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1.
J Neuromuscul Dis ; 11(2): 315-326, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38217607

RESUMO

Background: Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of muscle mass and muscle function. Previous work from our lab demonstrated that skeletal muscles from a mouse model of ALS show elevated intracellular calcium (Ca2+) levels and heightened endoplasmic reticulum (ER) stress. Objective: To investigate whether overexpression of sarcoplasmic reticulum (SR) Ca2+ ATPase 1 (SERCA1) in skeletal muscle would improve intracellular Ca2+ handling, attenuate ER stress, and improve motor function ALS transgenic mice. Methods: B6SJL-Tg (SOD1*G93A)1Gur/J (ALS-Tg) mice were bred with skeletal muscle α-actinin SERCA1 overexpressing mice to generate wild type (WT), SERCA1 overexpression (WT/+SERCA1), ALS-Tg, and SERCA1 overexpressing ALS-Tg (ALS-Tg/+SERCA1) mice. Motor function (grip test) was assessed weekly and skeletal muscles were harvested at 16 weeks of age to evaluate muscle mass, SR-Ca2+ ATPase activity, levels of SERCA1 and ER stress proteins - protein disulfide isomerase (PDI), Grp78/BiP, and C/EBP homologous protein (CHOP). Single muscle fibers were also isolated from the flexor digitorum brevis muscle to assess changes in resting and peak Fura-2 ratios. Results: ALS-Tg/+SERCA1 mice showed improved motor function, delayed onset of disease, and improved muscle mass compared to ALS-Tg. Further, ALS-Tg/+SERCA1 mice returned levels of SERCA1 protein and SR-Ca2+ ATPase activity back to levels in WT mice. Unexpectedly, SERCA-1 overexpression increased levels of the ER stress maker Grp78/BiP in both WT and ALS-Tg mice, while not altering protein levels of PDI or CHOP. Lastly, single muscle fibers from ALS-Tg/+SERCA1 had similar resting but lower peak Fura-2 levels (at 30 Hz and 100 Hz) compared to ALS-Tg mice. Conclusions: These data indicate that SERCA1 overexpression attenuates the progressive loss of muscle mass and maintains motor function in ALS-Tg mice while not lowering resting Ca2+ levels or ER stress.


Assuntos
Esclerose Amiotrófica Lateral , Camundongos , Animais , Chaperona BiP do Retículo Endoplasmático , Cálcio/metabolismo , Fura-2/metabolismo , Músculo Esquelético , Camundongos Transgênicos , Atrofia Muscular/metabolismo , ATPases Transportadoras de Cálcio/metabolismo
2.
J Agric Food Chem ; 72(2): 1276-1291, 2024 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-38179648

RESUMO

Microorganisms rely on diverse ion transport and trace elements to sustain growth, development, and secondary metabolism. Manganese (Mn2+) is essential for various biological processes and plays a crucial role in the metabolism of human cells, plants, and yeast. In Aspergillus flavus, we confirmed that Pmr1 localized in cis- and medial-Golgi compartments was critical in facilitating Mn2+ transport, fungal growth, development, secondary metabolism, and glycosylation. In comparison to the wild type, the Δpmr1 mutant displayed heightened sensitivity to environmental stress, accompanied by inhibited synthesis of aflatoxin B1, kojic acid, and a substantial reduction in pathogenicity toward peanuts and maize. Interestingly, the addition of exogenous Mn2+ effectively rectified the developmental and secondary metabolic defects in the Δpmr1 mutant. However, Mn2+ supplement failed to restore the growth and development of the Δpmr1Δgdt1 double mutant, which indicated that the Gdt1 compensated for the functional deficiency of pmr1. In addition, our results showed that pmr1 knockout leads to an upregulation of O-glycosyl-N-acetylglucose (O-GlcNAc) and O-GlcNAc transferase (OGT), while Mn2+ supplementation can restore the glycosylation in A. flavus. Collectively, this study indicates that the pmr1 regulates Mn2+ via Golgi and maintains growth and metabolism functions of A. flavus through regulation of the glycosylation.


Assuntos
ATPases Transportadoras de Cálcio , Proteínas de Saccharomyces cerevisiae , Humanos , ATPases Transportadoras de Cálcio/metabolismo , Aflatoxina B1/metabolismo , Aspergillus flavus/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo
3.
Artigo em Inglês | MEDLINE | ID: mdl-37977241

RESUMO

Polycyclic aromatic hydrocarbons (PAHs) are embryo- and cardiotoxic to fish that might be associated with improper intracellular Ca2+ management. Since sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a major regulator of intracellular Ca2+, the SERCA activity and the contractile properties of rainbow trout (Oncorhynchus mykiss) ventricle were measured in the presence of 3- and 4-cyclic PAHs. In unfractionated ventricular homogenates, acute exposure of SERCA to 0.1-1.0 µM phenanthrene (Phe), retene (Ret), fluoranthene (Flu), or pyrene (Pyr) resulted in concentration-dependent increase in SERCA activity, except for the Flu exposure, with maximal effects of 49.7-83 % at 1 µM. However, PAH mixture did not affect the contractile parameters of trout ventricular strips. Similarly, all PAHs, except Ret, increased the myotomal SERCA activity, but with lower effect (27.8-40.8 % at 1 µM). To investigate the putative chronic effects of PAHs on SERCA, the atp2a2a gene encoding trout cardiac SERCA was expressed in human embryonic kidney (HEK) cells. Culture of HEK cells in the presence of 0.3-1.0 µM Phe, Ret, Flu, and Pyr for 4 days suppressed SERCA expression in a concentration-dependent manner, with maximal inhibition of 49 %, 65 %, 39 % (P < 0.05), and 18 % (P > 0.05), respectively at 1 µM. Current findings indicate divergent effects of submicromolar PAH concentrations on SERCA: stimulation of SERCA activity in acute exposure and inhibition of SERCA expression in chronic exposure. The depressed expression of SERCA is likely to contribute to the embryo- and cardiotoxicity of PAHs by depressing muscle function and altering gene expression.


Assuntos
Oncorhynchus mykiss , Fenantrenos , Hidrocarbonetos Policíclicos Aromáticos , Animais , Humanos , Oncorhynchus mykiss/metabolismo , Fenantrenos/toxicidade , Fenantrenos/metabolismo , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Hidrocarbonetos Policíclicos Aromáticos/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Estresse do Retículo Endoplasmático , Cálcio/metabolismo
4.
Acta Derm Venereol ; 103: adv10436, 2023 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-38014829

RESUMO

Hailey-Hailey disease is a rare hereditary skin disease caused by mutations in the ATP2C1 gene encoding the secretory pathway Ca2+/Mn2+-ATPase 1 (SPCA1) protein. Extracutaneous manifestations of Hailey-Hailey disease are plausible but still largely unknown. The aim of this study was to explore the association between Hailey-Hailey disease and diabetes. A population-based cohort study of 347 individuals with Hailey-Hailey  disease was performed to assess the risks of type 1  diabetes and type 2 diabetes, using Swedish nationwide registries. Pedigrees from 2 Swedish families with Hailey-Hailey disease were also investigated: 1 with concurrent type 1 diabetes and HLA-DQ3, the other with type 2 diabetes. Lastly, a clinical cohort with 23 individuals with Hailey-Hailey disease and matched healthy controls was evaluated regarding diabetes. In the register data males with Hailey-Hailey disease had a 70% elevated risk of type 2 diabetes, whereas no  excess risk among women could be confirmed. In both pedigrees an unusually high inheritance for diabetes was observed. In the clinical cohort, individuals with Hailey-Hailey disease displayed a metabolic phenotype indicative of type 2 diabetes. Hailey-Hailey disease seems to act as a synergistic risk factor for diabetes. This study indicates, for the first time, an association between Hailey-Hailey disease and diabetes and represents human evidence that SPCA1 and the Golgi apparatus may be implicated in diabetes pathophysiology.


Assuntos
Diabetes Mellitus Tipo 2 , Pênfigo Familiar Benigno , Masculino , Humanos , Feminino , Pênfigo Familiar Benigno/diagnóstico , Pênfigo Familiar Benigno/epidemiologia , Pênfigo Familiar Benigno/genética , Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Linhagem , Estudos de Coortes , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Mutação
5.
Neuroscience ; 532: 103-112, 2023 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-37778690

RESUMO

At the vertebrate neuromuscular junction (NMJ), presynaptic homeostatic potentiation (PHP) refers to an increase in neurotransmitter release that restores the strength of synaptic transmission following a blockade of nicotinic acetylcholine receptors (nAChRs). Mechanisms informing the presynaptic terminal of the loss of postsynaptic receptivity remain poorly understood. Previous research at the mouse NMJ suggests that extracellular protons may function as a retrograde signal that triggers an upregulation of neurotransmitter output (measured by quantal content, QC) through the activation of acid-sensing ion channels (ASICs). We further investigated the pH-dependency of PHP in an ex-vivo mouse muscle preparation. We observed that increasing the buffering capacity of the perfusion saline with HEPES abolishes PHP and that acidifying the saline from pH 7.4 to pH 7.2-7.1 increases QC, demonstrating the necessity and sufficiency of extracellular acidification for PHP. We then sought to uncover how the blockade of nAChRs leads to the pH decrease. Plasma-membrane calcium ATPase (PMCA), a calcium-proton antiporter, is known to alkalize the synaptic cleft following neurotransmission in a calcium-dependent manner. We hypothesize that since nAChR blockade reduces postsynaptic calcium entry, it also reduces the alkalizing activity of the PMCA, thereby causing acidosis, ASIC activation, and QC upregulation. In line with this hypothesis, we found that pharmacological inhibition of the PMCA with carboxyeosin induces QC upregulation and that this effect requires functional ASICs. We also demonstrated that muscles pre-treated with carboxyeosin fail to generate PHP. These findings suggest that reduced PMCA activity causes presynaptic homeostatic potentiation by activating ASICs at the mouse NMJ.


Assuntos
Cálcio , Junção Neuromuscular , Animais , Camundongos , Cálcio/metabolismo , Junção Neuromuscular/metabolismo , Transmissão Sináptica , Terminações Pré-Sinápticas/metabolismo , Canais Iônicos Sensíveis a Ácido , Neurotransmissores/farmacologia , Concentração de Íons de Hidrogênio , ATPases Transportadoras de Cálcio/farmacologia
6.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 48(5): 641-647, 2023 May 28.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-37539566

RESUMO

OBJECTIVES: Application of ultrashort wave (USW) to rats with cerebral ischemia and reperfusion injury could inhibit the decrease of expression of secretory pathway Ca2+-ATPase 1 (SPCA1), an important participant in Golgi stress, reduce the damage of Golgi apparatus and the apoptosis of neuronal cells, thereby alleviating cerebral ischemia-reperfusion injury. This study aims to investigate the effect of USW on oxygen-glucose deprivation/reperfusion (OGD/R) injury and the expression of SPCA1 at the cellular level. METHODS: N2a cells were randomly divided into a control (Con) group, an OGD/R group, and an USW group. The cells in the Con group were cultured without exposure to OGD. The cells in the OGD/R group were treated with OGD/R. The cells in the USW group were treated with USW after OGD/R. Cell morphology was observed under the inverted phase-contrast optical microscope, cell activity was detected by cell counting kit-8 (CCK-8), apoptosis was detected by flow cytometry, and SPCA1 expression was detected by Western blotting. RESULTS: Most of the cells in the Con group showed spindle shape with a clear outline and good adhesion. In the OGD/R group, cells were wrinkled, with blurred outline, poor adhesion, and lots of suspended dead cells appeared; compared with the OGD/R group, the cell morphology and adherence were improved, with clearer outlines and fewer dead cells in the USW group. Compared with the Con group, the OGD/R group showed decreased cell activity, increased apoptotic rate, and down-regulating SPCA1 expression with significant differences (all P<0.001); compared with the OGD/R group, the USW group showed increased cell activity, decreased apoptotic rate, and up-regulating SPCA1 expression with significant differences (P<0.01 or P<0.001). CONCLUSIONS: USW alleviates the injury of cellular OGD/R, and its protective effect may be related to its up-regulation of SPCA1 expression.


Assuntos
Isquemia Encefálica , ATPases Transportadoras de Cálcio , Traumatismo por Reperfusão , Animais , Ratos , Apoptose , Glucose/metabolismo , Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Ativação Transcricional , Regulação para Cima , ATPases Transportadoras de Cálcio/metabolismo
7.
Photodiagnosis Photodyn Ther ; 44: 103738, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37558191

RESUMO

Hailey-Hailey disease (HHD) is a rare genetic benign condition resulting in blisters predominantly on the skin folds. The inheritance is autosomal dominant with complete penetrance, but a variable expressivity in affected family members. It can be triggered by a vast variety of factors such as sweating, weight gain, infection, trauma, pregnancy, and ultraviolet radiation, but the major cause of the disease is a mutation in the ATP2C1 gene. The lesions are typically distributed symmetrically within intertriginous regions such as the retroarticular folds, axillae, inguinal, and perianal regions and presents as flaccid vesicles and blisters on erythematous skin, giving rise to erosions, fissures, and vegetations. There is no specific therapy for HHD. The therapeutic approach to HHD involves the control of exacerbating factors, secondary infections, and cutaneous inflammation. Because of the rarity of the disease, evidence of efficacy for topical or systemic therapies is mainly based on small observational studies, case reports, and clinical experience. We present a case of HHD successfully treated by photodynamic therapy (PDT) with a topical liposomal chlorin photosensitizer.


Assuntos
Pênfigo Familiar Benigno , Fotoquimioterapia , Humanos , Pênfigo Familiar Benigno/tratamento farmacológico , Pênfigo Familiar Benigno/genética , Pênfigo Familiar Benigno/patologia , Vesícula/tratamento farmacológico , Raios Ultravioleta , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/uso terapêutico , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/uso terapêutico
8.
Mol Cell Neurosci ; 126: 103886, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37567489

RESUMO

Polyglutamine (polyQ) induced neurodegeneration is one of the leading causes of progressive neurodegenerative disorders characterized clinically by deteriorating movement defects, psychiatric disability, and dementia. Calcium [Ca2+] homeostasis, which is essential for the functioning of neuronal cells, is disrupted under these pathological conditions. In this paper, we simulated Huntington's disease phenotype in the neuronal cells of the Drosophila eye and identified [Ca2+] pump, sarco-endoplasmic reticulum calcium ATPase (SERCA), as one of the genetic modifiers of the neurodegenerative phenotype. This paper shows genetic and molecular interaction between polyglutamine (polyQ) aggregates, SERCA and DIAP1. We present evidence that polyQ aggregates interact with SERCA and alter its dynamics, resulting in a decrease in cytosolic [Ca2+] and an increase in ER [Ca2+], and thus toxicity. Downregulating SERCA lowers the enhanced calcium levels in the ER and rescues, morphological and functional defects caused due to expanded polyQ repeats. Cell proliferation markers such as Yorkie (Yki), Scalloped (Sd), and phosphatidylinositol 3 kinases/protein kinase B (PI3K/Akt), also respond to varying levels of calcium due to genetic manipulations, adding to the amelioration of degeneration. These results imply that neurodegeneration due to expanded polyQ repeats is sensitive to SERCA activity, and its manipulation can be an important step toward its therapeutic measures.


Assuntos
Cálcio , Drosophila , Proteínas Inibidoras de Apoptose , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Animais , Apoptose , Cálcio/metabolismo , ATPases Transportadoras de Cálcio , Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Fosfatidilinositol 3-Quinases , Proteínas de Drosophila
9.
Biochimie ; 214(Pt B): 123-133, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37429409

RESUMO

We investigated the effects of environment calcium challenge and 1α,25(OH)2 vitamin D3 (1,25-D3) on 45Ca2+ influx in the intestine of zebrafish (ZF). In vitro45Ca2+ influx was analyzed using intestines from fed and fasted fish. ZF were held in water containing Ca2+ (0.02, 0.7, 2.0 mM) to analyze the ex vivo45Ca2+ influx in the intestine and for histology. Intestines from fish held in water with Ca2+ were incubated ex vivo to characterize ion channels, receptors, ATPases and ion exchangers that orchestrate 45Ca2+ influx. For in vitro studies, intestines were incubated with antagonists/agonist or inhibitors to study the mechanism of 1,25-D3 on 45Ca2+ influx. Fasted ZF reached a plateau for 45Ca2+ influx at 30 min. In vivo fish at high Ca2+ stimulated ex vivo45Ca2+ influx and increased the height of intestinal villi in low calcium. In the normal calcium, 45Ca2+ influx was maintained by the reverse-mode Na+/Ca2+ (NCX) activation, Na+/K+-ATPase pump and sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. However, Ca2+ hyperosmolarity is supported by L-type voltage-dependent calcium channels (L-VDCC), transient receptor potential vanilloid subfamily 1 (TRPV1) and Na+/K+-ATPase activity. The calcium challenge causes morphological alteration and changes the ion type-channels involved in the intestine to maintain hyperosmolarity. 1,25-D3 stimulates Ca2+ influx in normal osmolarity coordinated by L-VDCC activation and SERCA inhibition to keeps high intracellular calcium in intestine. Our data showed that the adult ZF regulates the calcium challenge (per se osmolarity), independently of the hormonal regulation to maintain the calcium balance through the intestine to support ionic adaptation.


Assuntos
Cálcio , Peixe-Zebra , Animais , Cálcio/metabolismo , Peixe-Zebra/metabolismo , Colecalciferol/farmacologia , Canais de Cálcio Tipo L , Canais Iônicos , ATPases Transportadoras de Cálcio , Intestinos , Água
10.
Cancer Control ; 30: 10732748231182787, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37306722

RESUMO

Zinc finger protein 384 (ZNF384) encodes a C2H2-type zinc finger protein that can function as a transcription factor. ZNF384 rearrangement in acute lymphoblastic leukemia (ALL) was first reported in 2002. More than 19 different ZNF384 fusion partners have been detected in ALL. These include E1A-binding protein P300 (EP300), CREB-binding protein (CREBBP), transcription factor 3 (TCF3), TATA-box binding protein associated factor 15 (TAF15), Ewing sarcoma breakpoint region 1 gene (EWSR1), AT-rich interactive domain-containing protein 1B (ARID1B), SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily A, member 4 (SMARCA4), SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily A, member 2 (SMARCA2), synergin gamma (SYNRG), clathrin heavy chain (CLTC), bone morphogenic protein 2-inducible kinase (BMP2K), Nipped-B-like protein (NIPBL), A Kinase Anchoring Protein 8 (AKAP8), Chromosome 11 Open Reading Frame 74 (C11orf74), DEAD-Box Helicase 42 (DDX42), ATP Synthase F1 Subunit Gamma (ATP2C1), Euchromatic Histone Lysine Methyltransferase 1 (EHMT1), Testic Expressed 41 (TEX41), etc. Patients diagnosed with ALL harboring ZNF384 rearrangements commonly had a good prognosis. The mechanisms, performance, and features of different ZNF384 rearrangements in acute lymphoblastic leukemia have been well evaluated.


Assuntos
Actinas , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Cromatina , Proteínas de Ciclo Celular , DNA Helicases , Proteínas Nucleares , Fatores de Transcrição , Transativadores , ATPases Transportadoras de Cálcio
11.
Microvasc Res ; 150: 104572, 2023 11.
Artigo em Inglês | MEDLINE | ID: mdl-37353069

RESUMO

Pathological cardiac hypertrophy is an adaptive reaction in response to pressure or volume overload. Autophagy is critical for damage caused by pathological cardiac hypertrophy. Vacuole membrane protein 1 (VMP1) is an endoplasmic reticulum (ER) transmembrane protein that is effective in activating autophagy. However, the role of VMP1 in pathological cardiac hypertrophy and its underlying mechanisms remain elusive. This study was designed to explore the potential mechanisms of VMP1 on pressure overload-induced pathological cardiac hypertrophy. In this work, abdominal aorta constriction (AAC) surgery was used to induce pathological cardiac hypertrophy in male C57BL/6 mice. H9C2 cardiomyocytes were treated with phenylephrine stimulation (PE) to induce the hypertrophic response. The in vivo results revealed that mice with AAC surgery caused pathological cardiac hypertrophy as evidenced by improved cardiac function according to multiple echocardiographic parameters. Moreover, elevated VMP1 expression was also observed in mice after AAC surgery. VMP1 knockdown aggravated changes in cardiac structure, cardiac dysfunction, and fibrosis. Meanwhile, VMP1 knockdown suppressed autophagy and endoplasmic reticulum calcium ATPase (SERCA) activity in heart tissues. H9C2 cardiomyocytes with VMP1 overexpression were used to investigate the specific mechanism of VMP1 in pathological cardiac hypertrophy, and VMP1 overexpression increased autophagic flux by upregulating SERCA activity. In conclusion, these findings revealed that VMP1 protected against pressure overload-induced pathological cardiac hypertrophy by inducing SERCA-regulated autophagic flux. Our results provide valuable insights regarding the pathophysiology of pathological cardiac hypertrophy and clues to a novel target for the treatment of pathological cardiac hypertrophy.


Assuntos
Cardiomegalia , Miócitos Cardíacos , Masculino , Camundongos , Animais , Camundongos Endogâmicos C57BL , Cardiomegalia/metabolismo , Miócitos Cardíacos/patologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Autofagia , ATPases Transportadoras de Cálcio/metabolismo , ATPases Transportadoras de Cálcio/farmacologia
12.
Biochim Biophys Acta Mol Cell Res ; 1870(7): 119508, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37290725

RESUMO

Calcium (Ca2+)-ATPases are ATP-dependent enzymes that transport Ca2+ ions against their electrochemical gradient playing the fundamental biological function of keeping the free cytosolic Ca2+ concentration in the submicromolar range to prevent cytotoxic effects. In plants, type IIB autoinhibited Ca2+-ATPases (ACAs) are localised both at the plasma membrane and at the endomembranes including endoplasmic reticulum (ER) and tonoplast and their activity is primarily regulated by Ca2+-dependent mechanisms. Instead, type IIA ER-type Ca2+-ATPases (ECAs) are present mainly at the ER and Golgi Apparatus membranes and are active at resting Ca2+. Whereas research in plants has historically focused on the biochemical characterization of these pumps, more recently the attention has been also addressed on the physiological roles played by the different isoforms. This review aims to highlight the main biochemical properties of both type IIB and type IIA Ca2+ pumps and their involvement in the shaping of cellular Ca2+ dynamics induced by different stimuli.


Assuntos
Adenosina Trifosfatases , ATPases Transportadoras de Cálcio , Adenosina Trifosfatases/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Plantas/metabolismo , Transdução de Sinais/fisiologia , Membrana Celular/metabolismo
13.
Mol Biol Cell ; 34(8): br12, 2023 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-37163315

RESUMO

Protein secretion is essential for epithelial tissue homoeostasis and therefore has to be tightly regulated. However, while the mechanisms regulating polarized protein sorting and trafficking have been widely studied in the past decade, those governing polarized secretion remain elusive. The calcium manganese pump SPCA1 and the calcium-binding protein Cab45 were recently shown to regulate the secretion of a subset of soluble cargoes in nonpolarized HeLa cells. Interestingly, we demonstrated that in polarized epithelial cells calcium levels in the trans-Golgi network (TGN), controlled by SPCA1, and Cab45 are critical for the apical sorting of glycosylphosphatidylinositol-anchored proteins (GPI-APs), a class of integral membrane proteins containing a soluble protein attached to the membrane by the GPI anchor, prompting us to investigate the mechanism regulating the polarized secretion of soluble cargoes. By reducing Cab45 expression level or overexpressing an inactive mutant of SPCA1, we found that Cab45 and calcium levels in the TGN drive the polarized apical secretion of a secretory form of placental alkaline phosphatase, exogenously expressed, and the endogenous soluble protein clusterin/Gp80 in Madin-Darby canine kidney (MDCK) cells. These data highlight the critical role of a calcium-dependent Cab45 mechanism regulating apical exocytosis in polarized MDCK cells.


Assuntos
Cálcio , Placenta , Feminino , Gravidez , Humanos , Animais , Cães , Células HeLa , Cálcio/metabolismo , Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , Via Secretória , Polaridade Celular , Membrana Celular/metabolismo
14.
Cell Res ; 33(7): 533-545, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37258749

RESUMO

Secretory-pathway Ca2+-ATPases (SPCAs) play critical roles in maintaining Ca2+ homeostasis, but the exact mechanism of SPCAs-mediated Ca2+ transport remains unclear. Here, we determined six cryo-electron microscopy (cryo-EM) structures of human SPCA1 (hSPCA1) in a series of intermediate states, revealing a near-complete conformational cycle. With the aid of molecular dynamics simulations, these structures offer a clear structural basis for Ca2+ entry and release in hSPCA1. We found that hSPCA1 undergoes unique conformational changes during ATP binding and phosphorylation compared to other well-studied P-type II ATPases. In addition, we observed a conformational distortion of the Ca2+-binding site induced by the separation of transmembrane helices 4L and 6, unveiling a distinct Ca2+ release mechanism. Particularly, we determined a structure of the long-sought CaE2P state of P-type IIA ATPases, providing valuable insights into the Ca2+ transport cycle. Together, these findings enhance our understanding of Ca2+ transport by hSPCA1 and broaden our knowledge of P-type ATPases.


Assuntos
ATPases Transportadoras de Cálcio , Cálcio , Humanos , Cálcio/metabolismo , Microscopia Crioeletrônica , ATPases Transportadoras de Cálcio/metabolismo , Adenosina Trifosfatases/metabolismo
15.
Int J Mol Sci ; 24(8)2023 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-37108240

RESUMO

[Ca2+]-dependent crystallization of the Ca2+-ATPase molecules in sarcoplasmic reticulum (SR) vesicles isolated from scallop striated muscle elongated the vesicles in the absence of ATP, and ATP stabilized the crystals. Here, to determine the [Ca2+]-dependence of vesicle elongation in the presence of ATP, SR vesicles in various [Ca2+] environments were imaged using negative stain electron microscopy. The images obtained revealed the following phenomena. (i) Crystal-containing elongated vesicles appeared at ≤1.4 µM Ca2+ and almost disappeared at ≥18 µM Ca2+, where ATPase activity reaches its maximum. (ii) At ≥18 µM Ca2+, almost all SR vesicles were in the round form and covered by tightly clustered ATPase crystal patches. (iii) Round vesicles dried on electron microscopy grids occasionally had cracks, probably because surface tension crushed the solid three-dimensional spheres. (iv) [Ca2+]-dependent ATPase crystallization was rapid (<1 min) and reversible. These data prompt the hypothesis that SR vesicles autonomously elongate or contract with the help of a calcium-sensitive ATPase network/endoskeleton and that ATPase crystallization may modulate physical properties of the SR architecture, including the ryanodine receptors that control muscle contraction.


Assuntos
Pectinidae , Retículo Sarcoplasmático , Animais , Retículo Sarcoplasmático/metabolismo , Adenosina Trifosfatases , ATPases Transportadoras de Cálcio/metabolismo , Contração Muscular , Pectinidae/metabolismo , Trifosfato de Adenosina , Cálcio/metabolismo
16.
Eur J Hum Genet ; 31(6): 716-720, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36922631

RESUMO

A prenatal second-hit genetic change that occurs on the wild-type allele in an embryo with a congenital pathogenic variant allele results in mosaicism of monoallelic and biallelic defect of the gene, which is called superimposed mosaicism. Superimposed mosaicism of Hailey-Hailey disease (HHD) has been demonstrated in one familial case. Here, we report two unrelated HHD cases with superimposed mosaicism: a congenital monoallelic pathogenic variant of ATP2C1, followed by a postzygotic copy-neutral loss of heterozygosity. Uniquely, neither patient had a family history of HHD at the time of presentation. In the first case, the congenital pathogenic variant had occurred de novo. In the second case, the father had the pathogenic variant but had not yet developed skin symptoms. Our cases showed that superimposed mosaicism in HHD can lack a family history and that genetic analysis is crucial to classify the type of mosaicism and evaluate the risk of familial occurrence.


Assuntos
Pênfigo Familiar Benigno , Humanos , Pênfigo Familiar Benigno/diagnóstico , Pênfigo Familiar Benigno/genética , Pênfigo Familiar Benigno/patologia , Mosaicismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Alelos
17.
EMBO Rep ; 24(5): e56134, 2023 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-36929574

RESUMO

Multisubunit Tethering Complexes (MTCs) are a set of conserved protein complexes that tether vesicles at the acceptor membrane. Interactions with other components of the trafficking machinery regulate MTCs through mechanisms that are partially understood. Here, we systematically investigate the interactome that regulates MTCs. We report that P4-ATPases, a family of lipid flippases, interact with MTCs that participate in the anterograde and retrograde transport at the Golgi, such as TRAPPIII. We use the P4-ATPase Drs2 as a paradigm to investigate the mechanism and biological relevance of this interplay during transport of Atg9 vesicles. Binding of Trs85, the sole-specific subunit of TRAPPIII, to the N-terminal tail of Drs2 stabilizes TRAPPIII on membranes loaded with Atg9 and is required for Atg9 delivery during selective autophagy, a role that is independent of P4-ATPase canonical functions. This mechanism requires a conserved I(S/R)TTK motif that also mediates the interaction of the P4-ATPases Dnf1 and Dnf2 with MTCs, suggesting a broader role of P4-ATPases in MTC regulation.


Assuntos
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , ATPases Transportadoras de Cálcio/química , ATPases Transportadoras de Cálcio/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo
18.
Biochim Biophys Acta Biomembr ; 1865(5): 184143, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36863681

RESUMO

Ca2+-ATPases are membrane pumps that transport calcium ions across the cell membrane and are dependent on ATP. The mechanism of Listeria monocytogenes Ca2+-ATPase (LMCA1) in its native environment remains incompletely understood. LMCA1 has been investigated biochemically and biophysically with detergents in the past. This study characterizes LMCA1 using the detergent-free Native Cell Membrane Nanoparticles (NCMNP) system. As demonstrated by ATPase activity assays, the NCMNP7-25 polymer is compatible with a broad pH range and Ca2+ ions. This result suggests that NCMNP7-25 may have a wider array of applications in membrane protein research.


Assuntos
Adenosina Trifosfatases , ATPases Transportadoras de Cálcio , Adenosina Trifosfatases/metabolismo , ATPases Transportadoras de Cálcio/química , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Membranas/metabolismo
19.
Sci Adv ; 9(9): eadd9742, 2023 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-36867705

RESUMO

Secretory pathway Ca2+/Mn2+ ATPase 1 (SPCA1) actively transports cytosolic Ca2+ and Mn2+ into the Golgi lumen, playing a crucial role in cellular calcium and manganese homeostasis. Detrimental mutations of the ATP2C1 gene encoding SPCA1 cause Hailey-Hailey disease. Here, using nanobody/megabody technologies, we determined cryo-electron microscopy structures of human SPCA1a in the ATP and Ca2+/Mn2+-bound (E1-ATP) state and the metal-free phosphorylated (E2P) state at 3.1- to 3.3-Å resolutions. The structures revealed that Ca2+ and Mn2+ share the same metal ion-binding pocket with similar but notably different coordination geometries in the transmembrane domain, corresponding to the second Ca2+-binding site in sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). In the E1-ATP to E2P transition, SPCA1a undergoes similar domain rearrangements to those of SERCA. Meanwhile, SPCA1a shows larger conformational and positional flexibility of the second and sixth transmembrane helices, possibly explaining its wider metal ion specificity. These structural findings illuminate the unique mechanisms of SPCA1a-mediated Ca2+/Mn2+ transport.


Assuntos
Adenosina Trifosfatases , Complexo de Golgi , Humanos , Microscopia Crioeletrônica , Sítios de Ligação , Trifosfato de Adenosina , ATPases Transportadoras de Cálcio
20.
Exp Dermatol ; 32(6): 787-798, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36789506

RESUMO

Hailey-Hailey disease (HHD) is a rare autosomal dominantly inherited disorder caused by mutations in the ATP2C1 gene that encodes an adenosine triphosphate (ATP)-powered calcium channel pump. HHD is characterized by impaired epidermal cell-to-cell adhesion and defective keratinocyte growth/differentiation. The mechanism by which mutant ATP2C1 causes HHD is unknown and current treatments for affected individuals do not address the underlying defects and are ineffective. Notch signalling is a direct determinant of keratinocyte growth and differentiation. We found that loss of ATP2C1 leads to impaired Notch1 signalling, thus deregulation of the Notch signalling response is therefore likely to contribute to HHD manifestation. NOTCH1 is a transmembrane receptor and upon ligand binding, the intracellular domain (NICD) translocates to the nucleus activating its target genes. In the context of HHD, we found that loss of ATP2C1 function promotes upregulation of the active NOTCH1 protein (NICD-Val1744). Here, deeply exploring this aspect, we observed that NOTCH1 activation is not associated with the transcriptional enhancement of its targets. Moreover, in agreement with these results, we found a cytoplasmic localization of NICD-Val1744. We have also observed that ATP2C1-loss is associated with the degradation of NICD-Val1744 through the lysosomal/proteasome pathway. These results show that ATP2C1-loss could promote a mechanism by which NOTCH1 is endocytosed and degraded by the cell membrane. The deregulation of this phenomenon, finely regulated in physiological conditions, could in HHD lead to the deregulation of NOTCH1 with alteration of skin homeostasis and disease manifestation.


Assuntos
Pênfigo Familiar Benigno , Humanos , Pênfigo Familiar Benigno/genética , Pênfigo Familiar Benigno/metabolismo , Pele/metabolismo , Queratinócitos/metabolismo , Mutação , Epiderme/metabolismo , ATPases Transportadoras de Cálcio/genética , ATPases Transportadoras de Cálcio/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo
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