcium homeostasis by regulating the localization of PMCA.
Sujet(s)
Calcium , Protéines de transport , Plasma Membrane Calcium-Transporting ATPases , Jonctions adhérentes/métabolisme , Animaux , Calcium/métabolisme , Protéines de transport/métabolisme , Cellules cultivées , Cellules HeLa , Humains , Souris , Plasma Membrane Calcium-Transporting ATPases/génétique , Plasma Membrane Calcium-Transporting ATPases/métabolisme , Isoformes de protéines/métabolismeRÉSUMÉ
Plasma membrane calcium ATPases (PMCAs) are a family of transmembrane proteins responsible for the extrusion of cytosolic Ca2+ to the extracellular milieu. They are important players of the calcium homeostasis possibly implicated in some important diseases. The reference inhibitors of PMCA extruding activity are on one hand ortho-vanadate (IC50 in the 30 mM range), and on the other a series of 12- to 20-mer peptides named caloxins (IC50 in the 100 µM scale). As for all integral membrane proteins, biochemistry and pharmacology are difficult to study on isolated and/or purified proteins. Using a series of reference blockers, we assessed a pharmacological window with which we could study the functionality of PMCAs in living cells. Using this system, we screened for alternative versions of caloxins, aiming at shortening the peptide backbone, introducing non-natural amino acids, and overall trying to get a glimpse at the structure-activity relationship between those new peptides and the protein in a cellular context. We describe a short series of equipotent 5-residue long analogues with IC50 in the low µM range.