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1.
Nature ; 579(7798): 279-283, 2020 03.
Artículo en Inglés | MEDLINE | ID: mdl-32132708

RESUMEN

Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation-all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment-reversing hepatic steatosis and glucose intolerance-were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes.


Asunto(s)
Glucagón/farmacología , Gluconeogénesis/efectos de los fármacos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Hígado/efectos de los fármacos , Acetilcoenzima A/metabolismo , Tejido Adiposo/efectos de los fármacos , Animales , Diabetes Mellitus Tipo 2/fisiopatología , Activación Enzimática/efectos de los fármacos , Glucagón/sangre , Receptores de Inositol 1,4,5-Trifosfato/genética , Lipasa/metabolismo , Lipólisis/efectos de los fármacos , Lipólisis/genética , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/fisiopatología , Oxidación-Reducción/efectos de los fármacos
2.
Genes Dev ; 31(16): 1655-1665, 2017 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-28903979

RESUMEN

Starvation induces liver autophagy, which is thought to provide nutrients for use by other organs and thereby maintain whole-body homeostasis. Here we demonstrate that O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT) is required for glucagon-stimulated liver autophagy and metabolic adaptation to starvation. Genetic ablation of OGT in mouse livers reduces autophagic flux and the production of glucose and ketone bodies. Upon glucagon-induced calcium signaling, calcium/calmodulin-dependent kinase II (CaMKII) phosphorylates OGT, which in turn promotes O-GlcNAc modification and activation of Ulk proteins by potentiating AMPK-dependent phosphorylation. These findings uncover a signaling cascade by which starvation promotes autophagy through OGT phosphorylation and establish the importance of O-GlcNAc signaling in coupling liver autophagy to nutrient homeostasis.


Asunto(s)
Autofagia , Señalización del Calcio , Hígado/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Fenómenos Fisiológicos de la Nutrición , Adaptación Biológica , Animales , Proteína 5 Relacionada con la Autofagia/fisiología , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Células Cultivadas , Glucagón/farmacología , Células HEK293 , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Hígado/efectos de los fármacos , Hígado/enzimología , Ratones Endogámicos C57BL , N-Acetilglucosaminiltransferasas/fisiología
3.
Proc Natl Acad Sci U S A ; 117(29): 17389-17398, 2020 07 21.
Artículo en Inglés | MEDLINE | ID: mdl-32632005

RESUMEN

Wolfram syndrome is a rare multisystem disease characterized by childhood-onset diabetes mellitus and progressive neurodegeneration. Most cases are attributed to pathogenic variants in a single gene, Wolfram syndrome 1 (WFS1). There currently is no disease-modifying treatment for Wolfram syndrome, as the molecular consequences of the loss of WFS1 remain elusive. Because diabetes mellitus is the first diagnosed symptom of Wolfram syndrome, we aimed to further examine the functions of WFS1 in pancreatic ß cells in the context of hyperglycemia. Knockout (KO) of WFS1 in rat insulinoma (INS1) cells impaired calcium homeostasis and protein kinase B/Akt signaling and, subsequently, decreased cell viability and glucose-stimulated insulin secretion. Targeting calcium homeostasis with reexpression of WFS1, overexpression of WFS1's interacting partner neuronal calcium sensor-1 (NCS1), or treatment with calpain inhibitor and ibudilast reversed deficits observed in WFS1-KO cells. Collectively, our findings provide insight into the disease mechanism of Wolfram syndrome and highlight new targets and drug candidates to facilitate the development of a treatment for this disorder and similar diseases.


Asunto(s)
Antineoplásicos/farmacología , Proteínas de Unión a Calmodulina/metabolismo , Inhibidores de Cisteína Proteinasa/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Piridinas/farmacología , Síndrome de Wolfram/tratamiento farmacológico , Animales , Calcio/metabolismo , Proteínas de Unión a Calmodulina/genética , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Glicoproteínas , Homeostasis , Hiperglucemia , Secreción de Insulina , Proteínas de la Membrana/genética , Receptor de Insulina , Transcriptoma , Síndrome de Wolfram/genética
4.
FASEB J ; 35(10): e21873, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34499766

RESUMEN

Calcium (Ca2+ ) signaling is critical for neuronal functioning and requires the concerted interplay of numerous Ca2+ -binding proteins, including neuronal calcium sensor 1 (NCS1). Although an important role of NCS1 in neuronal processes and in neurodevelopmental and neurodegenerative diseases has been established, the underlying mechanisms remain enigmatic. Here, we systematically investigated the functions of NCS1 in the brain. Using Golgi-Cox staining, we observed a reduction in dendritic complexity and spine density in the prefrontal cortex and the dorsal hippocampus of Ncs1-/- mice, which may underlie concomitantly observed deficits in memory acquisition. Subsequent RNA sequencing of Ncs1-/- and Ncs1+/+ mouse brain tissues revealed that NCS1 modulates gene expression related to neuronal morphology and development. Investigation of developmental databases further supported a molecular role of NCS1 during brain development by identifying temporal gene expression patterns. Collectively, this study provides insights into NCS1-dependent signaling and lays the foundation for a better understanding of NCS1-associated diseases.


Asunto(s)
Calcio/metabolismo , Hipocampo/patología , Enfermedades del Sistema Nervioso/patología , Proteínas Sensoras del Calcio Neuronal/fisiología , Neuronas/patología , Neuropéptidos/fisiología , Animales , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/metabolismo , Neuronas/metabolismo , RNA-Seq , Transducción de Señal , Transcriptoma
5.
Mol Pharmacol ; 100(3): 258-270, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34321341

RESUMEN

Neuronal calcium sensor 1 (NCS1), a calcium-binding protein, and transient receptor potential V4 (TRPV4), a plasma membrane calcium channel, are fundamental in the regulation of calcium homeostasis. The interactions of these proteins and their regulation by paclitaxel (PTX) were investigated using biochemical, pharmacological, and electrophysiological approaches in both a breast cancer epithelial cell model and a neuronal model. TRPV4 and NCS1 reciprocally immunoprecipitated each other, suggesting that they make up a signaling complex. The functional consequence of this physical association was that TRPV4 currents increased with increased NCS1 expression. Calcium fluxes through TRPV4 correlated with the magnitude of TRPV4 currents, and these calcium fluxes depended on NCS1 expression levels. Exposure to PTX amplified the acute effects of TRPV4 expression, currents, and calcium fluxes but decreased the expression of NCS1. These findings augment the understanding of the properties of TRPV4, the role of NCS1 in the regulation of TRPV4, and the cellular mechanisms of PTX-induced neuropathy. SIGNIFICANCE STATEMENT: TRPV4 and NCS1 physically and functionally interact. Increased expression of NCS1 enhances TRPV4-dependent currents, which are further amplified by treatment with the chemotherapeutic drug paclitaxel, an effect associated with adverse effects of chemotherapy, including neuropathy.


Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Paclitaxel/farmacología , Canales Catiónicos TRPV/metabolismo , Antineoplásicos Fitogénicos/efectos adversos , Calcio/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fenómenos Electrofisiológicos , Humanos , Proteínas Sensoras del Calcio Neuronal/genética , Neuropéptidos/genética , Paclitaxel/efectos adversos , Transducción de Señal/efectos de los fármacos , Canales Catiónicos TRPV/genética
6.
J Biol Chem ; 294(49): 18923-18933, 2019 12 06.
Artículo en Inglés | MEDLINE | ID: mdl-31659121

RESUMEN

Inositol 1,4,5-trisphosphate receptors (InsP3Rs) are endoplasmic reticulum-localized channels that mediate Ca2+ release from the endoplasmic reticulum into the cytoplasm. We previously reported that an EF-hand Ca2+-binding protein, neuronal calcium sensor 1 (NCS1), binds to the InsP3R and thereby increases channel open probability, an event associated with chemotherapy-induced peripheral neuropathy. However, the exact NCS1-binding site on InsP3R remains unknown. Using protein docking, co-immunoprecipitation, and blocking peptides, we mapped the NCS1-binding site to residues 66-110 on the suppressor domain of InsP3R type 1 (InsP3R1). We also identified Leu-89, a residue in the hydrophobic pocket of NCS1, as being critical for facilitating the NCS1-InsP3R1 interaction. Overexpression of WT NCS1 in MDA-MB231 breast cancer cells increased Ca2+ signaling and survival, whereas overexpression of Leu-89 NCS1 variants decreased Ca2+ signaling and survival, further suggesting the importance of this residue in the NCS1-InsP3R1 interaction. In conclusion, we show that NCS1-InsP3R1 interaction enhances intracellular Ca2+ signaling in cells and can be modulated by altering or occluding the hydrophobic pocket of NCS1. This improved understanding of the NCS1-InsP3R1 interaction may facilitate the development of management strategies for diseases resulting from aberrant NCS1 expression.


Asunto(s)
Neoplasias de la Mama/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Sitios de Unión , Femenino , Humanos , Unión Proteica
7.
Am J Physiol Heart Circ Physiol ; 318(6): H1516-H1524, 2020 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-32383992

RESUMEN

Engineered heart tissues (EHTs) have emerged as a robust in vitro model to study cardiac physiology. Although biomimetic culture environments have been developed to better approximate in vivo conditions, currently available methods do not permit full recapitulation of the four phases of the cardiac cycle. We have developed a bioreactor which allows EHTs to undergo cyclic loading sequences that mimic in vivo work loops. EHTs cultured under these working conditions exhibited enhanced concentric contractions but similar isometric contractions compared with EHTs cultured isometrically. EHTs that were allowed to shorten cyclically in culture had increased capacity for contractile work when tested acutely. Increased work production was correlated with higher levels of mitochondrial proteins and mitochondrial biogenesis; this effect was eliminated when tissues were cyclically shortened in the presence of a myosin ATPase inhibitor. Leveraging our novel in vitro method to precisely apply mechanical loads in culture, we grew EHTs under two loading regimes prescribing the same work output but with different associated afterloads. These groups showed no difference in mitochondrial protein expression. In loading regimes with the same afterload but different work output, tissues subjected to higher work demand exhibited elevated levels of mitochondrial protein. Our findings suggest that regulation of mitochondrial mass in cultured human EHTs is potently modulated by the mechanical work the tissue is permitted to perform in culture, presumably communicated through ATP demand. Precise application of mechanical loads to engineered heart tissues in culture represents a novel in vitro method for studying physiological and pathological cardiac adaptation.NEW & NOTEWORTHY In this work, we present a novel bioreactor that allows for active length control of engineered heart tissues during extended tissue culture. Specific length transients were designed so that engineered heart tissues generated complete cardiac work loops. Chronic culture with various work loops suggests that mitochondrial mass and biogenesis are directly regulated by work output.


Asunto(s)
Mitocondrias Cardíacas/metabolismo , Proteínas Mitocondriales/metabolismo , Contracción Miocárdica/fisiología , Miocardio/metabolismo , Humanos , Ingeniería de Tejidos
8.
FASEB J ; 33(4): 4802-4813, 2019 04.
Artículo en Inglés | MEDLINE | ID: mdl-30592625

RESUMEN

Increased levels of the calcium-binding protein neuronal calcium sensor 1 (NCS1) predict an unfavorable patient outcome in several aggressive cancers, including breast and liver tumors. Previous studies suggest that NCS1 overexpression facilitates metastatic spread of these cancers. To investigate this hypothesis, we explored the effects of NCS1 overexpression on cell proliferation, survival, and migration patterns in vitro in 2- and 3-dimensional (2/3-D). Furthermore, we translated our results into an in vivo mouse xenograft model. Cell-based proliferation assays were used to demonstrate the effects of overexpression of NCS1 on growth rates. In vitro colony formation and wound healing experiments were performed and 3-D migration dynamics were studied using collagen gels. Nude mice were injected with breast cancer cells to monitor NCS1-dependent metastasis formation over time. We observed that increased NCS1 levels do not change cellular growth rates, but do significantly increase 2- and 3-D migration dynamics in vitro. Likewise, NCS1-overexpressing cells have an increased capacity to form distant metastases and demonstrate better survival and less necrosis in vivo. We found that NCS1 preferentially localizes to the leading edge of cells and overexpression increases the motility of cancer cells. Furthermore, this phenotype is correlated with an increased number of metastases in a xenograft model. These results lay the foundation for exploring the relevance of an NCS1-mediated pathway as a metastatic biomarker and as a target for pharmacologic interventions.-Apasu, J. E., Schuette, D., LaRanger, R., Steinle, J. A., Nguyen, L. D., Grosshans, H. K., Zhang, M., Cai, W. L., Yan, Q., Robert, M. E., Mak, M., Ehrlich, B. E. Neuronal calcium sensor 1 (NCS1) promotes motility and metastatic spread of breast cancer cells in vitro and in vivo.


Asunto(s)
Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Proteínas Sensoras del Calcio Neuronal/metabolismo , Neuropéptidos/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Proliferación Celular/fisiología , Femenino , Humanos , Ratones , Ratones Desnudos , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Ensayos Antitumor por Modelo de Xenoinjerto
9.
Proc Natl Acad Sci U S A ; 119(43): e2214826119, 2022 10 25.
Artículo en Inglés | MEDLINE | ID: mdl-36215521
10.
Support Care Cancer ; 31(8): 486, 2023 07 22.
Artículo en Inglés | MEDLINE | ID: mdl-37480366

Asunto(s)
Litio , Humanos
11.
Pflugers Arch ; 469(11): 1507-1517, 2017 11.
Artículo en Inglés | MEDLINE | ID: mdl-28762163

RESUMEN

Although autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple kidney cysts, the most frequent cause of death in ADPKD patients is cardiovascular disease. ADPKD is linked to mutations in PKD1 or pkd2, the genes that encode for the proteins polycystin 1 and polycystin 2 (PC1 and PC2, respectively). The cardiovascular complications have been assumed to be a consequence of renal hypertension and activation of renin/angiotensin/aldosterone (RAAS) pathway. However, the expression of PC1 and PC2 in cardiac tissue suggests additional direct effects of these proteins on cardiac function. We previously reported that zebrafish lacking PC2 develop heart failure, and that heterozygous Pkd2+/- mice are hypersensitive to acute ß-adrenergic receptor (ßAR) stimulation. Here, we investigate the effect of cardiac stress (prolonged continuous ßAR stimulus) on Pkd2+/- mice. After ßAR stimulation for 7 days, wild-type (WT) mice had increased left ventricular mass and natriuretic peptide (ANP and BNP) mRNA levels. The WT mice also had upregulated levels of PC2 and chromogranin B (CGB, an upstream regulator of BNP). Conversely, Pkd2+/- mice had increased left ventricular mass, but natriuretic peptide and CGB expression levels remained constant. Reversal of the increased cardiac mass was observed in WT mice 3 days after cessation of the ßAR stimulation, but not in Pkd2+/- mice. We suggest that cardiac stress leads to upregulation of the PC2-CGB-BNP signaling axis, and this pathway regulates the production of cardio-protective natriuretic peptides. The lack of a PC2-dependent cardio-protective function may contribute to the severity of cardiac dysfunction in Pkd2+/- mice and in ADPKD patients.


Asunto(s)
Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Corazón/fisiología , Proproteína Convertasa 2/metabolismo , Animales , Heterocigoto , Ratones , Ratones Endogámicos C57BL , Riñón Poliquístico Autosómico Dominante/metabolismo , Riñón Poliquístico Autosómico Dominante/fisiopatología , ARN Mensajero/metabolismo , Transducción de Señal/fisiología , Canales Catiónicos TRPP/metabolismo , Regulación hacia Arriba/fisiología
12.
Biochem J ; 473(3): 285-96, 2016 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-26574436

RESUMEN

PC2 (polycystin-2) forms a Ca(2+)-permeable channel in the cell membrane and its function is regulated by cytosolic Ca(2+) levels. Mutations in the C-terminal tail of human PC2 (HPC2 Cterm) lead to autosomal dominant polycystic kidney disease. The HPC2 Cterm protein contains a Ca(2+)-binding site responsible for channel gating and function. To provide the foundation for understanding how Ca(2+) regulates the channel through the HPC2 Cterm, we characterized Ca(2+) binding and its conformational and dynamic responses within the HPC2 Cterm. By examining hydrogen-deuterium (H-D) exchange profiles, we show that part of the coiled-coil domain in the HPC2 Cterm forms a stable helix bundle regardless of the presence of Ca(2+). The HPC2 L1EF construct contains the Ca(2+)-binding EF-hand and the N-terminal linker 1 region without the downstream coiled coil. We show that the linker stabilizes the Ca(2+)-bound conformation of the EF-hand, thus enhancing its Ca(2+)-binding affinity to the same level as the HPC2 Cterm. In comparison, the coiled coil is not required for the high-affinity binding. By comparing the conformational dynamics of the HPC2 Cterm and HPC2 L1EF with saturating Ca(2+), we show that the HPC2 Cterm and HPC2 L1EF share a similar increase in structural stability upon Ca(2+) binding. Nevertheless, they have different profiles of H-D exchange under non-saturating Ca(2+) conditions, implying their different conformational exchange between the Ca(2+)-bound and -unbound states. The present study, for the first time, provides a complete map of dynamic responses to Ca(2+)-binding within the full-length HPC2 Cterm. Our results suggest mechanisms for functional regulation of the PC2 channel and PC2's roles in the pathophysiology of polycystic kidney disease.


Asunto(s)
Calcio/metabolismo , Canales Catiónicos TRPP/química , Canales Catiónicos TRPP/metabolismo , Secuencia de Aminoácidos , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , Canales Catiónicos TRPP/genética
13.
Proc Natl Acad Sci U S A ; 111(46): 16604-9, 2014 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-25368166

RESUMEN

Cardiac disorders are the main cause of mortality in autosomal-dominant polycystic kidney disease (ADPKD). However, how mutated polycystins predispose patients with ADPKD to cardiac pathologies before development of renal dysfunction is unknown. We investigate the effect of decreased levels of polycystin 2 (PC2), a calcium channel that interacts with the ryanodine receptor, on myocardial function. We hypothesize that heterozygous PC2 mice (Pkd2(+/-)) undergo cardiac remodeling as a result of changes in calcium handling, separate from renal complications. We found that Pkd2(+/-) cardiomyocytes have altered calcium handling, independent of desensitized calcium-contraction coupling. Paradoxically, in Pkd2(+/-) mice, protein kinase A (PKA) phosphorylation of phospholamban (PLB) was decreased, whereas PKA phosphorylation of troponin I was increased, explaining the decoupling between calcium signaling and contractility. In silico modeling supported this relationship. Echocardiography measurements showed that Pkd2(+/-) mice have increased left ventricular ejection fraction after stimulation with isoproterenol (ISO), a ß-adrenergic receptor (ßAR) agonist. Blockers of ßAR-1 and ßAR-2 inhibited the ISO response in Pkd2(+/-) mice, suggesting that the dephosphorylated state of PLB is primarily by ßAR-2 signaling. Importantly, the Pkd2(+/-) mice were normotensive and had no evidence of renal cysts. Our results showed that decreased PC2 levels shifted the ßAR pathway balance and changed expression of calcium handling proteins, which resulted in altered cardiac contractility. We propose that PC2 levels in the heart may directly contribute to cardiac remodeling in patients with ADPKD in the absence of renal dysfunction.


Asunto(s)
Señalización del Calcio/fisiología , Acoplamiento Excitación-Contracción/fisiología , Miocitos Cardíacos/fisiología , Receptores Adrenérgicos beta 1/fisiología , Receptores Adrenérgicos beta 2/fisiología , Canales Catiónicos TRPP/deficiencia , Agonistas Adrenérgicos beta/farmacología , Antagonistas Adrenérgicos beta/farmacología , Animales , Presión Sanguínea/genética , Cafeína/farmacología , Señalización del Calcio/efectos de los fármacos , Proteínas de Unión al Calcio/metabolismo , Estimulación Cardíaca Artificial , Acoplamiento Excitación-Contracción/efectos de los fármacos , Acoplamiento Excitación-Contracción/genética , Heterocigoto , Isoproterenol/farmacología , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/biosíntesis , Receptores Adrenérgicos beta 1/efectos de los fármacos , Receptores Adrenérgicos beta 2/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Transducción de Señal/fisiología , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/fisiología , Troponina I/metabolismo , Remodelación Ventricular/efectos de los fármacos , Remodelación Ventricular/genética , Remodelación Ventricular/fisiología
14.
Proc Natl Acad Sci U S A ; 111(39): 14283-8, 2014 Sep 30.
Artículo en Inglés | MEDLINE | ID: mdl-25228769

RESUMEN

Mutations in polycystin 1 and 2 (PC1 and PC2) cause the common genetic kidney disorder autosomal dominant polycystic kidney disease (ADPKD). It is unknown how these mutations result in renal cysts, but dysregulation of calcium (Ca(2+)) signaling is a known consequence of PC2 mutations. PC2 functions as a Ca(2+)-activated Ca(2+) channel of the endoplasmic reticulum. We hypothesize that Ca(2+) signaling through PC2, or other intracellular Ca(2+) channels such as the inositol 1,4,5-trisphosphate receptor (InsP3R), is necessary to maintain renal epithelial cell function and that disruption of the Ca(2+) signaling leads to renal cyst development. The cell line LLC-PK1 has traditionally been used for studying PKD-causing mutations and Ca(2+) signaling in 2D culture systems. We demonstrate that this cell line can be used in long-term (8 wk) 3D tissue culture systems. In 2D systems, knockdown of InsP3R results in decreased Ca(2+) transient signals that are rescued by overexpression of PC2. In 3D systems, knockdown of either PC2 or InsP3R leads to cyst formation, but knockdown of InsP3R type 1 (InsP3R1) generated the largest cysts. InsP3R1 and InsP3R3 are differentially localized in both mouse and human kidney, suggesting that regional disruption of Ca(2+) signaling contributes to cystogenesis. All cysts had intact cilia 2 wk after starting 3D culture, but the cells with InsP3R1 knockdown lost cilia as the cysts grew. Studies combining 2D and 3D cell culture systems will assist in understanding how mutations in PC2 that confer altered Ca(2+) signaling lead to ADPKD cysts.


Asunto(s)
Señalización del Calcio , Riñón Poliquístico Autosómico Dominante/metabolismo , Animales , Técnicas de Cultivo de Célula , Cilios/metabolismo , Cilios/patología , Medios de Cultivo , Técnicas de Silenciamiento del Gen , Humanos , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inhibidores , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Riñón/metabolismo , Riñón/patología , Células LLC-PK1 , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/patología , Porcinos , Canales Catiónicos TRPP/deficiencia , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo
15.
J Physiol ; 594(15): 4141-9, 2016 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-26857659

RESUMEN

Mutations in polycystin-2 (PC2) lead to autosomal dominant polycystic kidney disease (ADPKD). The molecular mechanism linking mutations in PC2 and the pathogenesis of ADPKD is not well understood. Therefore, understanding the functional regulation of PC2 and its interaction with other proteins under both physiological and pathogenic conditions is important for elucidating the disease mechanism and identifying potential molecular targets for treatment. Normally, PC2 functions as a calcium-permeable channel whose activity is regulated by calcium binding to the C-terminal domain of PC2 (PC2 Cterm). The PC2 Cterm is also involved in the PC2 channel assembly and hetero-oligomerization with other binding partners in cells. Different functional domains of the PC2 Cterm have been studied using structural approaches. Within the PC2 Cterm, there is a calcium-binding EF-hand domain, crucial for the calcium-dependent activity of the PC2 channel. Downstream of the EF-hand domain lies a coiled-coil region, which is involved in the assembly and hetero-interaction of the PC2 protein. The PC2 Cterm can form an oligomer, mediated by the coiled-coil region. Although PC2 Cterm has been extensively studied for its relationship with ADPKD and its importance in PC2 regulation, there are misunderstandings with respect to the definition of the domain topology within the PC2 Cterm and the functional role of each domain. Here, we review previous studies that connect the molecular properties of the domains of PC2 Cterm to distinct aspects of PC2 functions and regulation.


Asunto(s)
Canales Catiónicos TRPP/química , Canales Catiónicos TRPP/metabolismo , Calcio/metabolismo , Humanos , Conformación Proteica
16.
J Biol Chem ; 290(10): 6419-27, 2015 Mar 06.
Artículo en Inglés | MEDLINE | ID: mdl-25609250

RESUMEN

The GxGD proteases function to cleave protein substrates within the membrane. As these proteases contain multiple transmembrane domains typical of ion channels, we examined if GxGD proteases also function as ion channels. We tested the putative dual function by examining two archeobacterial GxGD proteases (PSH and FlaK), with known three-dimensional structures. Both are in the same GxGD family as presenilin, a protein mutated in Alzheimer Disease. Here, we demonstrate that PSH and FlaK form cation channels in lipid bilayers. A mutation that affected the enzymatic activity of FlaK rendered the channel catalytically inactive and altered the ion selectivity, indicating that the ion channel and the catalytic activities are linked. We report that the GxGD proteases, PSH and FlaK, are true "chanzymes" with interdependent ion channel and protease activity conferred by a single structural domain embedded in the membrane, supporting the proposal that higher-order proteases, including presenilin, have channel function.


Asunto(s)
Canales de Calcio/química , Proteínas de la Membrana/química , Péptido Hidrolasas/química , Presenilinas/química , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Animales , Archaea/enzimología , Canales de Calcio/genética , Dominio Catalítico , Cristalografía por Rayos X , Humanos , Membrana Dobles de Lípidos/química , Proteínas de la Membrana/metabolismo , Ratones , Péptido Hidrolasas/genética , Péptido Hidrolasas/metabolismo , Presenilinas/metabolismo , Estructura Terciaria de Proteína
17.
J Biol Chem ; 290(16): 10544-54, 2015 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-25716316

RESUMEN

Polycystin-2 (PC2) belongs to the transient receptor potential (TRP) family and forms a Ca(2+)-regulated channel. The C-terminal cytoplasmic tail of human PC2 (HPC2 Cterm) is important for PC2 channel assembly and regulation. In this study, we characterized the oligomeric states and Ca(2+)-binding profiles in the C-terminal tail using biophysical approaches. Specifically, we determined that HPC2 Cterm forms a trimer in solution with and without Ca(2+) bound, although TRP channels are believed to be tetramers. We found that there is only one Ca(2+)-binding site in the HPC2 Cterm, located within its EF-hand domain. However, the Ca(2+) binding affinity of the HPC2 Cterm trimer is greatly enhanced relative to the intrinsic binding affinity of the isolated EF-hand domain. We also employed the sea urchin PC2 (SUPC2) as a model for biophysical and structural characterization. The sea urchin C-terminal construct (SUPC2 Ccore) also forms trimers in solution, independent of Ca(2+) binding. In contrast to the human PC2, the SUPC2 Ccore contains two cooperative Ca(2+)-binding sites within its EF-hand domain. Consequently, trimerization does not further improve the affinity of Ca(2+) binding in the SUPC2 Ccore relative to the isolated EF-hand domain. Using NMR, we localized the Ca(2+)-binding sites in the SUPC2 Ccore and characterized the conformational changes in its EF-hand domain due to trimer formation. Our study provides a structural basis for understanding the Ca(2+)-dependent regulation of the PC2 channel by its cytosolic C-terminal domain. The improved methodology also serves as a good strategy to characterize other Ca(2+)-binding proteins.


Asunto(s)
Calcio/metabolismo , Erizos de Mar/metabolismo , Canales Catiónicos TRPP/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Humanos , Activación del Canal Iónico , Transporte Iónico , Modelos Moleculares , Datos de Secuencia Molecular , Agregado de Proteínas , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Erizos de Mar/química , Alineación de Secuencia , Especificidad de la Especie , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo , Termodinámica
18.
FASEB J ; 28(5): 2332-46, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24558196

RESUMEN

Polycystin 2 (PC2) is a calcium-dependent calcium channel, and mutations to human PC2 (hPC2) are associated with polycystic kidney disease. The C-terminal tail of hPC2 contains 2 EF hand motifs, but only the second binds calcium. Here, we investigate whether these EF hand motifs serve as a calcium sensor responsible for the calcium dependence of PC2 function. Using NMR and bioinformatics, we show that the overall fold is highly conserved, but in evolutionarily earlier species, both EF hands bind calcium. To test whether the EF hand motif is truly a calcium sensor controlling PC2 channel function, we altered the number of calcium binding sites in hPC2. NMR studies confirmed that modified hPC2 binds an additional calcium ion. Single-channel recordings demonstrated a leftward shift in the calcium dependence, and imaging studies in cells showed that calcium transients were enhanced compared with wild-type hPC2. However, biophysics and functional studies showed that the first EF hand can only bind calcium and be functionally active if the second (native) calcium-binding EF hand is intact. These results suggest that the number and location of calcium-binding sites in the EF hand senses the concentration of calcium required for PC2 channel activity and cellular function.


Asunto(s)
Motivos EF Hand , Canales Catiónicos TRPP/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Calcio/metabolismo , Canales de Calcio/metabolismo , Línea Celular , Biología Computacional , Humanos , Riñón/metabolismo , Membrana Dobles de Lípidos/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Filogenia , Plásmidos/metabolismo , Unión Proteica , Erizos de Mar , Homología de Secuencia de Aminoácido , Transducción de Señal , Porcinos
19.
Am J Physiol Endocrinol Metab ; 307(11): E1057-64, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25315698

RESUMEN

Inositol 1,4,5-trisphosphate receptor type II (InsP3R-II) is the most prevalent isoform of the InsP3R in hepatocytes and is concentrated under the canalicular membrane, where it plays an important role in bile secretion. We hypothesized that altered calcium (Ca(2+)) signaling may be involved in metabolic dysfunction, as InsP3R-mediated Ca(2+) signals have been implicated in the regulation of hepatic glucose homeostasis. Here, we find that InsP3R-II, but not InsP3R-I, is reduced in the livers of obese mice. In our investigation of the functional consequences of InsP3R-II deficiency, we found that organic anion secretion at the canalicular membrane and Ca(2+) signals were impaired. However, mice lacking InsP3R-II showed no deficits in energy balance, glucose production, glucose tolerance, or susceptibility to hepatic steatosis. Thus, our results suggest that reduced InsP3R-II expression is not sufficient to account for any disruptions in metabolic homeostasis that are observed in mouse models of obesity. We conclude that metabolic homeostasis is maintained independently of InsP3R-II. Loss of InsP3R-II does impair secretion of bile components; therefore, we suggest that conditions of obesity would lead to a decrease in this Ca(2+)-sensitive process.


Asunto(s)
Homeostasis/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones Obesos/metabolismo , Animales , Bilis/metabolismo , Composición Corporal/fisiología , Señalización del Calcio/genética , Colesterol/metabolismo , Dieta Alta en Grasa , Hígado Graso/genética , Hígado Graso/metabolismo , Prueba de Tolerancia a la Glucosa , Hepatocitos/fisiología , Masculino , Ratones , Ratones Noqueados
20.
Mol Cell Neurosci ; 56: 102-14, 2013 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-23624073

RESUMEN

Chromogranin B (CGB) is a high capacity, low affinity calcium binding protein in the endoplasmic reticulum (ER) that binds to the inositol 1,4,5 trisphosphate receptor (InsP3R) and amplifies calcium release from ER stores. Recently, it was discovered that levels of CGB-derived peptides are decreased in the cerebrospinal fluid of multiple sclerosis (MS) patients. One of the mechanisms by which neurodegeneration in MS is thought to occur is through increased levels of intra-axonal calcium. The combination of excess intracellular calcium and dysregulated levels of CGB in MS led us to hypothesize that CGB may be involved in MS pathophysiology. Here, we show in a mouse model of MS that CGB levels are elevated in neurons prior to onset of symptoms. Once symptoms develop, CGB protein levels increase with disease severity. Additionally, we show that elevated levels of CGB may have a role in the pathophysiology of MS and suggest that the initial elevation of CGB, prior to symptom onset, is due to inflammatory processes. Upon development of symptoms, CGB accumulation in neurons results from decreased ubiquitination and decreased secretion. Furthermore, we show that calpain activity is increased and levels of InsP3R are decreased. From these results, we suggest that the elevated levels of CGB and altered InsP3R levels may contribute to the axonal/neuronal damage and dysregulated calcium homeostasis observed in MS. Additionally, we propose that CGB can be a biomarker that predicts the onset and severity of disease in patients with MS.


Asunto(s)
Cromogranina B/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Esclerosis Múltiple/metabolismo , Animales , Calpaína/genética , Calpaína/metabolismo , Cromogranina B/genética , Exocitosis , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/inducido químicamente , Neuronas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Ubiquitinación
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