-Induced Decrease in Membrane Expression of Na,K-ATPase Leads to Gastric Injury.

is a highly prevalent human gastric pathogen that causes gastritis, ulcer disease, and gastric cancer. It is not yet fully understood how injures the gastric epithelium. The Na,K-ATPase, an essential transporter found in virtually all mammalian cells, has been shown to be important for maintaining the barrier function of lung and kidney epithelia.

Contribution of septins to human platelet structure and function.

Although septins have been well-studied in nucleated cells, their role in anucleate blood platelets remains obscure. Here, we elucidate the contribution of septins to human platelet structure and functionality. We show that Septin-2 and Septin-9 are predominantly distributed at the periphery of resting platelets and co-localize strongly with microtubules.

Maturation of the Na,K-ATPase in the Endoplasmic Reticulum in Health and Disease.

The Na,K-ATPase establishes the electrochemical gradient of cells by driving an active exchange of Na and K ions while consuming ATP. The minimal functional transporter consists of a catalytic α-subunit and a β-subunit with chaperon activity. The Na,K-ATPase also functions as a cell adhesion molecule and participates in various intracellular signaling pathways.

infection impairs chaperone-assisted maturation of Na-K-ATPase in gastric epithelium.

infection always induces gastritis, which may progress to ulcer disease or cancer. The mechanisms underlying mucosal injury by the bacteria are incompletely understood. Here, we identify a novel pathway for -induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase.

Hypercapnia Impairs Na,K-ATPase Function by Inducing Endoplasmic Reticulum Retention of the β-Subunit of the Enzyme in Alveolar Epithelial Cells.

Alveolar edema, impaired alveolar fluid clearance, and elevated CO levels (hypercapnia) are hallmarks of the acute respiratory distress syndrome (ARDS). This study investigated how hypercapnia affects maturation of the Na,K-ATPase (NKA), a key membrane transporter, and a cell adhesion molecule involved in the resolution of alveolar edema in the endoplasmic reticulum (ER). Exposure of human alveolar epithelial cells to elevated CO concentrations caused a significant retention of NKA-β in the ER and, thus, decreased levels of the transporter in the Golgi apparatus.

A Functional Interaction Between Na,K-ATPase β-Subunit/AMOG and NF2/Merlin Regulates Growth Factor Signaling in Cerebellar Granule Cells.

The Na,K-ATPase, consisting of a catalytic α-subunit and a regulatory β-subunit, is a ubiquitously expressed ion pump that carries out the transport of Na and K across the plasma membranes of most animal cells. In addition to its pump function, Na,K-ATPase serves as a signaling scaffold and a cell adhesion molecule. Of the three β-subunit isoforms, β is found in almost all tissues, while β expression is mostly restricted to brain and muscle.

Fatal dysfunction and disintegration of thrombin-stimulated platelets.

Platelets play a key role in the formation of hemostatic clots and obstructive thrombi as well as in other biological processes. In response to physiological stimulants, including thrombin, platelets change shape, express adhesive molecules, aggregate, and secrete bioactive substances, but their subsequent fate is largely unknown. Here we examined late-stage structural, metabolic, and functional consequences of thrombin-induced platelet activation.

Reorganization of Septins Modulates Synaptic Transmission at Neuromuscular Junctions.

Septins (Sept) are highly conserved Guanosine-5'-triphosphate (GTP)-binding cytoskeletal proteins involved in neuronal signaling in the central nervous system but their involvement in signal transmission in peripheral synapses remains unclear. Sept5 and Sept9 proteins were detected in mouse peripheral neuromuscular junctions by immunofluorescence with a greater degree of co-localization with presynaptic than postsynaptic membranes. Preincubation of neuromuscular junction preparations with the inhibitor of Sept dynamics, forchlorfenuron (FCF), decreased co-localization of Sept with presynaptic membranes.

Measurement of Internal pH in Helicobacter pylori by Using Green Fluorescent Protein Fluorimetry.

is an organism known to colonize the normal human stomach. Previous studies have shown that the bacterium does this by elevating its periplasmic pH via the hydrolysis of urea. However, the value of the periplasmic pH was calculated indirectly from the proton motive force equation.

HIF and HOIL-1L-mediated PKCζ degradation stabilizes plasma membrane Na,K-ATPase to protect against hypoxia-induced lung injury.

Organisms have evolved adaptive mechanisms in response to stress for cellular survival. During acute hypoxic stress, cells down-regulate energy-consuming enzymes such as Na,K-ATPase. Within minutes of alveolar epithelial cell (AEC) exposure to hypoxia, protein kinase C zeta (PKCζ) phosphorylates the α-Na,K-ATPase subunit and triggers it for endocytosis, independently of the hypoxia-inducible factor (HIF).

The cryo-EM structure of gastric H,K-ATPase with bound BYK99, a high-affinity member of K-competitive, imidazo[1,2-a]pyridine inhibitors.

The gastric proton pump H,K-ATPase acidifies the gastric lumen, and thus its inhibitors, including the imidazo[1,2-a]pyridine class of K-competitive acid blockers (P-CABs), have potential application as acid-suppressing drugs. We determined the electron crystallographic structure of H,K-ATPase at 6.5 Å resolution in the E2P state with bound BYK99, a potent P-CAB with a restricted ring structure.

FXYD5 Is an Essential Mediator of the Inflammatory Response during Lung Injury.

The alveolar epithelium secretes cytokines and chemokines that recruit immune cells to the lungs, which is essential for fighting infections but in excess can promote lung injury. Overexpression of FXYD5, a tissue-specific regulator of the Na,K-ATPase, in mice, impairs the alveolo-epithelial barrier, and FXYD5 overexpression in renal cells increases C-C chemokine ligand-2 (CCL2) secretion in response to lipopolysaccharide (LPS). The aim of this study was to determine whether FXYD5 contributes to the lung inflammation and injury.

Analysis of N- and O-Glycosylation of Lysosomal Glycoproteins.

The vast majority of lysosomal proteins are heavily glycosylated. The present protocol describes the method of analyzing N- and O-linked glycans in lysosomal proteins of interest. The method is based on using deglycosylating enzymes, endoglycosidases, and exoglycosidases.

Septins: Regulators of Protein Stability.

Septins are small GTPases that play a role in several important cellular processes. In this review, we focus on the roles of septins in protein stabilization. Septins may regulate protein stability by: (1) interacting with proteins involved in degradation pathways, (2) regulating the interaction between transmembrane proteins and cytoskeletal proteins, (3) affecting the mobility of transmembrane proteins in lipid bilayers, and (4) modulating the interaction of proteins with their adaptor or signaling proteins.

Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart: DISTINCT FUNCTIONAL PROPERTIES AND ISOFORM-SELECTIVE INHIBITORS.

The Na,K-ATPase α subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca, whereas α has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α in the heart is related to selective assembly with a particular one of the three β isoforms.

The O-glycosylated ectodomain of FXYD5 impairs adhesion by disrupting cell-cell trans-dimerization of Na,K-ATPase β1 subunits.

FXYD5 (also known as dysadherin), a regulatory subunit of the Na,K-ATPase, impairs intercellular adhesion by a poorly understood mechanism. Here, we determined whether FXYD5 disrupts the trans-dimerization of Na,K-ATPase molecules located in neighboring cells. Mutagenesis of the Na,K-ATPase β1 subunit identified four conserved residues, including Y199, that are crucial for the intercellular Na,K-ATPase trans-dimerization and adhesion.

Septin oligomerization regulates persistent expression of ErbB2/HER2 in gastric cancer cells.

Septins are a family of cytoskeletal GTP-binding proteins that assemble into membrane-associated hetero-oligomers and organize scaffolds for recruitment of cytosolic proteins or stabilization of membrane proteins. Septins have been implicated in a diverse range of cancers, including gastric cancer, but the underlying mechanisms remain unclear. The hypothesis tested here is that septins contribute to cancer by stabilizing the receptor tyrosine kinase ErbB2, an important target for cancer treatment.

Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis: LAMP-2 deficient mice develop pancreatitis.

Background & Aims: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated membrane proteins (LAMPs) in pancreatitis.

High CO2 Leads to Na,K-ATPase Endocytosis via c-Jun Amino-Terminal Kinase-Induced LMO7b Phosphorylation.

The c-Jun amino-terminal kinase (JNK) plays a role in inflammation, proliferation, apoptosis, and cell adhesion and cell migration by phosphorylating paxillin and β-catenin. JNK phosphorylation downstream of AMP-activated protein kinase (AMPK) activation is required for high CO2 (hypercapnia)-induced Na,K-ATPase endocytosis in alveolar epithelial cells. Here, we provide evidence that during hypercapnia, JNK promotes the phosphorylation of LMO7b, a scaffolding protein, in vitro and in intact cells.

Septin dynamics are essential for exocytosis.

Septins are a family of 14 cytoskeletal proteins that dynamically form hetero-oligomers and organize membrane microdomains for protein complexes. The previously reported interactions with SNARE proteins suggested the involvement of septins in exocytosis. However, the contradictory results of up- or down-regulation of septin-5 in various cells and mouse models or septin-4 in mice suggested either an inhibitory or a stimulatory role for these septins in exocytosis.

Recruitment of septin cytoskeletal proteins by botulinum toxin A protease determines its remarkable stability.

Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of botulinum neurotoxin type A (LCA) results in a blockade of neurotransmitter release that persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both the proteolytic and neuroparalytic effects of the neurotoxin in mice. To elucidate the cellular mechanism for LCA longevity, we studied the effects of L428A/L429A mutation on the interactome, localization and stability of LCA expressed in cultured neuronal cells.

Helicobacter pylori impedes acid-induced tightening of gastric epithelial junctions.

Gastric infection by Helicobacter pylori is the most common cause of ulcer disease and gastric cancer. The mechanism of progression from gastritis and inflammation to ulcers and cancer in a fraction of those infected is not definitively known. Significant acidity is unique to the gastric environment and is required for ulcer development.

Subunit isoform selectivity in assembly of Na,K-ATPase α-β heterodimers.

To catalyze ion transport, the Na,K-ATPase must contain one α and one β subunit. When expressed by transfection in various expression systems, each of the four α subunit isoforms can assemble with each of the three β subunit isoforms and form an active enzyme, suggesting the absence of selective α-β isoform assembly. However, it is unknown whether in vivo conditions the α-β assembly is random or isoform-specific.

Identification of the amino acid region involved in the intercellular interaction between the β1 subunits of Na+/K+ -ATPase.

Epithelial junctions depend on intercellular interactions between β(1) subunits of the Na(+)/K(+)-ATPase molecules of neighboring cells. The interaction between dog and rat subunits is less effective than the interaction between two dog β(1) subunits, indicating the importance of species-specific regions for β(1)-β(1) binding. To identify these regions, the species-specific amino acid residues were mapped on a high-resolution structure of the Na(+)/K(+)-ATPase β(1) subunit to select those exposed towards the β(1) subunit of the neighboring cell.