The Experts below are selected from a list of 291 Experts worldwide ranked by ideXlab platform
Franz X Heinz - One of the best experts on this subject based on the ideXlab platform.
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Proteolytic Activation of tick borne encephalitis virus by furin
Journal of Virology, 1997Co-Authors: Konrad Stadler, Steven L Allison, Juliane Schalich, Franz X HeinzAbstract:Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological Activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pHinduced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage Activation of flaviviruses. The surface glycoproteins of many enveloped viruses are initially synthesized as inactive precursors, and Proteolytic cleavage is often required for maturation and full functional activity. In several virus families, this processing step is carried out by cellular proprotein convertases (reviewed in reference 16), most commonly furin, a component of the constitutive secretory pathway of many different types of cells (7, 35). Furin is a membrane-bound, calcium-dependent subtilisin-like protease whose primary site of action is the trans-Golgi network (TGN), although cycling of furin between the exocytic and endocytic pathways and the plasma membrane has also been demonstrated (4, 21). This enzyme is also secreted from cells in an active soluble form which is produced by self-cleavage in the TGN (39, 41).
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Proteolytic Activation of tick borne encephalitis virus by furin
Journal of Virology, 1997Co-Authors: Konrad Stadler, Steven L Allison, Juliane Schalich, Franz X HeinzAbstract:Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological Activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pH-induced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage Activation of flaviviruses.
Ryuichiro Sato - One of the best experts on this subject based on the ideXlab platform.
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piperine induces hepatic low density lipoprotein receptor expression through Proteolytic Activation of sterol regulatory element binding proteins
PLOS ONE, 2015Co-Authors: Ayasa Ochiai, Jun Inoue, Makoto Shimizu, Shingo Miyata, Ryuichiro SatoAbstract:Elevated plasma low-density lipoprotein (LDL) cholesterol is considered as a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) uptakes plasma lipoproteins and lowers plasma LDL cholesterol, the Activation of LDLR is a promising drug target for atherosclerosis. In the present study, we identified the naturally occurring alkaloid piperine, as an inducer of LDLR gene expression by screening the effectors of human LDLR promoter. The treatment of HepG2 cells with piperine increased LDLR expression at mRNA and protein levels and stimulated LDL uptake. Subsequent luciferase reporter gene assays revealed that the mutation of sterol regulatory element-binding protein (SREBP)-binding element abolished the piperine-mediated induction of LDLR promoter activity. Further, piperine treatments increased mRNA levels of several SREBP targets and mature forms of SREBPs. However, the piperine-mediated induction of the mature forms of SREBPs was not observed in SRD–15 cells, which lack insulin-induced gene–1 (Insig–1) and Insig–2. Finally, the knockdown of SREBPs completely abolished the piperine-meditated induction of LDLR gene expression in HepG2 cells, indicating that piperine stimulates the Proteolytic Activation of SREBP and subsequent induction of LDLR expression and activity.
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Resveratrol increases the expression and activity of the low density lipoprotein receptor in hepatocytes by the Proteolytic Activation of the sterol regulatory element-binding proteins
Atherosclerosis, 2011Co-Authors: Takuya Yashiro, Jun Inoue, Makoto Shimizu, Manami Nanmoku, Ryuichiro SatoAbstract:Abstract Objective The hepatocyte low density lipoprotein receptor (LDLR) plays a pivotal role in lipoprotein metabolism by lowering plasma LDL-cholesterol, a risk factor for atherosclerosis. The present study was conducted to investigate the effects of grape polyphenols on LDLR gene expression in human hepatocyte models. Methods and results Among the 14 phenolic compounds in red wine, we found that a stilbene trans -resveratrol most strongly up-regulated LDLR gene expression in HepG2 cells. Trans -resveratrol increased the LDLR protein and uptake of fluorescent-labeled LDL. Moreover, it enhanced LDLR gene promoter activity through the Proteolytic Activation of the sterol regulatory element-binding protein-2 (SREBP-2) as well as SREBP-1. However, sterols completely abolished trans -resveratrol-induced SREBP Activation and LDLR gene expression. Finally, AMP-activated protein kinase (AMPK) knockdown analyses by siRNA revealed that AMPK Activation was unnecessary for the effects of trans -resveratrol. Conclusions Trans -resveratrol up-regulated hepatic LDLR expression via Proteolytic Activation of SREBPs. We concluded that trans -resveratrol exhibits the anti-atherogenic effect, at least in part, by increased hepatic LDLR expression and subsequent LDL uptake.
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Proteolytic Activation of SREBPs during adipocyte differentiation.
Biochemical and biophysical research communications, 2001Co-Authors: Jun Inoue, Hidetoshi Kumagai, Tomoyuki Terada, Masatomo Maeda, Makoto Shimizu, Ryuichiro SatoAbstract:A member of sterol regulatory element-binding protein (SREBP) family, SREBP-1, is a key regulator of adipocyte differentiation. Expression of the SREBP-1 gene is induced during adipocyte differentiation, but Proteolytic Activation of the synthesized precursor form of SREBP-1 has not been well analyzed. The Proteolytic processing of SREBPs is severely suppressed in sterol loaded culture cells. Here we report that a splicing isoform, SREBP-1a, is predominantly expressed in 3T3-L1 preadipocytes and adipocytes, and that the nuclear active form of SREBP-1 protein increases in adipocyte differentiation. We further show that the amount of nuclear SREBP-2 protein also increases despite no increase in SREBP-2 mRNA, suggesting that Proteolytic cleavage of SREBPs is induced in lipid loaded adipocytes. Northern blot analyses reveal that mRNA levels for SREBP cleavage-activating protein (SCAP), Site-1 protease (S1P), and Site-2 protease (S2P), which participate in the Proteolytic processing of SREBPs, are relatively unaffected in adipogenesis. These results demonstrate that SREBP-2 appears to promote adipocyte differentiation as well as SREBP-1 and that the Proteolytic Activation of SREBPs may be induced by an as-yet unidentified mechanism in lipid loaded adipocytes.
Anumantha G. Kanthasamy - One of the best experts on this subject based on the ideXlab platform.
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Role of Proteolytic Activation of protein kinase Cδ in the pathogenesis of prion disease.
Prion, 2014Co-Authors: Dilshan S. Harischandra, Vellareddy Anantharam, Arthi Kanthasamy, Naveen Kondru, Dustin P. Martin, Huajun Jin, Anumantha G. KanthasamyAbstract:Prion diseases are infectious and inevitably fatal neurodegenerative diseases characterized by prion replication, widespread protein aggregation and spongiform degeneration of major brain regions controlling motor function. Oxidative stress has been implicated in prion-related neuronal degeneration, but the molecular mechanisms underlying prion-induced oxidative damage are not well understood. In this study, we evaluated the role of oxidative stress-sensitive, pro-apoptotic protein kinase Cδ (PKCδ) in prion-induced neuronal cell death using cerebellar organotypic slice cultures (COSC) and mouse models of prion diseases. We found a significant upregulation of PKCδ in RML scrapie-infected COSC, as evidenced by increased levels of both PKCδ protein and its mRNA. We also found an enhanced regulatory phosphorylation of PKCδ at its two regulatory sites, Thr505 in the Activation loop and Tyr311 at the caspase-3 cleavage site. The prion infection also induced Proteolytic Activation of PKCδ in our COSC model. Immu...
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Tyrosine Phosphorylation Regulates the Proteolytic Activation of Protein Kinase Cδ in Dopaminergic Neuronal Cells
The Journal of biological chemistry, 2005Co-Authors: Siddharth Kaul, Vellareddy Anantharam, Arthi Kanthasamy, Yongjie Yang, Christopher J. Choi, Anumantha G. KanthasamyAbstract:Oxidative stress is a key apoptotic stimulus in neuronal cell death and has been implicated in the pathogenesis of many neurodegenerative disorders, including Parkinson disease (PD). Recently, we demonstrated that protein kinase C-delta (PKCdelta) is an oxidative stress-sensitive kinase that can be activated by caspase-3-dependent Proteolytic cleavage to induce apoptotic cell death in cell culture models of Parkinson disease (Kaul, S., Kanthasamy, A., Kitazawa, M., Anantharam, V., and Kanthasamy, A. G. (2003) Eur. J. Neurosci. 18, 1387-1401 and Kanthasamy, A. G., Kitazawa, M., Kanthasamy, A., and Anantharam, V. (2003) Antioxid. Redox. Signal. 5, 609-620). Here we showed that the phosphorylation of a tyrosine residue in PKCdelta can regulate the Proteolytic Activation of the kinase during oxidative stress, which consequently influences the apoptotic cell death in dopaminergic neuronal cells. Exposure of a mesencephalic dopaminergic neuronal cell line (N27 cells) to H(2)O(2)(0-300 microm) induced a dose-dependent increase in cytotoxicity, caspase-3 Activation and PKCdelta cleavage. H(2)O(2)-induced Proteolytic Activation of PKC was delta mediated by the Activation of caspase-3. Most interestingly, both the general Src tyrosine kinase inhibitor genistein (25 microm) and the p60(Src) tyrosine-specific kinase inhibitor (TSKI; 5 microm) dramatically inhibited H(2)O(2) and the Parkinsonian toxin 1-methyl-4-phenylpyridinium-induced PKCdelta cleavage, kinase Activation, and apoptotic cell death. H(2)O(2) treatment also increased phosphorylation of PKCdelta at tyrosine site 311, which was effectively blocked by co-treatment with TSKI. Furthermore, N27 cells overexpressing a PKCdelta(Y311F) mutant protein exhibited resistance to H(2)O(2)-induced PKCdelta cleavage, caspase Activation, and apoptosis. To our knowledge, these data demonstrate for the first time that phosphorylation of Tyr-311 on PKCdelta can regulate the Proteolytic Activation and proapoptotic function of the kinase in dopaminergic neuronal cells.
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Blockade of PKCδ Proteolytic Activation by Loss of Function Mutants Rescues Mesencephalic Dopaminergic Neurons from Methylcyclopentadienyl Manganese Tricarbonyl (MMT)‐Induced Apoptotic Cell Death
Annals of the New York Academy of Sciences, 2004Co-Authors: Vellareddy Anantharam, Masashi Kitazawa, Arthi Kanthasamy, C. Latchoumycandane, Anumantha G. KanthasamyAbstract:The use of methylcyclopentadienyl manganese tricarbonyl (MMT) as a gasoline additive has raised health concerns and increased interest in understanding the neurotoxic effects of manganese. Chronic exposure to inorganic manganese causes Manganism, a neurological disorder somewhat similar to Parkinson's disease. However, the cellular mechanism by which MMT, an organic manganese compound, induces neurotoxicity in dopaminergic neuronal cells remains unclear. Therefore, we systematically investigated apoptotic cell-signaling events following exposure to 3-200 microM MMT in mesencephalic dopaminergic neuronal (N27) cells. MMT treatment resulted in a time- and dose-dependent increase in reactive oxygen species generation and cell death in N27 cells. The cell death was preceded by sequential Activation of mitochondrial-dependent proapoptotic events including cytochrome c release, caspase-3 Activation, and DNA fragmentation, indicating that the mitochondrial-dependent apoptotic cascade primarily triggers MMT-induced apoptotic cell death. Importantly, MMT induced Proteolytic cleavage of protein kinase Cdelta (PKCdelta), resulting in persistently increased kinase activity. The Proteolytic Activation of PKCdelta was suppressed by treatment with 100 microM Z-VAD-FMK and 100 microM Z-DEVD-FMK, suggesting that caspase-3 mediates the Proteolytic Activation of PKCdelta. Pretreatment with 100 microM Z-DEVD-FMK and 5 microM rottlerin (a PKCdelta inhibitor) also significantly attenuated MMT-induced DNA fragmentation. Furthermore, overexpression of either the kinase inactive dominant negative PKCdelta(K376R) mutant or the caspase cleavage resistant PKCdelta(D327A) mutant rescued N27 cells from MMT-induced DNA fragmentation. Collectively, these results demonstrate that the mitochondrial-dependent apoptotic cascade mediates apoptosis via Proteolytic Activation of PKCdelta in MMT-induced dopaminergic degeneration and suggest that PKCdelta may serve as an attractive therapeutic target in Parkinson-related neurological diseases.
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Suppression of caspase-3-dependent Proteolytic Activation of protein kinase Cδ by small interfering RNA prevents MPP+-induced dopaminergic degeneration
Molecular and cellular neurosciences, 2004Co-Authors: Yongjie Yang, Siddharth Kaul, Vellareddy Anantharam, Danhui Zhang, Anumantha G. KanthasamyAbstract:Abstract The cellular mechanisms underlying the neurodegenerative process in Parkinson's disease are not well understood. Using RNA interference (RNAi), we demonstrate that caspase-3-dependent Proteolytic Activation of protein kinase Cδ (PKCδ) contributes to the degenerative process in dopaminergic neurons. The Parkinsonian toxin MPP + activated caspase-3 and Proteolytically cleaved PKCδ into catalytic and regulatory subunits, resulting in persistent kinase Activation in mesencephalic dopaminergic neuronal cells. The caspase-3 inhibitor Z-DEVD-FMK and the caspase-9 inhibitor Z-LEHD-FMK effectively blocked MPP + -induced PKCδ Proteolytic Activation. To characterize the functional role of PKCδ Activation in MPP + -induced dopaminergic cell death, RNAi-mediated gene knockdown was performed. Among four siRNAs designed against PKCδ, two specifically suppressed PKCδ expression. The application of siRNA abolished the MPP + -induced PKCδ Activation, DNA fragmentation, and tyrosine hydroxylase (TH)-positive neuronal loss. Together, these results suggest that Proteolytic Activation of PKCδ may be a critical downstream event in the degenerative process of Parkinson's disease.
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caspase 3 dependent Proteolytic Activation of protein kinase cδ mediates and regulates 1 methyl 4 phenylpyridinium mpp induced apoptotic cell death in dopaminergic cells relevance to oxidative stress in dopaminergic degeneration
European Journal of Neuroscience, 2003Co-Authors: Siddharth Kaul, Vellareddy Anantharam, Masashi Kitazawa, Arthi Kanthasamy, Anumantha G. KanthasamyAbstract:1-Methyl-4-phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine), induces apoptosis in dopaminergic neurons; however, the cellular mechanisms underlying the degenerative process are not well understood. In the present study, we demonstrate that caspase-3 mediated Proteolytic Activation of protein kinase C delta (PKC delta) is critical in MPP+-induced oxidative stress and apoptosis. MPP+ exposure in rat dopaminergic neuronal cells resulted in time-dependent increases in reactive oxygen species generation, cytochrome c release, and caspase-9 and caspase-3 Activation. Interestingly, MPP+ induced Proteolytic cleavage of PKC delta (72-74 kDa) into a 41-kDa catalytic and a 38-kDa regulatory subunit, resulting in persistently increased kinase activity. The caspase-3 inhibitor Z-DEVD-fmk effectively blocked MPP+-induced PKC delta cleavage and kinase activity, suggesting that the Proteolytic Activation is caspase-3 mediated. Similar results were seen in MPP+-treated rat midbrain slices. Z-DEVD-fmk and the PKC delta specific inhibitor rottlerin almost completely blocked MPP+-induced DNA fragmentation. The superoxide dismutase mimetic, MnTBAP also effectively attenuated MPP+-induced caspase-3 Activation, PKC delta cleavage, and DNA fragmentation. Furthermore, rottlerin attenuated MPP+-induced caspase-3 activity without affecting basal activity, suggesting positive feedback Activation of caspase-3 by PKC delta. Intracellular delivery of catalytically active recombinant PKC delta significantly increased caspase-3 activity, further indicating that PKC delta regulates caspase-3 activity. Finally, over-expression of a kinase inactive PKC delta K376R mutant prevented MPP+-induced caspase Activation and DNA fragmentation, confirming the pro-apoptotic function of PKC delta in dopaminergic cell death. Together, we demonstrate for the first time that MPP+-induced oxidative stress Proteolytically activates PKC delta in a caspase-3-dependent manner to induce apoptosis and up-regulate the caspase cascade in dopaminergic neuronal cells.
Konrad Stadler - One of the best experts on this subject based on the ideXlab platform.
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Proteolytic Activation of tick borne encephalitis virus by furin
Journal of Virology, 1997Co-Authors: Konrad Stadler, Steven L Allison, Juliane Schalich, Franz X HeinzAbstract:Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological Activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pHinduced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage Activation of flaviviruses. The surface glycoproteins of many enveloped viruses are initially synthesized as inactive precursors, and Proteolytic cleavage is often required for maturation and full functional activity. In several virus families, this processing step is carried out by cellular proprotein convertases (reviewed in reference 16), most commonly furin, a component of the constitutive secretory pathway of many different types of cells (7, 35). Furin is a membrane-bound, calcium-dependent subtilisin-like protease whose primary site of action is the trans-Golgi network (TGN), although cycling of furin between the exocytic and endocytic pathways and the plasma membrane has also been demonstrated (4, 21). This enzyme is also secreted from cells in an active soluble form which is produced by self-cleavage in the TGN (39, 41).
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Proteolytic Activation of tick borne encephalitis virus by furin
Journal of Virology, 1997Co-Authors: Konrad Stadler, Steven L Allison, Juliane Schalich, Franz X HeinzAbstract:Flaviviruses are assembled intracellularly in an immature form containing heterodimers of two envelope proteins, E and prM. Shortly before the virion exits the cell, prM is cleaved by a cellular enzyme, and this processing step can be blocked by treatment with agents that raise the pH of exocytic compartments. We carried out in vivo and in vitro studies with tick-borne encephalitis (TBE) virus to investigate the possible role of furin in this process as well as the functional consequences of prM cleavage. We found that prM in immature virions can be correctly cleaved in vitro by recombinant bovine furin but that efficient cleavage occurs only after exposure of the virion to mildly acidic pH. The data suggest that exposure to an acidic environment induces an irreversible structural change that renders the cleavage site accessible to the enzyme. Cleavage by furin in vitro resulted in biological Activation, as shown by a 100-fold increase in specific infectivity, the acquisition of membrane fusion and hemagglutination activity, and the ability of the envelope proteins to undergo low-pH-induced structural rearrangements characteristic of mature virions. In vivo, prM cleavage was blocked by a furin inhibitor, and infection of the furin-deficient cell line LoVo yielded only immature virions, suggesting that furin is essential for cleavage Activation of flaviviruses.
Silke Haerteis - One of the best experts on this subject based on the ideXlab platform.
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Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments
Journal of visualized experiments : JoVE, 2014Co-Authors: Matteus Krappitz, Christoph Korbmacher, Silke HaerteisAbstract:The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic Activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel’s γ-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (ΔIami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of γENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of Proteolytic Activation of ENaC-mediated whole-cell currents correlates with the appearance of a γENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel Activation. Moreover, they confirm the concept that a cleavage event in γENaC is required as a final step in Proteolytic channel Activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.
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Proteolytic Activation of the human epithelial sodium channel by trypsin iv and trypsin i involves distinct cleavage sites
Journal of Biological Chemistry, 2014Co-Authors: Silke Haerteis, Annabel Krappitz, Matteus Krappitz, Jane E Murphy, Marko Bertog, Bettina Krueger, Regina Nacken, Hyunjae Chung, Morley D Hollenberg, Wolfgang KnechtAbstract:Proteolytic Activation is a unique feature of the epithelial sodium channel (ENaC). However, the underlying molecular mechanisms and the physiologically relevant proteases remain to be identified. The serine protease trypsin I can activate ENaC in vitro but is unlikely to be the physiologically relevant activating protease in ENaC-expressing tissues in vivo. Herein, we investigated whether human trypsin IV, a form of trypsin that is co-expressed in several extrapancreatic epithelial cells with ENaC, can activate human ENaC. In Xenopus laevis oocytes, we monitored Proteolytic Activation of ENaC currents and the appearance of γENaC cleavage products at the cell surface. We demonstrated that trypsin IV and trypsin I can stimulate ENaC heterologously expressed in oocytes. ENaC cleavage and Activation by trypsin IV but not by trypsin I required a critical cleavage site (Lys-189) in the extracellular domain of the γ-subunit. In contrast, channel Activation by trypsin I was prevented by mutating three putative cleavage sites (Lys-168, Lys-170, and Arg-172) in addition to mutating previously described prostasin (RKRK178), plasmin (Lys-189), and neutrophil elastase (Val-182 and Val-193) sites. Moreover, we found that trypsin IV is expressed in human renal epithelial cells and can increase ENaC-mediated sodium transport in cultured human airway epithelial cells. Thus, trypsin IV may regulate ENaC function in epithelial tissues. Our results show, for the first time, that trypsin IV can stimulate ENaC and that trypsin IV and trypsin I activate ENaC by cleavage at distinct sites. The presence of distinct cleavage sites may be important for ENaC regulation by tissue-specific proteases.
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Proteolytic Activation of the epithelial sodium channel enac by the cysteine protease cathepsin s
Pflügers Archiv: European Journal of Physiology, 2012Co-Authors: Silke Haerteis, Annabel Krappitz, Matteus Krappitz, Jane E Murphy, Marko Bertog, Vera Baraznenok, Ian Henderson, Erik Lindstrom, Nigel W Bunnett, Christoph KorbmacherAbstract:Proteolytic processing of the amiloride-sensitive epithelial sodium channel (ENaC) by serine proteases is known to be important for channel Activation. Inappropriate ENaC Activation by proteases may contribute to the pathophysiology of cystic fibrosis and could be involved in sodium retention and the pathogenesis of arterial hypertension in the context of renal disease. We hypothesized that in addition to serine proteases, cathepsin proteases may activate ENaC. Cathepsin proteases belong to the group of cysteine proteases and play a pathophysiological role in inflammatory diseases. Under pathophysiological conditions, cathepsin-S (Cat-S) may reach ENaC in the apical membrane of epithelial cells. The aim of this study was to investigate the effect of purified Cat-S on human ENaC heterologously expressed in Xenopus laevis oocytes and on ENaC-mediated sodium transport in cultured M-1 mouse renal collecting duct cells. We demonstrated that Cat-S activates amiloride-sensitive whole-cell currents in ENaC-expressing oocytes. The stimulatory effect of Cat-S was preserved at pH 5. ENaC stimulation by Cat-S was associated with the appearance of a γENaC cleavage fragment at the plasma membrane indicating Proteolytic channel Activation. Mutating two valine residues (V182 and V193) in the critical region of γENaC prevented Proteolytic Activation of ENaC by Cat-S. Pre-incubation of the oocytes with the Cat-S inhibitor morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS) prevented the stimulatory effect of Cat-S on ENaC. In contrast, LHVS had no effect on ENaC Activation by the prototypical serine proteases trypsin and chymotrypsin. Cat-S also stimulated ENaC in differentiated renal epithelial cells. These findings demonstrate that the cysteine protease Cat-S can activate ENaC which may be relevant under pathophysiological conditions.
