Hepatitis B virus X(HBx)protein plays a pivotal role in the development of hepatitis B virus(HBV)-associated hepatocellular carcinoma.Although regulation of cytosolic calcium is essential for HBV replication and is me...Hepatitis B virus X(HBx)protein plays a pivotal role in the development of hepatitis B virus(HBV)-associated hepatocellular carcinoma.Although regulation of cytosolic calcium is essential for HBV replication and is mediated by HBx protein,the mechanism of HBx protein regulating intracellular calcium level remains poorly understood.The present study examined whether HBx protein elevated the intracellular calcium through interacting with storeoperated calcium entry(SOCE)components,Orai1 and stromal interaction molecule 1,and then identified the targets of HBx protein,with an attempt to understand the mechanism of HBx protein upsetting intracellular calcium homeostasis.By employing co-immunoprecipitation and GST-pull-down assay,we found that Orai1 protein interacted with HBx protein,and the C-terminus of Orai1 was implicated in the interaction.Confocal microscopy also revealed that HBx protein could co-localize with full-length Orai1 protein in HEK293 cells.Moreover,live cell calcium imaging exhibited that HBx protein elevated intracellular calcium,possibly by binding to SOCE components.Our results suggest that HBx protein binds to STIM1-Orai1 complexes to positively regulate the activity of plasma membrane store-operated calcium channels.展开更多
Autosomal dominant polycystic kidney disease （ADPKD） is the most common inherited monogenic kidney disease. Characterized by the development and growth of cysts that cause progressive kidney enlargement, it ultimate...Autosomal dominant polycystic kidney disease （ADPKD） is the most common inherited monogenic kidney disease. Characterized by the development and growth of cysts that cause progressive kidney enlargement, it ultimately leads to end-stage renal disease. Approximately 85% of ADPKD cases are caused by mutations in the PKD1 gene, while mutations in the PKD2 gene account for the remaining 15% of cases. The PKD1 gene encodes for polycystin-1 （PC1）, a large multi-functional memb-rane receptor protein able to regulate ion channel complexes, whereas polycystin-2 （PC2）, encoded by the PKD2 gene, is an integral membrane protein that functions as a calcium-permeable cation channel, located mainly in the endoplasmic reticulum （ER）. In the primary cilia of the epithelial cells, PC1 interacts with PC2 to form a polycystin complex that acts as a mechanosensor, regulating signaling pathways involved in the differentiation of kidney tubular epithelial cells. Despite progress in understanding the function of these proteins, the molecular mechanisms associated with the pathogenesis of ADPKD remain unclear. In this review we discuss how an imbalance between functional PC1 and PC2 proteins may disrupt calcium channel activities in the cilium, plasma membrane and ER, thereby altering intracellular calcium signaling and leading to the aberrant cell proliferation and apoptosis associated with the development and growth of renal cysts. Research in this feld could lead to the discovery of new molecules able to rebalance intracellular calcium, thereby normalizing cell proliferation and reducing kidney cyst progression.展开更多
The formation of axonal spheroid is a common feature following spinal cord injury.To further understand the source of Ca^(2+)that mediates axonal spheroid formation,we used our previously characterized ex vivo mouse s...The formation of axonal spheroid is a common feature following spinal cord injury.To further understand the source of Ca^(2+)that mediates axonal spheroid formation,we used our previously characterized ex vivo mouse spinal cord model that allows precise perturbation of extracellular Ca^(2+).We performed twophoton excitation imaging of spinal cords isolated from Thy1YFP+transgenic mice and applied the lipophilic dye,Nile red,to record dynamic changes in dorsal column axons and their myelin sheaths respectively.We selectively released Ca^(2+)from internal stores using the Ca^(2+)ionophore ionomycin in the presence or absence of external Ca^(2+).We reported that ionomycin dose-dependently induces pathological changes in myelin and pronounced axonal spheroid formation in the presence of normal 2 m M Ca^(2+)artificial cerebrospinal fluid.In contrast,removal of external Ca^(2+)significantly decreased ionomycin-induced myelin and axonal spheroid formation at 2 hours but not at 1 hour after treatment.Using mice that express a neuron-specific Ca^(2+)indicator in spinal cord axons,we confirmed that ionomycin induced significant increases in intra-axonal Ca^(2+),but not in the absence of external Ca^(2+).Periaxonal swelling and the resultant disruption in the axo-myelinic interface often precedes and is negatively correlated with axonal spheroid formation.Pretreatment with YM58483(500 n M),a well-established blocker of store-operated Ca^(2+)entry,significantly decreased myelin injury and axonal spheroid formation.Collectively,these data reveal that ionomycin-induced depletion of internal Ca^(2+)stores and subsequent external Ca^(2+)entry through store-operated Ca^(2+)entry contributes to pathological changes in myelin and axonal spheroid formation,providing new targets to protect central myelinated fibers.展开更多
基金supported by grants from the National Natural Science Foundation of China(No.81001063)the Fundamental Research Funds for the Central Universities(No.2015QN150)
文摘Hepatitis B virus X(HBx)protein plays a pivotal role in the development of hepatitis B virus(HBV)-associated hepatocellular carcinoma.Although regulation of cytosolic calcium is essential for HBV replication and is mediated by HBx protein,the mechanism of HBx protein regulating intracellular calcium level remains poorly understood.The present study examined whether HBx protein elevated the intracellular calcium through interacting with storeoperated calcium entry(SOCE)components,Orai1 and stromal interaction molecule 1,and then identified the targets of HBx protein,with an attempt to understand the mechanism of HBx protein upsetting intracellular calcium homeostasis.By employing co-immunoprecipitation and GST-pull-down assay,we found that Orai1 protein interacted with HBx protein,and the C-terminus of Orai1 was implicated in the interaction.Confocal microscopy also revealed that HBx protein could co-localize with full-length Orai1 protein in HEK293 cells.Moreover,live cell calcium imaging exhibited that HBx protein elevated intracellular calcium,possibly by binding to SOCE components.Our results suggest that HBx protein binds to STIM1-Orai1 complexes to positively regulate the activity of plasma membrane store-operated calcium channels.
基金Supported by University of Ferrara local funds:FAR 2012,2013,2014 and Regione Emilia Romagna grant(Ricerca Regione-Università)2007-2009
文摘Autosomal dominant polycystic kidney disease （ADPKD） is the most common inherited monogenic kidney disease. Characterized by the development and growth of cysts that cause progressive kidney enlargement, it ultimately leads to end-stage renal disease. Approximately 85% of ADPKD cases are caused by mutations in the PKD1 gene, while mutations in the PKD2 gene account for the remaining 15% of cases. The PKD1 gene encodes for polycystin-1 （PC1）, a large multi-functional memb-rane receptor protein able to regulate ion channel complexes, whereas polycystin-2 （PC2）, encoded by the PKD2 gene, is an integral membrane protein that functions as a calcium-permeable cation channel, located mainly in the endoplasmic reticulum （ER）. In the primary cilia of the epithelial cells, PC1 interacts with PC2 to form a polycystin complex that acts as a mechanosensor, regulating signaling pathways involved in the differentiation of kidney tubular epithelial cells. Despite progress in understanding the function of these proteins, the molecular mechanisms associated with the pathogenesis of ADPKD remain unclear. In this review we discuss how an imbalance between functional PC1 and PC2 proteins may disrupt calcium channel activities in the cilium, plasma membrane and ER, thereby altering intracellular calcium signaling and leading to the aberrant cell proliferation and apoptosis associated with the development and growth of renal cysts. Research in this feld could lead to the discovery of new molecules able to rebalance intracellular calcium, thereby normalizing cell proliferation and reducing kidney cyst progression.
文摘The formation of axonal spheroid is a common feature following spinal cord injury.To further understand the source of Ca^(2+)that mediates axonal spheroid formation,we used our previously characterized ex vivo mouse spinal cord model that allows precise perturbation of extracellular Ca^(2+).We performed twophoton excitation imaging of spinal cords isolated from Thy1YFP+transgenic mice and applied the lipophilic dye,Nile red,to record dynamic changes in dorsal column axons and their myelin sheaths respectively.We selectively released Ca^(2+)from internal stores using the Ca^(2+)ionophore ionomycin in the presence or absence of external Ca^(2+).We reported that ionomycin dose-dependently induces pathological changes in myelin and pronounced axonal spheroid formation in the presence of normal 2 m M Ca^(2+)artificial cerebrospinal fluid.In contrast,removal of external Ca^(2+)significantly decreased ionomycin-induced myelin and axonal spheroid formation at 2 hours but not at 1 hour after treatment.Using mice that express a neuron-specific Ca^(2+)indicator in spinal cord axons,we confirmed that ionomycin induced significant increases in intra-axonal Ca^(2+),but not in the absence of external Ca^(2+).Periaxonal swelling and the resultant disruption in the axo-myelinic interface often precedes and is negatively correlated with axonal spheroid formation.Pretreatment with YM58483(500 n M),a well-established blocker of store-operated Ca^(2+)entry,significantly decreased myelin injury and axonal spheroid formation.Collectively,these data reveal that ionomycin-induced depletion of internal Ca^(2+)stores and subsequent external Ca^(2+)entry through store-operated Ca^(2+)entry contributes to pathological changes in myelin and axonal spheroid formation,providing new targets to protect central myelinated fibers.
