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A significant number of patients with coronavirus disease 2019 (COVID-19) develop acute respiratory distress syndrome (ARDS) that is associated with a poor outcome. The molecular mechanisms driving failure of the alveolar barrier upon severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection remain incompletely understood. The Na,K-ATPase is an adhesion molecule and a plasma membrane transporter that is critically required for proper alveolar epithelial function by both promoting barrier integrity and resolution of excess alveolar fluid, thus enabling appropriate gas exchange. However, numerous SARS-CoV-2-mediated and COVID-19-related signals directly or indirectly impair the function of the Na,K-ATPase, thereby potentially contributing to disease progression. In this Perspective, we highlight some of the putative mechanisms of SARS-CoV-2-driven dysfunction of the Na,K-ATPase, focusing on expression, maturation, and trafficking of the transporter. A therapeutic mean to selectively inhibit the maladaptive signals that impair the Na,K-ATPase upon SARS-CoV-2 infection might be effective in reestablishing the alveolar epithelial barrier and promoting alveolar fluid clearance and thus advantageous in patients with COVID-19-associated ARDS.

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, β1 is found in almost all tissues, while β2 expression is mostly restricted to brain and muscle. In cerebellar granule cells, the β2-subunit, also known as adhesion molecule on glia (AMOG), has been linked to neuron-astrocyte adhesion and granule cell migration, suggesting its role in cerebellar development. Nevertheless, little is known about molecular pathways that link the β2-subunit to its cellular functions. Using cerebellar granule precursor cells, we found that the β2-subunit, but not the β1-subunit, negatively regulates the expression of a key activator of the Hippo/YAP signaling pathway, Merlin/neurofibromin-2 (NF2). The knockdown of the β2-subunit resulted in increased Merlin/NF2 expression and affected downstream targets of Hippo signaling, i.e., increased YAP phosphorylation and decreased expression of N-Ras. Further, the β2-subunit knockdown altered the kinetics of epidermal growth factor receptor (EGFR) signaling in a Merlin-dependent mode and impaired EGF-induced reorganization of the actin cytoskeleton. Therefore, our studies for the first time provide a functional link between the Na,K-ATPase β2-subunit and Merlin/NF2 and suggest a role for the β2-subunit in regulating cytoskeletal dynamics and Hippo/YAP signaling during neuronal differentiation.

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. Using a combination of confocal microscopy, scanning and transmission electron microscopy, flow cytometry, biochemical and biomechanical measurements, we showed that thrombin-induced activation is followed by time-dependent platelet dysfunction and disintegration. After ~30 minutes of incubation with thrombin, unlike with collagen or ADP, human platelets disintegrated into cellular fragments containing organelles, such as mitochondria, glycogen granules, and vacuoles. This platelet fragmentation was preceded by Ca2+ influx, integrin αIIbβ3 activation and phosphatidylserine exposure (activation phase), followed by mitochondrial depolarization, generation of reactive oxygen species, metabolic ATP depletion and impairment of platelet contractility along with dramatic cytoskeletal rearrangements, concomitant with platelet disintegration (death phase). Coincidentally with the platelet fragmentation, thrombin caused calpain activation but not activation of caspases 3 and 7. Our findings indicate that the late functional and structural damage of thrombin-activated platelets comprise a calpain-dependent platelet death pathway that shares some similarities with the programmed death of nucleated cells, but is unique to platelets, therefore representing a special form of cellular destruction. Fragmentation of activated platelets suggests that there is an underappreciated pathway of enhanced elimination of platelets from the circulation in (pro)thrombotic conditions once these cells have performed their functions.

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. Activation of platelets by thrombin causes clustering of septins and impairs their association with microtubules. Inhibition of septin dynamics with forchlorfenuron (FCF) reduces thrombin-induced densification of septins and lessens their colocalization with microtubules in resting and activated platelets. Exposure to FCF alters platelet shape, suggesting that septins stabilize platelet cytoskeleton. FCF suppresses platelet integrin αIIbβ3 activation, promotes phosphatidylserine exposure on activated platelets, and induces P-selectin expression on resting platelets, suggesting septin involvement in these processes. Inhibition of septin dynamics substantially reduces platelet contractility and abrogates their spreading on fibrinogen-coated surfaces. Overall, septins strongly contribute to platelet structure, activation and biomechanics.

Elevated carbon dioxide levels (hypercapnia) are frequently observed in patients with acute and chronic lung diseases. Na,K-ATPase is a sodium transporter and junctional molecule that is critically required for the maintenance of optimal alveolar fluid balance and thus gas exchange. Here, we explored how hypercapnia affects maturation of the Na,K-ATPase in the endoplasmic reticulum (ER). Murine precision-cut lung slices and human alveolar epithelial cells exposed to elevated CO2 levels showed a rapid and dose-dependent reduction of the Na,K-ATPase β-subunit in the ER and decreased Na,K-ATPase plasma membrane abundance. By combining chemical and genetic approaches, we identified that hypercapnia activated inositol-requiring enzyme 1α (IRE1α) and initiated a specific signaling pathway that resulted in ER-associated degradation (ERAD) of the Na,K-ATPase β-subunit. ERAD of the β-subunit prevented assembly with the α-subunit of the enzyme and thus downregulated formation of functional transporter molecules as well as delivery of the complex to the cell surface. Moreover, we observed that IRE1α activation was triggered by changes in ER and cytosolic calcium levels, and were dependent on the activity of the inositol trisphosphate ER receptor (IP3R). Treatment with the IP3R inhibitor, 2-aminoethoxydiphenyl borate (2-APB) prevented calcium dysregulation and increased plasma membrane abundance of the Na,K-ATPase α/β-complex. Accordingly, our results suggest that elevated CO2 levels decrease Na,K-ATPase plasma membrane abundance by altering ER calcium homeostasis and enhancing degradation of the Na,K-ATPase β-subunit in the ER.

Helicobacter pylori 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 H. pylori-induced gastric injury, the impairment of maturation of the essential transport enzyme and cell adhesion molecule, Na-K-ATPase. Na-K-ATPase comprises α- and β-subunits that assemble in the endoplasmic reticulum (ER) before trafficking to the plasma membrane. Attachment of H. pylori to gastric epithelial cells increased Na-K-ATPase ubiquitylation, decreased its surface and total levels, and impaired ion balance. H. pylori did not alter degradation of plasmalemma-resident Na-K-ATPase subunits or their mRNA levels. Infection decreased association of α- and β-subunits with ER chaperone BiP and impaired assembly of α/β-heterodimers, as was revealed by quantitative mass spectrometry and immunoblotting of immunoprecipitated complexes. The total level of BiP was not altered, and the decrease in interaction with BiP was not observed for other BiP client proteins. The H. pylori-induced decrease in Na-K-ATPase was prevented by BiP overexpression, stopping protein synthesis, or inhibiting proteasomal, but not lysosomal, protein degradation. The results indicate that H. pylori impairs chaperone-assisted maturation of newly made Na-K-ATPase subunits in the ER independently of a generalized ER stress and induces their ubiquitylation and proteasomal degradation. The decrease in Na-K-ATPase levels is also seen in vivo in the stomachs of gerbils and chronically infected children. Further understanding of H. pylori-induced Na-K-ATPase degradation will provide insights for protection against advanced disease. NEW & NOTEWORTHY This work provides evidence that Helicobacter pylori decreases levels of Na-K-ATPase, a vital transport enzyme, in gastric epithelia, both in acutely infected cultured cells and in chronically infected patients and animals. The bacteria interfere with BiP-assisted folding of newly-made Na-K-ATPase subunits in the endoplasmic reticulum, accelerating their ubiquitylation and proteasomal degradation and decreasing efficiency of the assembly of native enzyme. Decreased Na-K-ATPase expression contributes to H. pylori-induced gastric injury.

The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas α1 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 α2 in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with β2 in T-tubules of cardiac myocytes, forming α2β2 heterodimers. We have expressed human α1β1, α2β1, α2β2, and α2β3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2β2 and α2β3 differ significantly from both α2β1 and α1β1 in having a higher K0.5K+ and lower K0.5Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1P-E2P conformational equilibrium toward E1P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2β2 and α2β3 over α1β1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2β2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2β2 for cardiac muscle contractility. The high sensitivity of α2β2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2β2-selective digoxin derivatives for reducing cardiotoxicity.

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 three β-subunit isoforms, β(1) is found almost in all tissues, while β(2) expression is mostly restricted to brain and muscle. In cerebellar granule cells, the β(2)-subunit, also known as Adhesion Molecule on Glia (AMOG), has been linked to neuron-astrocyte adhesion and granule cell migration, suggesting its role in cerebellar development. Nevertheless, little is known about molecular pathways that link the β(2)-subunit to its cellular functions. Using cerebellar granule precursor cells, we found that the β(2)-subunit, but not the β(1)-subunit, negatively regulates expression of a key activator of the Hippo/YAP signaling pathway, Merlin/neurofibromin-2 (NF2). The knockdown of the β(2)-subunit resulted in increased Merlin/NF2 expression and affected down-stream targets of Hippo signaling, i.e. increased YAP phosphorylation and decreased expression of N-Ras. Further, the β(2)-subunit knockdown altered the kinetics of Epidermal Growth Factor Receptor (EGFR) signaling in a Merlin-dependent mode and impaired EGF-induced reorganization of the actin cytoskeleton. Therefore, our studies for the first time provide a functional link between the Na,K-ATPase β(2)-subunit and Merlin/NF2 and suggest a role for the β(2)-subunit in regulating cytoskeletal dynamics and Hippo/YAP signaling during neuronal differentiation.

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. FCF introduced ex vivo or in vivo had no effect on the amplitude of the spontaneous endplate currents (EPCs), indicating the absence of postsynaptic effects of FCF. However, FCF decreased acetylcholine (ACh) quantal release in response to nerve stimulation, reduced the amplitude of evoked quantal currents and decreased the number of quanta with long synaptic delays, demonstrating the presynaptic action of FCF. Nevertheless, FCF had no effect on the amplitude of calcium transient in nerve terminals, as detected by calcium-sensitive dye, and slightly decreased the ratio of the second response amplitude to the first one in paired-pulse experiments. These results suggest that FCF-induced decrease in ACh quantal secretion is not due to a decrease in Ca2+ influx but is likely related to the impairment of later stages occurring after Ca2+ entry, such as trafficking, docking or membrane fusion of synaptic vesicles. Therefore, Sept9 and Sept5 are abundantly expressed in presynaptic membranes, and disruption of Sept dynamics suppresses the evoked synchronous and delayed asynchronous quantal release of ACh, strongly suggesting an important role of Sept in the regulation of neurotransmission in peripheral synapses.

Helicobacter pylori 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. To measure the periplasmic pH directly in H. pylori , we fused enhanced green fluorescent protein (EGFP) to the predicted twin-arginine signal peptides of HydA and KapA from H. pylori and TorA from Escherichia coli . The fusion proteins were expressed in the H. pylori genome under the control of the cagA promoter. Confocal microscopic and cell fractionation/immunoblotting analyses detected TorA-EGFP in the periplasm and KapA-EGFP in both the periplasm and cytoplasm, while the mature form of HydA-EGFP was seen at low levels in the periplasm, with major cytoplasmic retention of the precursor form. With H. pylori expressing TorA-EGFP, we established a system to directly measure periplasmic pH based on the pH-sensitive fluorimetry of EGFP. These measurements demonstrated that the addition of 5 mM urea has little effect on the periplasmic pH at a medium pH higher than pH 6.5 but rapidly increases the periplasmic pH to pH 6.1 at an acidic medium pH (pH 5.0), corresponding to the opening of the proton-gated channel, UreI, and confirming the basis of gastric colonization. Measurements of the periplasmic pH in an HP0244 (FlgS)-deficient mutant of H. pylori expressing TorA-EGFP revealed a significant loss of the urea-dependent increase in the periplasmic pH at an acidic medium pH, providing additional evidence that FlgS is responsible for recruitment of urease to the inner membrane in association with UreI. IMPORTANCE Helicobacter pylori has been identified as the major cause of chronic superficial gastritis and peptic ulcer disease. In addition, persistent infection with H. pylori , which, if untreated, lasts for the lifetime of an infected individual, predisposes one to gastric malignancies, such as adenocarcinoma and mucosa-associated lymphoid tissue (MALT) lymphoma. A unique feature of the neutralophilic bacterium H. pylori is its ability to survive in the extremely acidic environment of the stomach through its acid acclimation mechanism. The presented results on measurements of periplasmic pH in H. pylori based on fluorimetry of fully active green fluorescent protein fusion proteins exported with the twin-arginine translocase system provide a reliable and rapid tool for the investigation of acid acclimation in H. pylori .

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. LMO7b phosphorylation was blocked by exposing the cells to the JNK inhibitor SP600125 and by infecting cells with dominant-negative JNK or AMPK adenovirus. The knockdown of the endogenous LMO7b or overexpression of mutated LMO7b with alanine substitutions of five potential JNK phosphorylation sites (LMO7b-5SA) or only Ser-1295 rescued both LMO7b phosphorylation and the hypercapnia-induced Na,K-ATPase endocytosis. Moreover, high CO2 promoted the colocalization and interaction of LMO7b and the Na,K-ATPase α1 subunit at the plasma membrane, which were prevented by SP600125 or by transfecting cells with LMO7b-5SA. Collectively, our data suggest that hypercapnia leads to JNK-induced LMO7b phosphorylation at Ser-1295, which facilitates the interaction of LMO7b with Na,K-ATPase at the plasma membrane promoting the endocytosis of Na,K-ATPase in alveolar epithelial cells.

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. Methods: We analyzed changes in LAMPs in experimental models and human pancreatitis, and the underlying mechanisms: LAMP deglycosylation and degradation. LAMP cleavage by cathepsin B (CatB) was analyzed by mass spectrometry. We used mice deficient in LAMP-2 to assess its role in pancreatitis. Results: Pancreatic levels of LAMP-1 and LAMP-2 greatly decrease across various pancreatitis models and in human disease. Pancreatitis does not trigger the LAMPsâ bulk deglycosylation but induces their degradation via CatB-mediated cleavage of the LAMP molecule close to the boundary between luminal and transmembrane domains. LAMP-2 null mice spontaneously develop pancreatitis that begins with acinar cell vacuolization due to impaired autophagic flux, and progresses to severe pancreas damage characterized by trypsinogen activation, macrophage-driven inflammation, and acinar cell death. LAMP-2 deficiency causes a decrease in pancreatic digestive enzymes content, and stimulates the basal and inhibits cholecystokinin-induced amylase secretion by acinar cells. The effects of LAMP-2 knockout and acute cerulein pancreatitis overlap, which corroborates the pathogenic role of LAMP decrease in experimental pancreatitis models. Conclusions: The results indicate a critical role for LAMPs, particularly LAMP-2, in maintaining pancreatic acinar cell homeostasis and provide evidence that defective lysosomal function, resulting in impaired autophagy, leads to pancreatitis. Mice with LAMP-2 deficiency present a novel genetic model of human pancreatitis caused by lysosomal/autophagic dysfunction. Keywords: Amylase Secretion, Autophagy, Cathepsin B, Cerulein

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 α1-Na,K-ATPase subunit and triggers it for endocytosis, independently of the hypoxia-inducible factor (HIF). However, the Na,K-ATPase activity is essential for cell homeostasis. HIF induces the heme-oxidized IRP2 ubiquitin ligase 1L (HOIL-1L), which leads to PKCζ degradation. Here we report a mechanism of prosurvival adaptation of AECs to prolonged hypoxia where PKCζ degradation allows plasma membrane Na,K-ATPase stabilization at ∼50% of normoxic levels, preventing its excessive down-regulation and cell death. Mice lacking HOIL-1L in lung epithelial cells (CreSPC/HOIL-1Lfl/fl) were sensitized to hypoxia because they express higher levels of PKCζ and, consequently, lower plasma membrane Na,K-ATPase levels, which increased cell death and worsened lung injury. In AECs, expression of an α1-Na,K-ATPase construct bearing an S18A (α1-S18A) mutation, which precludes PKCζ phosphorylation, stabilized the Na,K-ATPase at the plasma membrane and prevented hypoxia-induced cell death even in the absence of HOIL-1L. Adenoviral overexpression of the α1-S18A mutant Na,K-ATPase in vivo rescued the enhanced sensitivity of CreSPC/HOIL-1Lfl/fl mice to hypoxic lung injury. These data suggest that stabilization of Na,K-ATPase during severe hypoxia is a HIF-dependent process involving PKCζ degradation. Accordingly, we provide evidence of an important adaptive mechanism to severe hypoxia, whereby halting the exaggerated down-regulation of plasma membrane Na,K-ATPase prevents cell death and 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. Exposure of alveolar epithelial cells (AEC) to LPS increased FXYD5 levels at the plasma membrane, and FXYD5 silencing prevented both the activation of NF-κB and the secretion of cytokines in response to LPS. Intratracheal instillation of LPS into mice increased FXYD5 levels in the lung. FXYD5 overexpression increased the recruitment of interstitial macrophages and classical monocytes to the lung in response to LPS. FXYD5 silencing decreased CCL2 levels, number of cells, and protein concentration in bronchoalveolar lavage fluid (BALF) after LPS treatment, indicating that FXYD5 is required for the NF-κB-stimulated epithelial production of CCL2, the influx of immune cells, and the increase in alveolo-epithelial permeability in response to LPS. Silencing of FXYD5 also prevented the activation of NF-κB and cytokine secretion in response to interferon α and TNF-α, suggesting that pro-inflammatory effects of FXYD5 are not limited to the LPS-induced pathway. Furthermore, in the absence of other stimuli, FXYD5 overexpression in AEC activated NF-κB and increased cytokine production, while FXYD5 overexpression in mice increased cytokine levels in BALF, indicating that FXYD5 is sufficient to induce the NF-κB-stimulated cytokine secretion by the alveolar epithelium. The FXYD5 overexpression also increased cell counts in BALF, which was prevented by silencing the CCL2 receptor (CCR2), or by treating mice with a CCR2-blocking antibody, confirming that FXYD5-induced CCL2 production leads to the recruitment of monocytes to the lung. Taken together, the data demonstrate that FXYD5 is a key contributor to inflammatory lung injury.

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. The interplay between gastric acidity and H. pylori pathogenesis is important in progression to advanced disease. The aim of this study was to characterize the impact of acid on gastric epithelial integrity and cytokine release and how H. pylori infection alters these responses. Human gastric epithelial (HGE-20) cells were grown on porous inserts, and survival, barrier function, and cytokine release were studied at various apical pH levels in the presence and absence of H. pylori. With apical acidity, gastric epithelial cells demonstrate increased barrier function, as evidenced by increased transepithelial electrical resistance (TEER) and decreased paracellular permeability. This effect is reduced in the presence of wild-type, but not urease knockout, H. pylori. The epithelial inflammatory response is also modulated by acidity and H. pylori infection. Without H. pylori, epithelial IL-8 release decreases in acid, while IL-6 release increases. In the presence of H. pylori, acidic pH diminishes the magnitude of the previously reported increase in IL-8 and IL-6 release. H. pylori interferes with the gastric epithelial response to acid, contributing to altered barrier function and inflammatory response. H. pylori diminishes acid-induced tightening of cell junctions in a urease-dependent manner, suggesting that local pH elevation promotes barrier compromise and progression to mucosal damage.

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. Endoglycosidases catalyze the cleavage of an internal bond in an oligosaccharide, while exoglycosidases remove terminal carbohydrates from glycans. Different types of carbohydrate residues or chains can be removed by specific glycosidases. Removing oligosaccharides with glycosidases increases the electrophoretic mobility of a protein. This increase in mobility depends on the size and number of removed carbohydrate chains. Therefore, the treatment of lysosomal proteins with specific glycosidases followed by a western blot analysis of a protein of interest provides a way to determine which types of glycans are present in the protein by comparing the gel mobility before and after treatment.

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. In this context, we discuss the role of septins in protecting four different proteins from degradation. First we consider botulinum neurotoxin serotype A (BoNT/A) and the contribution of septins to its extraordinarily long intracellular persistence. Next, we discuss the role of septins in stabilizing the receptor tyrosine kinases EGFR and ErbB2. Finally, we consider the contribution of septins in protecting hypoxia-inducible factor 1α (HIF-1α) from degradation.

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. The α(2)-β(2) complex was selectively immunoprecipitated by both anti-α(2) and anti-β(2) antibodies from extracts of mouse brain, which contains cells co-expressing multiple Na,K-ATPase isoforms. Neither α(1)-β(2) nor α(2)-β(1) complexes were detected in the immunoprecipitates. Furthermore, in MDCK cells co-expressing α(1), β(1), and β(2) isoforms, a greater fraction of the β(2) subunits was unassembled with α(1) as compared with that of the β(1) subunits, indicating preferential association of the α(1) isoform with the β(1) isoform. In addition, the α(1)-β(2) complex was less resistant to various detergents than the α(1)-β(1) complex isolated from MDCK cells or the α(2)-β(2) complex isolated from mouse brain. Therefore, the diversity of the α-β Na,K-ATPase heterodimers in vivo is determined not only by cell-specific co-expression of particular isoforms, but also by selective association of the α and β subunit isoforms.

N-Glycans of the Na,K-ATPase β1 subunit are important for intercellular adhesion in epithelia, suggesting that epithelial junctions depend on N-glycan-mediated interactions between the β1 subunits of neighboring cells. The level of co-immunoprecipitation of the endogenous β1 subunit with various YFP-linked β1 subunits expressed in Madin-Darby canine kidney cells was used to assess β1-β1 interactions. The amount of co-precipitated endogenous dog β1 was greater with dog YFP-β1 than with rat YFP-β1, showing that amino acid-mediated interactions are important for β1-β1 binding. Co-precipitation of β1 was also less with the unglycosylated YFP-β1 than with glycosylated YFP-β1, indicating a role for N-glycans. Mixing cells expressing dog YFP-β1 with non-transfected cells increased the amount of co-precipitated β1, confirming the presence of intercellular (YFP-β1)-β1 complexes. Accordingly, disruption of intercellular junctions decreased the amount of co-precipitated β1 subunits. The decrease in β1 co-precipitation both with rat YFP-β1 and unglycosylated YFP-β1 was associated with decreased detergent stability of junctional proteins and increased paracellular permeability. Reducing N-glycan branching by specific inhibitors increased (YFP-β1)-β1 co-precipitation and strengthened intercellular junctions. Therefore, interactions between the β1 subunits of neighboring cells maintain integrity of intercellular junctions, and alterations in the β1 subunit N-glycan structure can regulate stability and tightness of intercellular junctions.

Introduction: Platelets play a key role in formation of protective hemostatic blood clots and pathological obstructive thrombi. Under (patho)physiological conditions, chemically activated platelets change their morphology, express adhesive molecules, undergo aggregation, secrete procoagulant substances, and induce mechanical contraction (retraction) of the blood clots. Despite the vital importance of these platelet functions, the subsequent fate of activated platelets is largely unknown. We hypothesize that activated platelets undergo late alterations that determine their fate and may have a pathogenic importance in thrombotic and hemostatic disorders. Methods: We used a combination of confocal microscopy, immunofluorescence, scanning and transmission electron microscopy, flow cytometry, biochemical and biomechanical measurements to study deferred structural, metabolic, and functional consequences of thrombin-induced activation of viable human platelets, either suspended in platelet-rich plasma or isolated by gel-filtration. Results: Visualized by confocal microscopy, fluorescently labeled platelets in thrombin-induced plasma clots initially underwent shape changes characteristic of platelet activation, but in about 30 min many platelets and platelet aggregates broke up into organelle-containing vesicular fragments. There were two types of platelet-derived vesicles differing in their size and cellular origin: one smaller type was shedding from the tips of filopodia, while the other type resulted from fragmentation of platelet bodies. Concurrently with the fragmentation, thrombin-activated platelets displayed dramatically altered intracellular distribution of F-actin and septins detected as intense fluorescent clusters with a ~2-fold increase in the intensity of septins 2 and 9 and a ~300-fold increase in the F-actin staining. Synchronously with the structural alterations, thrombin induced a time-dependent reduction of the mitochondrial membrane potential (Δψm) in platelets. The overall fluorescence intensity of the Δψm-sensitive MitoTracker dye in freshly formed thrombin-initiated plasma clots dropped 2- and 4-fold after 60 min and 90 min, respectively. A drop of Δψm inversely correlated with an increase of the fraction of disintegrated platelets (r=-0.93, p Conclusions: Our findings indicate that following thrombin-induced platelet activation, a substantial fraction of platelets undergo time-dependent dysfunction and structural disintegration into subcellular particles. The fragmentation of platelets is accompanied by dramatic rearrangements of platelet cytoskeletal components, including polymerization, clustering, and redistribution of actin and septins. Thrombin-induced platelet fragmentation is concurrent with severe impairment of platelet functionality, including mitochondrial depolarization, ATP depletion, and loss of platelet contractility. The lack of caspase activity and increased calpain activity in energetically exhausted thrombin-treated platelets undergoing fragmentation suggests a calpain-dependent platelet death pathway. These studies indicate that such a form of platelet death may be an underappreciated mechanism for enhanced elimination of platelets from the circulation in (pro)thrombotic conditions or under other conditions once they have performed their functions. Work supported by the Program for Competitive Growth at KFU and AHA grant 17SDG33680177. Disclosures No relevant conflicts of interest to declare.

The endoplasmic reticulum (ER) lumen contains high concentrations of molecular chaperones and folding enzymes that allow newly synthesized proteins to acquire their native conformation. Nevertheless, a large fraction of ER-synthesized proteins fail to fold properly on the first attempt. Cells overcome this problem by employing an ER-resident protein quality control system that includes a variety of proteins and operates on levels of protein synthesis, folding, and assembly. By preventing the premature exit of non-native conformers and incompletely assembled proteins from the ER, the quality control system extends exposure of substrates to the folding machinery in the ER lumen and hence improves the chances of correct maturation. If proper folding or full assembly is not achieved even after repeated attempts, proteins are then moved from the ER to the cytosol and destroyed via the ER-associated degradation (ERAD)1 pathway (1–3). Hence, the ER quality control system ensures that only properly folded and assembled proteins are exported to the Golgi to begin their travel to their final destinations. The importance of the ER quality control system in maintaining the fidelity of cellular functions is evidenced by many diseases, including cystic fibrosis, antitrypsin deficiency, Alzheimer’s disease, and Huntington’s disease, that are linked to defects in this system (3, 4). There are two major groups of ER chaperones, heat shock protein homologues (e.g., BiP, Hsp40, and Hsp90) and lectins (e.g., calnexin, calreticulin, and EDEM-1) (5–9). Correct folding of nonglycosylated proteins is assisted by the ER heat shock protein homologues that, in conjunction with luminal thiol oxidoreductases, mediate disulfide bond formation. These non-lectin chaperones also mediate quality control of nonglycosylated proteins by retarding the premature exit of incompletely folded or unassembled proteins and by targeting of misfolded proteins to ERAD (5). In addition to these non-lectin chaperones, N-glycosylated proteins, which represent the majority of ER-synthesized proteins, also utilize the lectin chaperone system to help mediate folding and quality control (5–9). Lectin chaperones use the N-glycans of glycoproteins as molecular recognition sites to guide the newly synthesized proteins through highly ordered steps of maturation and quality control. Every lectin-assisted maturation step is coupled with a particular modification of the N-glycan structure that is catalyzed by ER-resident enzymes. In eukaryotic cells, glycoproteins acquire the N-linked glycans during the process of translocation and elongation of the polypeptide chain. First, a 14-saccharide core is transferred from the dolichol phosphate precursor to the asparagine (Asn) residue within the N-glycosylation site, a consensus Asn-X-Thr/Ser (X is any amino acid residue except Pro) sequence of a nascent protein. Immediately after covalent linkage of this core oligosaccharide, its terminal glucose residues are sequentially trimmed by ER glucosidases. Removal of two of the three glucose residues enables N-glycan binding to a membrane-bound lectin, calnexin, or its soluble homologue, calreticulin. Binding to calnexin (calreticulin) can occur cotranslationally while the protein is still associated with the translocon. Calnexin forms a complex with the oxidoreductase, ERp57, which catalyzes formation of disulfide bonds in the protein (5, 6). When the newly synthesized protein is released from calnexin, it meets one of three possible fates. If it is folded correctly, is exported to the Golgi, or assembles with its partner subunits, if any, and then is exported. If the protein is terminally misfolded, it is often recognized by EDEMS and/or by a non-lectin chaperone, BiP, that facilitates translocation of the protein to the cytoplasm and proteasome-mediated degradation (5). If the protein is slightly misfolded, it is reglucosylated by UGGT1 and binds again to calnexin for refolding. This cycle can be repeated several times (5, 6). Thus,UGGT1 acts as a conformation sensor. Proteomic analysis of calnexin-interacting proteins in embryonic fibroblasts from UGGT1-null mice shows that many proteins do not require UGGT1 for maturation and complete their folding program in one calnexin binding event (10). Other proteins exhibit accelerated dissociation from calnexin and impaired folding in UGGT1-null fibroblasts, indicating that these proteins normally undergo several cycles of interaction with calnexin, mediated by UGGT1(10). Some of these proteins must be essential for mouse viability since disruption of the UGGT1 gene results in embryonic lethality (11). Here, we present data showing that one of these essential proteins that requires post-translational calnexin binding for proper folding is the β2 subunit of the Na,K-ATPase. The Na,K-ATPase is a vital transport enzyme expressed in all animal tissues where it generates ion gradients responsible for membrane potential and solute symport or antiport, as well as mediating intercellular junctions and signal transduction (12–18). The Na,K-ATPase consists of an α subunit that is responsible for catalysis of ion transport and an N-glycosylated β subunit that is implicated in maturation and membrane targeting of the enzyme. There are four isoforms of the Na,K-ATPase α subunit (α1, α2, α3, and α4) and three isoforms of the Na,K-ATPase β subunit (β1, β2, and β3) (19, 20). Assembly of the α and β subunits that occurs in the ER soon after their biosynthesis is essential for formation of the functional enzyme (21) and for the export of both subunits from the ER to the Golgi (22–25). Expression in Xenopus oocytes showed that α1, β1, and β3 subunits of the Na,K-ATPase interact with BiP and this interaction is important for retention of unassembled α1, β1, and β3 subunits in the ER (26), implying the involvement of this non-lectin chaperone system in the ER quality control of these particular isoforms of Na,K-ATPase. The presence of three, eight, and two N-glycans on the Na,K-ATPase β1, β2, and β3 subunits, respectively, also suggests the possible involvement of an ER lectin chaperone system in the folding of these subunits, their assembly with the α subunit, and quality control of the α–β complex. However, prevention of N-glycosylation of the β1 subunit by tunicamycin or by mutations of all three N-glycosylation sites only slightly impairs assembly of the α1–β1 complex (26, 27), trafficking of the pump to the plasma membrane (16, 28), or Na,K-ATPase activity (26, 28–30). Similarly, prevention of N-glycosylation of the β3 subunit by tunicamycin or by mutations of both N-glycosylation sites does not impair trafficking of this subunit to the plasma membrane (31). These results suggest that N-glycans and hence glycan–lectin interactions are not critical for folding of the β1 and β3 subunits. On the other hand, native N-glycosylation has been shown to be essential for normal trafficking of the homologous H,K-ATPase β subunit from the ER (32). The Na,K-ATPase β2 subunit is more homologous to the H,K-ATPase β subunit than to the Na,K-ATPase β1 and β3 subunits. Similar to the H,K-ATPase β subunit, the Na,K-ATPase β2 subunit has multiple N-glycosylation sites. However, the contribution of glycan–lectin interactions to folding, maturation, and quality control of the Na,K-ATPase β2 subunit has not been investigated. In this study, canine renal MDCK cells were used as the expression system for YFP-linked β1 and β2 subunit isoforms of the Na,K-ATPase and their N-glycosylation-deficient mutants to test whether the ER lectin quality control system is involved in maturation of the proteins. The results indicate that at least two of the eight N-glycans of the β2 subunit as well as the hydrophobic amino acid cluster downstream to the seventh N-glycan are critical both for calnexin binding and for normal maturation and ER exit of the subunit, suggesting the involvement of UGGT1-induced post-translational calnexin binding in folding and quality control of this subunit. In contrast, maturation of the β1 subunit does not require post-translational calnexin binding.

The sodium pump is expressed in all animal tissues where it establishes concentration gradients for Na+ and K+ by pumping three Na+ from the cytoplasm in exchange for two extracellular K+. The ion gradients generated by the Na,K-ATPase are used in many essential cellular processes, such as osmoregulation, generation of plasma membrane potential and maintenance of intracellular pH and Ca2+ concentration, vectorial transport of many solutes, and excitability in muscle fibers and neurons. Further, the Na,K-ATPase serves as a functional signal transducer (1, 2). The pump is a heterodimer, consisting of an α subunit that is responsible for ion transport and a β subunit that is implicated in maturation and membrane targeting of the enzyme. There are four isoforms of the Na,K-ATPase α subunit (α1, α2, α3, and α4) and three isoforms of the Na,K-ATPase β subunit (β1, β2, and β3) (3, 4). Additional tissue-specific regulatory subunits of the Na,K-ATPase that belong to the FXYD family of small membrane proteins have also been identified (5). Most data have shown that α and β subunits are always present in equimolar amounts in the isolated Na,K-ATPase (6–9). Recent data claiming that the α–β ratio can be less (10) or more (11) than one are difficult to reconcile with the 3D high-resolution structure of the enzyme that shows two specific regions of interaction between the α and β subunits at a 1:1 stoichiometry (12, 13). Given these sites of interaction, stable recruitment of additional β subunits to the α subunit or vice versa is unlikely. ER assembly of the α with the β subunit is necessary for export of the α subunit of the Na,K-ATPase from the ER to the Golgi and formation of functional enzyme (14), suggesting that the β subunit acts as a molecular chaperone that facilitates maturation of the enzyme (15, 16). In addition, both β1 and β2 subunits have important non-enzymatic roles such as formation and maintenance of intercellular junctions and regulation of cell migration (16–23). It is not known whether the β subunits perform these functions only as components of the α–β complexes or perhaps as individual plasma membrane proteins. Results of studies addressing the question as to whether unassembled β subunits are present in the plasma membrane are ambiguous. A consecutive series of immunoprecipitation using the antibody against the α1 subunit resulted in depletion of MDCK cell lysates of the mature but not immature β1 subunits, indicating that the mature β1 subunits are all bound to the α1 subunit (10). Along with these data, overexpressed Xenopus oocyte Na,K-ATPase β1 and β3 subunits are retained in the ER in the absence of the α subunit in Xenopus oocytes (24–26). By contrast, the Torpedo californica β1 subunit expressed in Xenopus oocytes without the α subunit shows the mature N-glycosylation pattern, implying that it is able to traffic from the ER to the Golgi (27). Whether the unassembled Na, K-ATPase β2 subunit isoform is able to reach the plasma membrane is unknown. However, the H,K-ATPase β subunit that has more homology with the Na,K-ATPase β2 subunit than the other Na,K-ATPase β subunit isoforms is detected in the plasma membrane when expressed without the H,K-ATPase α subunit in various cell types (28–32). In this work, analysis of the nature of the N-linked glycans of endogenous and expressed β subunits of the Na,K-ATPase enables determination of their localization either in the ER or in the post-ER compartments. The presence of high-mannose-type N-glycans shows ER location of the β subunits, while the presence of hybrid- or complex-type N-glycans in the β subunits that can only be generated by Golgi-located glycosyltransferases shows successful export of the β subunits from the ER. Renal MDCK cells were used as an expression system for YFP-linked β1 and β2 subunit isoforms and β subunit mutants in order to determine whether assembly with the α subunit is essential for ER exit of either β1 or β2 subunits. Confocal microscopy, immunohistochemistry, immunoprecipitation, surface-selective biotinylation, and Western blot analysis were employed to evaluate trafficking and molecular interactions of endogenous and exogenous Na,K-ATPase subunits. The results show that (i) association with the α subunit at 1:1 stoichiometry is a prerequisite for the export of both the β1 and β2 subunits from the ER and (ii) the amount of the α subunit in the ER is a limiting factor for α–β assembly.

The gastric H,K-ATPase, a member of the P(2)-type ATPase family, is the integral membrane protein responsible for gastric acid secretion. It is an alpha,beta-heterodimeric enzyme that exchanges cytoplasmic hydronium with extracellular potassium. The catalytic alpha subunit has ten transmembrane segments with a cluster of intramembranal carboxylic amino acids located in the middle of the transmembrane segments TM4, TM5,TM6, and TM8. Comparison to the known structure of the SERCA pump, mutagenesis, and molecular modeling has identified these as constituents of the ion binding domain. The beta subunit has one transmembrane segment with N terminus in cytoplasmic region. The extracellular domain of the beta subunit contains six or seven N-linked glycosylation sites. N-glycosylation is important for the enzyme assembly, maturation, and sorting. The enzyme pumps acid by a series of conformational changes from an E(1) (ion site in) to an E(2) (ion site out) configuration following binding of MgATP and phosphorylation. Several experimental observations support the hypothesis that expulsion of the proton at 160 mM (pH 0.8) results from movement of lysine 791 into the ion binding site in the E(2)P configuration. Potassium access from the lumen depends on activation of a K and Cl conductance via a KCNQ1/KCNE2 complex and Clic6. K movement through the luminal channel in E(2)P is proposed to displace the lysine along with dephosphorylation to return the enzyme to the E(1) configuration. This enzyme is inhibited by the unique proton pump inhibitor class of drug, allowing therapy of acid-related diseases.

The majority of cell adhesion molecules are N-glycosylated, but the role of N-glycans in intercellular adhesion in epithelia remains ill-defined. Reducing N-glycan branching of cellular glycoproteins by swainsonine, the inhibitor of N-glycan processing, tightens and stabilizes cell-cell junctions as detected by a 3-fold decrease in the paracellular permeability and a 2-3-fold increase in the resistance of the adherens junction proteins to extraction by non-ionic detergent. In addition, exposure of cells to swainsonine inhibits motility of MDCK cells. Mutagenic removal of N-glycosylation sites from the Na,K-ATPase β1 subunit impairs cell-cell adhesion and decreases the effect of swainsonine on the paracellular permeability of the cell monolayer and also on detergent resistance of adherens junction proteins, indicating that the extent of N-glycan branching of this subunit is important for intercellular adhesion. The N-glycans of the Na,K-ATPase β1 subunit and E-cadherin are less complex in tight renal epithelia than in the leakier intestinal epithelium. The complexity of the N-glycans linked to these proteins gradually decreases upon the formation of a tight monolayer from dispersed MDCK cells. This correlates with a cell-cell adhesion-induced increase in expression of GnT-III (stops N-glycan branching) and a decrease in expression of GnTs IVC and V (promote N-glycan branching) as detected by real-time quantitative PCR. Consistent with these results, partial silencing of the gene encoding GnT-III increases branching of N-glycans linked to the Na,K-ATPase β1 subunit and other glycoproteins and results in a 2-fold increase in the paracellular permeability of MDCK cell monolayers. These results suggest epithelial cells can regulate tightness of cell junctions via remodeling of N-glycans, including those linked to the Na,K-ATPase β1-subunit.

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. Modulation of expression of FXYD5 or of the β1 subunit with intact or mutated β1–β1 binding sites demonstrated that the anti-adhesive effect of FXYD5 depends on the presence of Y199 in the β1 subunit. Immunodetection of the plasma membrane FXYD5 was prevented by the presence of O-glycans. Partial FXYD5 deglycosylation enabled antibody binding and showed that the protein level and the degree of O-glycosylation were greater in cancer than in normal cells. FXYD5-induced impairment of adhesion was abolished by both genetic and pharmacological inhibition of FXYD5 O-glycosylation. Therefore, the extracellular O-glycosylated domain of FXYD5 impairs adhesion by interfering with intercellular β1–β1 interactions, suggesting that the ratio between FXYD5 and α1–β1 heterodimer determines whether the Na,K-ATPase acts as a positive or negative regulator of intercellular adhesion.

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. Modulation of expression of FXYD5 or of the β1 subunit with intact or mutated β1-β1 binding sites demonstrated that the anti-adhesive effect of FXYD5 depends on the presence of Y199 in the β1 subunit. Immunodetection of the plasma membrane FXYD5 was prevented by the presence of O-glycans. Partial FXYD5 deglycosylation enabled antibody binding and showed that the protein level and the degree of O-glycosylation were greater in cancer than in normal cells. FXYD5-induced impairment of adhesion was abolished by both genetic and pharmacological inhibition of FXYD5 O-glycosylation. Therefore, the extracellular O-glycosylated domain of FXYD5 impairs adhesion by interfering with intercellular β1-β1 interactions, suggesting that the ratio between FXYD5 and α1-β1 heterodimer determines whether the Na,K-ATPase acts as a positive or negative regulator of intercellular adhesion.

Inhibition of gastric acid secretion is the mainstay of the treatment of gastroesophageal reflux disease and peptic ulceration; therapies to inhibit acid are among the best-selling drugs worldwide. Highly effective agents targeting the histamine H2 receptor were first identified in the 1970s. These were followed by the development of irreversible inhibitors of the parietal cell hydrogen-potassium ATPase (the proton pump inhibitors) that inhibit acid secretion much more effectively. Reviewed here are the chemistry, biological targets and pharmacology of these drugs, with reference to their current and evolving clinical utilities. Future directions in the development of acid inhibitory drugs include modifications of current agents and the emergence of a novel class of agents, the acid pump antagonists.

The Na,K-ATPases and the H,K-ATPases are two potassium-dependent homologous heterodimeric P2-type pumps that catalyze active transport of Na+ in exchange for K+ (Na,K-ATPase) or H+ in exchange for K+ (H,K-ATPase). The ubiquitous Na,K-ATPase maintains intracellular ion balance and membrane potential. The gastric H,K-ATPase is responsible for acid secretion by the parietal cell of the stomach. Both pumps consist of a catalytic α-subunit and a glycosylated β-subunit that is obligatory for normal pump maturation and trafficking. Individual N-glycans linked to the β-subunits of the Na,K-ATPase and H,K-ATPase are important for stable membrane integration of their respective α subunits, folding, stability, subunit assembly, and enzymatic activity of the pumps. They are also essential for the quality control of unassembled β-subunits that results in either the exit of the subunits from the ER or their ER retention and subsequent degradation. Overall, the importance of N-glycans for the␣maturation and quality control of the H,K-ATPase is greater than that of the Na,K-ATPase. The roles of individual N-glycans of the β-subunits in the post-ER trafficking, membrane targeting and plasma membrane retention of the Na,K-ATPase and H,K-ATPase are different. The Na,K-ATPase β 1-subunit is the major β-subunit isoform in cells with lateral location of the pump. All three N-glycans of the Na,K-ATPase β 1-subunit are important for the lateral membrane retention of the pump due to glycan-mediated interaction between the β 1-subunits of the two neighboring cells in the cell monolayer and cytosolic linkage of the α-subunit to the cytoskeleton. This intercellular β 1–β 1 interaction is also important for formation of cell–cell contacts. In contrast, the N-glycans unique to the Na,K-ATPase β 2-subunit,which has up to eight N-glycosylation sites, contain apical sorting information. This is consistent with the apical location of the Na,K-ATPase in normal and malignant epithelial cells with high abundance of the β 2-subunit. Similarly, all seven N-glycans of the gastric H,K-ATPase β-subunit determine apical sorting of this subunit.

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. Septins and ErbB2 were highly over-expressed in gastric cancer cells. Immunoprecipitation followed by MS analysis identified ErbB2 as a septin-interacting protein. Knockdown of septin-2 or cell exposure to forchlorfenuron (FCF), a well-established inhibitor of septin oligomerization, decreased surface and total levels of ErbB2. These treatments had no effect on epidermal growth factor receptor (EGFR), emphasizing the specificity and functionality of the septin–ErbB2 interaction. The level of ubiquitylated ErbB2 at the plasma membrane was elevated in cells treated with FCF, which was accompanied by a decrease in co-localization of ErbB2 with septins at the membrane. Cathepsin B inhibitor, but not bafilomycin or lactacystin, prevented FCF-induced decrease in total ErbB2 by increasing accumulation of ubiquitylated ErbB2 in lysosomes. Therefore, septins protect ErbB2 from ubiquitylation, endocytosis and lysosomal degradation. The FCF-induced degradation pathway is distinct from and additive with the degradation induced by inhibiting ErbB2 chaperone Hsp90. These results identify septins as novel regulators of ErbB2 expression that contribute to the remarkable stabilization of the receptor at the plasma membrane of cancer cells and may provide a basis for the development of new ErbB2-targeting anti-cancer therapies.

The fluorescent styryl dye RH421 was used to identify and investigate electrogenic reaction steps of the H,K-ATPase pump cycle. Equilibrium titration experiments were performed with membrane vesicles isolated from hog gastric mucosa, and cytoplasmic and luminal binding of K(+) and H(+) ions was studied. It was found that the binding and release steps of both ion species in both principal conformations of the ion pump, E(1) and P-E(2), are electrogenic, whereas the conformation transitions do not contribute significantly to a charge movement within the membrane dielectric. This behavior is in agreement with the transport mechanism found for the Na,K-ATPase and the sarcoplasmic reticulum Ca-ATPase. The data were analyzed on the basis of the Post-Albers reaction cycle. For proton binding, two pK values were found in both conformations: 6.7 and/=4.5 in the E(1) conformation; 6.7 and/=2 in the P-E(2) conformation. The equilibrium dissociation constants for K(+) binding on the cytoplasmic side were 11 and 16 mM. The respective equilibrium dissociation constants on the luminal side were obtained via K(+) concentration dependence of the enzyme activity and determined to be 0.11 mM for both luminal binding sites.

The gastric H,K-ATPase and the Na,K-ATPase both are stimulated by luminal K(+), but differ in sensitivity to K(+)-competitive inhibitors (ouabain and SCH28080), which implies a difference in structure near the luminal ion pathways in these two pumps. Knowledge of the amino acids in the H,K-ATPase that affect the mode of inhibition by SCH28080 and inhibitor affinity should provide insight into the regions of the membrane domain influencing the inhibitor selectivity and the luminal route to the ion transport site. Mutational scans in M4, 5, 6, and 8 have shown that amino acid residues affecting ion affinity (E343, K791, E795, E820, D824, E936) with either no or a lesser effect on the inhibitor affinity are located in the middle of the membrane domain. The residues significantly reducing inhibitor affinity, but not ion affinity (L809, P810, L811, T813, I816, Y925, T929), are located in the exoplasmic 5-6 loop and the luminal ends of M6 and M8. This suggests that the binding domain for SCH28080 contains the surface between L809 in the 5-6 loop and C813 at the luminal end of M6, approximately two helical turns out from the ion binding region, where it blocks an ion access pathway. The mutations that change inhibitor kinetics are on the opposing faces of M6 and M8 and apparently modify the normal ion pathway or, perhaps, create an alternate ion pathway.

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. The involvement of the ubiquitously expressed septin-2 or general septin polymerization in exocytosis has not been explored to date. Here, by nano-LC with tandem MS and immunoblot analyses of the septin-2 interactome in mouse brain, we identified not only SNARE proteins but also Munc-18-1 (stabilizes assembled SNARE complexes), N-ethylmaleimide-sensitive factor (NSF) (disassembles SNARE complexes after each membrane fusion event), and the chaperones Hsc70 and synucleins (maintain functional conformation of SNARE proteins after complex disassembly). Importantly, α-soluble NSF attachment protein (SNAP), the adaptor protein that mediates NSF binding to the SNARE complex, did not interact with septin-2, indicating that septins undergo reorganization during each exocytosis cycle. Partial depletion of septin-2 by siRNA or impairment of septin dynamics by forchlorfenuron inhibited constitutive and stimulated exocytosis of secreted and transmembrane proteins in various cell types. Forchlorfenuron impaired the interaction between SNAP-25 and its chaperone Hsc70, decreasing SNAP-25 levels in cultured neuroendocrine cells, and inhibited both spontaneous and stimulated acetylcholine secretion in mouse motor neurons. The results demonstrate a stimulatory role of septin-2 and the dynamic reorganization of septin oligomers in exocytosis.

Renal tissue hypoxia is a major cause of acute renal failure. The goal of this study was to elucidate the mechanism(s) underlying role of the Na,K-ATPase in the adaptive response of renal cells to a hypoxic stress. Kidney microsome membranes were isolated from mice exposed to 7% O2 (hypoxia) or to room air for three days, proteins were extracted by a non-ionic detergent, immunoprecipitated using anti-Na,K-ATPase antibodies, and analyzed by nLC-MS/MS and Western blot analyses. Hypoxia did not change the total amount of the Na,K-ATPase, as was assessed by comparing the Mascot scores and confirmed by a Western blot analysis. In contrast, hypoxia significantly decreased the amount of the Na,K-ATPase-co-immunoprecipitated ER chaperones and enzymes presumably involved in the maturation of the Na,K-ATPase, including BiP, Hsp40, calnexin, PDI, glucosidase, mannosidase and DnaJ. Consistent with these results, a significant retention of the Na,K-ATPase was observed in cultured epithelial cells exposed to hypoxia. T...

The gastric H,K-ATPase is inhibited selectively and K(+)-competitively from its luminal surface by protonated imidazo[1,2alpha]pyridines (e.g., SCH28080). Identification of the amino acids in the membrane domain that affect SCH28080 inhibition should provide a template for modeling a luminally directed vestibule in this enzyme, based on the crystal structure of the sr Ca-ATPase. Five conserved carboxylic residues, Glu343, Glu795, Glu820, Asp824, Glu936, and unique Lys791 in the H,K-ATPase were mutated, and the effects of mutations on the K(i) for SCH28080, V(max), and K(m,app)[NH(4)(+)] were measured. A kinetic analysis of the ATP hydrolysis data indicated that all of these residues significantly affect the interaction of NH(4)(+) ions with the protein but only three of them, Glu795, Glu936, and Lys791, greatly affected SCH28080 inhibition. A Glu795Asp mutation increased the K(i) from 64 +/- 11 to 700 +/- 110 nM. Since, however, the mutation Glu795Gln did not change the K(i) (86 +/- 31 nM), this site has a significant spatial effect on inhibitor kinetics. A Glu936Asp mutation resulted in noncompetitive kinetics while Gln substitution had no effect either on inhibitor affinity or on the nature of the kinetics, suggesting that the length of the Glu936 side chain is critical for the exclusive binding of the ion and SCH28080. Mutation of Lys791 to Ser, the residue present in the SCH28080-insensitive Na,K-ATPase, resulted in a 20-fold decrease in SCH28080 affinity, suggesting an important role of this residue in SCH28080 selectivity of the H,K-ATPase versus Na,K-ATPase. Mutations of Asp824, Glu343, and Glu820 increased the K(i) 2-3-fold, implying a relatively minor role for these residues in SCH28080 inhibition. It appears that the imidazopyridine moiety of SCH28080 in the protonated state interacts with residues near the negatively charged residues of the empty ion site from the luminal side (TM4, -5, -6, and -8) while the hydrophobic phenyl ring interacts with TM1 or TM2 (the latter conclusion based on previous data from photoaffinity labeling). The integrity of the SCH28080 binding site depends on the presence of Lys791, Glu936, and Glu795 in H,K-ATPase. A computer-generated model of this region illustrates the possible involvement of the residues previously shown to affect SCH28080 inhibition (Cys813, Ile816, Thr823, Met334, Val337) and may predict other residues that line the SCH28080 binding vestibule in the E(2) conformation of the pump.

This review focuses on the gastric acid pump as a therapeutic target for the control of acid secretion in peptic ulcer and gastro-oesophageal reflux disease. The mechanism of the proton pump inhibitors is discussed as well as their clinical use. The biology of Helicobacter pylori as a gastric denizen is then discussed, with special regard to its mechanisms of acid resistance. Here the properties of the products of the urease gene clusters, ureA, B and ureI, E, F, G and H are explored in order to explain the unique location of this pathogen. The dominant requirement for acid resistance is the presence of a proton gated urea transporter, UreI, which increases access of gastric juice urea to the intrabacterial urease 300-fold. This enables rapid and continuous buffering of the bacterial periplasm to approximately pH 6.0, allowing acid resistance and growth at acidic pH in the presence of 1 mM urea. A hypothesis for the basis of combination therapy for eradication is also presented.

The gastric H,K-ATPase is covalently inhibited by substituted pyridyl-methylsulfinyl-benzimidazoles, such as omeprazole, that convert to thiophilic probes of luminally accessible cysteines in the acid space. The K(+) competitive inhibitor, SCH28080, prevented inhibition of acid transport by omeprazole. In stably expressing HEK293 cells, the benzimidazole-reactive cysteines, Cys-321 (transmembrane helix (TM) 3), Cys-813 and Cys-822 (TM5/6), and Cys-892 (TM7/8) were mutated to the amino acids found in the SCH28080-resistant Na,K-ATPase and kinetic parameters of H,K-ATPase activity analyzed. Mutations of Cys-822 and Cys-892 had insignificant effects on the K(i(app)), K(m(app)) or V(max), but mutations of Cys-813 to threonine and Cys-321 to alanine decreased the affinity for SCH28080. Mutation of Cys-321 to alanine produced mixed kinetics of inhibition, still with higher affinity for the cation-free form of phosphoenzyme. Since the phenylmethoxy ring of the imidazo-pyridine inhibitors binds to TM1/2, as shown by earlier photoaffinity studies, and the mutations in TM6 (Cys-813 --Thr) as well as the end of TM3 (Cys-321 --Ala) decrease the affinity for SCH28080, the TM1/2, TM3, and TM6 helices lie within approximately 16 A of each other based on the size of the active, extended conformation of SCH28080.

Proteolytic cleavage of synaptosomal-associated protein 25 by the light chain of Botulinum neurotoxin type A (LCA), resulting in a blockade of neurotransmitter release, persists for several months in motor neurons. The L428A/L429A mutation in LCA is known to significantly shorten both 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. Mass spectrometry analysis of the LCA interactome showed that the mutation prevented the interaction of LCA with septins. The wild type LCA was concentrated in plasma membrane-associated clusters, co-localizing with septins-2 and septin-7, which accumulated in these clusters only in the presence of LCA. The L428A/L429A mutation decreased co-clustering of LCA and septins and accelerated proteasomal and non-proteasomal degradation of LCA. Similarly, the impairment of septin oligomerization by forchlorfenuron or silencing of septin-2 prevented LCA interaction and clustering with septins and increased LCA degradation. Therefore, the dileucine-mediated LCA-septin co-clustering is crucial for the long-lasting stabilization of LCA-related proteolytic and presumably neuroparalytic activity.

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. These exposed residues were mutated in both dog and rat YFP-linked β(1) subunits (YFP-β(1)) and also in the secreted extracellular domain of the dog β(1) subunit. Five rat-like mutations in the amino acid region spanning residues 198-207 of the dog YFP-β(1) expressed in Madin-Darby canine kidney (MDCK) cells decreased co-precipitation of the endogenous dog β(1) subunit with YFP-β(1) to the level observed between dog β(1) and rat YFP-β(1). In parallel, these mutations impaired the recognition of YFP-β(1) by the dog-specific antibody that inhibits cell adhesion between MDCK cells. Accordingly, dog-like mutations in rat YFP-β(1) increased both the (YFP-β(1))-β(1) interaction in MDCK cells and recognition by the antibody. Conversely, rat-like mutations in the secreted extracellular domain of the dog β(1) subunit increased its interaction with rat YFP-β(1) in vitro. In addition, these mutations resulted in a reduction of intercellular adhesion between rat lung epithelial cells following addition of the secreted extracellular domain of the dog β(1) subunit to a cell suspension. Therefore, the amino acid region 198-207 is crucial for both trans-dimerization of the Na(+)/K(+)-ATPase β(1) subunits and cell-cell adhesion.

The ion gradients generated by the Na-K-ATPase play a critical role in epithelia by driving transepithelial transport of various solutes. The efficiency of this Na-K-ATPase-driven vectorial transport depends on the integrity of epithelial junctions that maintain polar distribution of membrane transporters, including the basolateral sodium pump, and restrict paracellular diffusion of solutes. The review summarizes the data showing that, in addition to pumping ions, the Na-K-ATPase located at the sites of cell-cell junction acts as a cell adhesion molecule by interacting with the Na-K-ATPase of the adjacent cell in the intercellular space accompanied by anchoring to the cytoskeleton in the cytoplasm. The review also discusses the experimental evidence on the importance of a specific amino acid region in the extracellular domain of the Na-K-ATPase β1subunit for the Na-K-ATPase trans-dimerization and intercellular adhesion. Furthermore, a possible role of N-glycans linked to the Na-K-ATPase β1subunit in regulation of epithelial junctions by modulating β1-β1interactions is discussed.

Epithelial junctions depend on intercellular interactions between β1 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 β1 subunits, indicating the importance of species-specific regions for β1–β1 binding. To identify these regions, the species-specific amino acid residues were mapped on a high-resolution structure of the Na+/K+-ATPase β1 subunit to select those exposed towards the β1 subunit of the neighboring cell. These exposed residues were mutated in both dog and rat YFP-linked β1 subunits (YFP–β1) and also in the secreted extracellular domain of the dog β1 subunit. Five rat-like mutations in the amino acid region spanning residues 198–207 of the dog YFP–β1 expressed in Madin–Darby canine kidney (MDCK) cells decreased co-precipitation of the endogenous dog β1 subunit with YFP–β1 to the level observed between dog β1 and rat YFP–β1. In parallel, these mutations impaired the recognition of YFP–β1 by the dog-specific antibody that inhibits cell adhesion between MDCK cells. Accordingly, dog-like mutations in rat YFP–β1 increased both the (YFP–β1)–β1 interaction in MDCK cells and recognition by the antibody. Conversely, rat-like mutations in the secreted extracellular domain of the dog β1 subunit increased its interaction with rat YFP–β1 in vitro. In addition, these mutations resulted in a reduction of intercellular adhesion between rat lung epithelial cells following addition of the secreted extracellular domain of the dog β1 subunit to a cell suspension. Therefore, the amino acid region 198–207 is crucial for both trans-dimerization of the Na+/K+-ATPase β1 subunits and cell–cell adhesion.