The Experts below are selected from a list of 20604 Experts worldwide ranked by ideXlab platform
Rebecca Ellis Dutch - One of the best experts on this subject based on the ideXlab platform.
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a mature and fusogenic form of the nipah virus fusion protein requires Proteolytic Processing by cathepsin l
Virology, 2006Co-Authors: Cara T Pager, Willie Warren Craft, Jared R Patch, Rebecca Ellis DutchAbstract:The Nipah virus fusion (F) protein is Proteolytically processed to F1 + F2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic Processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form.
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endocytosis plays a critical role in Proteolytic Processing of the hendra virus fusion protein
Journal of Virology, 2005Co-Authors: Kelly Ann Meulendyke, Mark A Wurth, Richard O Mccann, Rebecca Ellis DutchAbstract:The Hendra virus fusion (F) protein is synthesized as a precursor protein, F0, which is Proteolytically processed to the mature form, F1+F2. Unlike the case for the majority of paramyxovirus F proteins, the Processing event is furin independent, does not require the addition of exogenous proteases, is not affected by reductions in intracellular Ca2+, and is strongly affected by conditions that raise the intracellular pH (C. T. Pager, M. A. Wurth, and R. E. Dutch, J. Virol. 78:9154-9163, 2004). The Hendra virus F protein cytoplasmic tail contains a consensus motif for endocytosis, YXXΦ. To analyze the potential role of endocytosis in the Processing and membrane fusion promotion of the Hendra virus F protein, mutation of tyrosine 525 to alanine (Hendra virus F Y525A) or phenylalanine (Hendra virus F Y525F) was performed. The rate of endocytosis of Hendra virus F Y525A was significantly reduced compared to that of the wild-type (wt) F protein, confirming the functional importance of the endocytosis motif. An intermediate level of endocytosis was observed for Hendra virus F Y525F. Surprisingly, dramatic reductions in the rate of Proteolytic Processing were observed for Hendra virus F Y525A, although initial transport to the cell surface was not affected. The levels of surface expression for both Hendra virus F Y525A and Hendra virus F Y525F were higher than that of the wt protein, and these mutants displayed enhanced syncytium formation. These results suggest that endocytosis is critically important for Hendra virus F protein cleavage, representing a new paradigm for Proteolytic Processing of paramyxovirus F proteins.
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cathepsin l is involved in Proteolytic Processing of the hendra virus fusion protein
Journal of Virology, 2005Co-Authors: Cara T Pager, Rebecca Ellis DutchAbstract:Proteolytic Processing of paramyxovirus fusion (F) proteins is essential for the generation of a mature and fusogenic form of the F protein. Although many paramyxovirus F proteins are Proteolytically processed by the cellular protease furin at a multibasic cleavage motif, cleavage of the newly emerged Hendra virus F protein occurs by a previously unidentified cellular protease following a single lysine at residue 109. We demonstrate here that the cellular protease cathepsin L is involved in converting the Hendra virus precursor F protein (F 0 ) to the active F 1 + F 2 disulfide-linked heterodimer. To initially identify the class of protease involved in Hendra virus F protein cleavage, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F (known to be Proteolytically processed by furin) were metabolically labeled and chased in the absence or presence of serine, cysteine, aspartyl, and metalloprotease inhibitors. Nonspecific and specific protease inhibitors known to decrease cathepsin activity inhibited Proteolytic Processing of Hendra virus F but had no effect on simian virus 5 F Processing. We next designed shRNA oligonucleotides to cathepsin L which dramatically reduced cathepsin L protein expression and enzyme activity. Cathepsin L shRNA-expressing Vero cells transfected with pCAGGS-Hendra F demonstrated a nondetectable amount of cleavage of the Hendra virus F protein and significantly decreased membrane fusion activity. Additionally, we found that purified human cathepsin L processed immunopurified Hendra virus F 0 into F 1 and F 2 fragments. These studies introduce a novel mechanism for primary Proteolytic Processing of viral glycoproteins and also suggest a previously unreported biological role for cathepsin L.
Thomas Braulke - One of the best experts on this subject based on the ideXlab platform.
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Proteolytic Processing of the γ subunit is associated with the failure to form glcnac 1 phosphotransferase complexes and mannose 6 phosphate residues on lysosomal enzymes in human macrophages
Journal of Biological Chemistry, 2010Co-Authors: Sandra Pohl, Stephan Tiede, Katrin Marschner, Marisa Encarnacao, Monica Castrichini, Katrin Kollmann, Nicole Muschol, K Ullrich, Sven Mullerloennies, Thomas BraulkeAbstract:GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, alpha(2)beta(2)gamma(2), that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3-10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the gamma-subunit but not the beta-subunit was found to be Proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the gamma-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that Proteolytic Processing of the gamma-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.
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Proteolytic Processing of the γ subunit is associated with the failure to form glcnac 1 phosphotransferase complexes and mannose 6 phosphate residues on lysosomal enzymes in human macrophages
Journal of Biological Chemistry, 2010Co-Authors: Sandra Pohl, Stephan Tiede, Katrin Marschner, Marisa Encarnacao, Monica Castrichini, Katrin Kollmann, Nicole Muschol, K Ullrich, Sven Mullerloennies, Thomas BraulkeAbstract:GlcNAc-1-phosphotransferase is a Golgi-resident 540-kDa complex of three subunits, α2β2γ2, that catalyze the first step in the formation of the mannose 6-phosphate (M6P) recognition marker on lysosomal enzymes. Anti-M6P antibody analysis shows that human primary macrophages fail to generate M6P residues. Here we have explored the sorting and intracellular targeting of cathepsin D as a model, and the expression of the GlcNAc-1-phosphotransferase complex in macrophages. Newly synthesized cathepsin D is transported to lysosomes in an M6P-independent manner in association with membranes whereas the majority is secreted. Realtime PCR analysis revealed a 3–10-fold higher GlcNAc-1-phosphotransferase subunit mRNA levels in macrophages than in fibroblasts or HeLa cells. At the protein level, the γ-subunit but not the β-subunit was found to be Proteolytically cleaved into three fragments which form irregular 97-kDa disulfide-linked oligomers in macrophages. Size exclusion chromatography showed that the γ-subunit fragments lost the capability to assemble with other GlcNAc-1-phosphotransferase subunits to higher molecular complexes. These findings demonstrate that Proteolytic Processing of the γ-subunit represents a novel mechanism to regulate GlcNAc-1-phosphotransferase activity and the subsequent sorting of lysosomal enzymes.
Harald Steiner - One of the best experts on this subject based on the ideXlab platform.
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Proteolytic Processing of neuregulin 1 type iii by three intramembrane cleaving proteases
Journal of Biological Chemistry, 2016Co-Authors: Daniel Fleck, Matthias Voss, Ben Brankatschk, Camilla Giudici, Heike Hampel, Benjamin M Schwenk, Dieter Edbauer, Akio Fukumori, Harald SteinerAbstract:Numerous membrane-bound proteins undergo regulated intramembrane proteolysis. Regulated intramembrane proteolysis is initiated by shedding, and the remaining stubs are further processed by intramembrane-cleaving proteases (I-CLiPs). Neuregulin 1 type III (NRG1 type III) is a major physiological substrate of β-secretase (β-site amyloid precursor protein-cleaving enzyme 1 (BACE1)). BACE1-mediated cleavage is required to allow signaling of NRG1 type III. Because of the hairpin nature of NRG1 type III, two membrane-bound stubs with a type 1 and a type 2 orientation are generated by Proteolytic Processing. We demonstrate that these stubs are substrates for three I-CLiPs. The type 1-oriented stub is further cleaved by γ-secretase at an ϵ-like site five amino acids N-terminal to the C-terminal membrane anchor and at a γ-like site in the middle of the transmembrane domain. The ϵ-cleavage site is only one amino acid N-terminal to a Val/Leu substitution associated with schizophrenia. The mutation reduces generation of the NRG1 type III β-peptide as well as reverses signaling. Moreover, it affects the cleavage precision of γ-secretase at the γ-site similar to certain Alzheimer disease-associated mutations within the amyloid precursor protein. The type 2-oriented membrane-retained stub of NRG1 type III is further processed by signal peptide peptidase-like proteases SPPL2a and SPPL2b. Expression of catalytically inactive aspartate mutations as well as treatment with 2,2'-(2-oxo-1,3-propanediyl)bis[(phenylmethoxy)carbonyl]-l-leucyl-l-leucinamide ketone inhibits formation of N-terminal intracellular domains and the corresponding secreted C-peptide. Thus, NRG1 type III is the first protein substrate that is not only cleaved by multiple sheddases but is also processed by three different I-CLiPs.
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the biological and pathological function of the presenilin 1 deltaexon 9 mutation is independent of its defect to undergo Proteolytic Processing
Journal of Biological Chemistry, 1999Co-Authors: Harald Steiner, Ralf Baumeister, Helmut Romig, Melissa G Grim, Uwe Philipp, Brigitte Pesold, Martin Citron, Christian HaassAbstract:The two homologous presenilins are key factors for the generation of amyloid beta-peptide (Abeta), since Alzheimer's disease (AD)-associated mutations enhance the production of the pathologically relevant 42-amino acid Abeta (Abeta42), and a gene knockout of presenilin-1 (PS1) significantly inhibits total Abeta production. Presenilins undergo Proteolytic Processing within the domain encoded by exon 9, a process that may be closely related to their biological and pathological activity. An AD-associated mutation within the PS1 gene deletes exon 9 (PS1Deltaexon9) due to a splicing error and results in the accumulation of the uncleaved full-length protein. We now demonstrate the unexpected finding that the pathological activity of PS1Deltaexon9 is independent of its lack to undergo Proteolytic Processing, but is rather due to a point mutation (S290C) occurring at the aberrant exon 8/10 splice junction. Mutagenizing the cysteine residue at position 290 to the original serine residue completely inhibits the pathological activity in regard to the elevated production of Abeta42. Like PS1Deltaexon9, the resulting presenilin variant (PS1Deltaexon9 C290S) accumulates as an uncleaved protein and fully replaces endogenous presenilin fragments. Moreover, PS1Deltaexon9 C290S exhibits a significantly increased biological activity in a highly sensitive in vivo assay as compared with the AD-associated mutation. Therefore not only the increased Abeta42 production but also the decreased biological function of PS1Deltaexon9 is due to a point mutation and independent of the lack of Proteolytic Processing.
Julio Escribano - One of the best experts on this subject based on the ideXlab platform.
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bicarbonate dependent secretion and Proteolytic Processing of recombinant myocilin
PLOS ONE, 2013Co-Authors: Josedaniel Arocaaguilar, Miguel Cocaprados, Francisco Martinezredondo, Alba Martingil, Jesus Pintor, Julio EscribanoAbstract:Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular Proteolytic Processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the Proteolytic Processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc.) affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular Proteolytic Processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.
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functional role of Proteolytic Processing of recombinant myocilin in self aggregation
Investigative Ophthalmology & Visual Science, 2010Co-Authors: Josedaniel Arocaaguilar, Julio Escribano, Francisco Sanchezsanchez, Miguel Cocaprados, Francisco MartinezredondoAbstract:Myocilin is an extracellular glycoprotein present in muscular and ocular tissues such as iris, ciliary body (CB), and trabecular meshwork (TM).1–6 It forms large molecular aggregates in the aqueous humor (AH).7–9 Mutations in the myocilin (MYOC) gene cause autosomal dominant juvenile glaucoma, and they are also present in a reduced proportion (3%–5%) of adult-onset primary open-angle glaucoma (POAG) in different populations.10–12 POAG is a heterogeneous disease originated by the progressive apoptosis of the optic nerve,13 which constitutes a leading cause of blindness in developed countries. Myocilin presents a modular design composed of the N-terminal region (amino acids 1-202), the central linker domain (amino acids 202-244),14,15 and the compact16 C-terminal olfactomedin-like domain (amino acids 244-504).3 The N-terminal domain contains two coiled-coil (CC) domains17 and a leucine zipper-like motif3 in the second CC. These three modules are encoded by exons 1, 2, and 3 and coincide approximately with three independent folding domains.17 We have reported that recombinant myocilin undergoes an intracellular endoProteolytic cleavage by calpain II in the central linker domain of the polypeptide chain.15 The Processing takes place in the endoplasmic reticulum and releases two fragments that contain the N- and C-terminal structural domains. The C-terminal fragment has been detected in both human and bovine CB and the AH, indicating that the Proteolytic Processing also occurs in ocular tissues.15 Of note, photomedin-1 and gliomedin, two olfactomedin-like domain–containing proteins, are cleaved in a similar fashion.18,19 Pathogenic mutations located in the olfactomedin-like domain reduce the specific cleavage of the recombinant protein.15 Despite many efforts having being made since the discovery of MYOC as a glaucoma gene in 1997,10 the function of this protein in normal and glaucomatous eyes remains poorly understood. Similarly, the functional meaning of the Proteolytic Processing of myocilin is currently unknown, although it has been suggested to contribute to the modulation of myocilin interactions.15 In the present study, the specific Proteolytic cleavage of recombinant myocilin reduced its extracellular covalent aggregates. In addition, the results revealed the existence of noncovalent interactions between myocilin aggregates, which may play an important role in the extracellular function of the protein.
Thomas R Kleyman - One of the best experts on this subject based on the ideXlab platform.
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Proteolytic Processing of the epithelial sodium channel gamma subunit has a dominant role in channel activation
Journal of Biological Chemistry, 2008Co-Authors: Marcelo D Carattino, Rebecca P Hughey, Thomas R KleymanAbstract:Maturation of the epithelial sodium channel (ENaC) involves furin-dependent cleavage at two extracellular sites within the α subunit and at a single extracellular site within the γ subunit. Channels lacking furin Processing of the α subunit have very low activity. We recently identified a prostasin-dependent cleavage site (RKRK186) in the γ subunit. We also demonstrated that the tract α D206-R231, between the two furin cleavage sites in the α subunit, as well as the tract γ E144-K186, between the furin and prostasin cleavage sites in the γ subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of the α and γ subunit inhibitory tracts, respectively. We examined whether release of either theα orγ inhibitory tract has a dominant role in activating the channel. Co-expression of prostasin and either wild type channels or mutant channels lacking furin cleavage of the α subunit (αR205A,R208A,R231Aβγ) in Xenopus laevis oocytes led to increases in whole cell currents to similar levels. In an analogous manner and independent of the Proteolytic Processing of theα subunit, amiloride-sensitive currents in oocytes expressing channels carrying γ subunits with both a mutation in the furin cleavage site and a deletion of the inhibitory tract (αβγR143A,ΔE144-K186 and αR205A,R208A,R231AβγR143A, ΔE144-K186) were significantly higher than those from oocytes expressing wild type ENaC. When channels lacked the α and γ subunit inhibitory tracts, α subunit cleavage was required for channels to be fully active. Channels lacking both furin cleavage and the inhibitory tract in theγ subunit (αβγR143A,ΔE144-K186) showed a significant reduction in the efficacy of block by the syntheticα-26 inhibitory peptide representing the tract αD206-R231. Our data indicate that removal of the inhibitory tract from the γ subunit, in the absence ofα subunit cleavage, results in nearly full activation of the channel.
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Proteolytic Processing of the epithelial sodium channel γ subunit has a dominant role in channel activation
Journal of Biological Chemistry, 2008Co-Authors: Marcelo D Carattino, Rebecca P Hughey, Thomas R KleymanAbstract:Maturation of the epithelial sodium channel (ENaC) involves furin-dependent cleavage at two extracellular sites within the α subunit and at a single extracellular site within the γ subunit. Channels lacking furin Processing of the α subunit have very low activity. We recently identified a prostasin-dependent cleavage site (RKRK186) in the γ subunit. We also demonstrated that the tract α D206-R231, between the two furin cleavage sites in the α subunit, as well as the tract γ E144-K186, between the furin and prostasin cleavage sites in the γ subunit, are inhibitory domains. ENaC cleavage by furin, and subsequently by prostasin, leads to a stepwise increase in the open probability of the channel as a result of release of the α and γ subunit inhibitory tracts, respectively. We examined whether release of either theα orγ inhibitory tract has a dominant role in activating the channel. Co-expression of prostasin and either wild type channels or mutant channels lacking furin cleavage of the α subunit (αR205A,R208A,R231Aβγ) in Xenopus laevis oocytes led to increases in whole cell currents to similar levels. In an analogous manner and independent of the Proteolytic Processing of theα subunit, amiloride-sensitive currents in oocytes expressing channels carrying γ subunits with both a mutation in the furin cleavage site and a deletion of the inhibitory tract (αβγR143A,ΔE144-K186 and αR205A,R208A,R231AβγR143A, ΔE144-K186) were significantly higher than those from oocytes expressing wild type ENaC. When channels lacked the α and γ subunit inhibitory tracts, α subunit cleavage was required for channels to be fully active. Channels lacking both furin cleavage and the inhibitory tract in theγ subunit (αβγR143A,ΔE144-K186) showed a significant reduction in the efficacy of block by the syntheticα-26 inhibitory peptide representing the tract αD206-R231. Our data indicate that removal of the inhibitory tract from the γ subunit, in the absence ofα subunit cleavage, results in nearly full activation of the channel.
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maturation of the epithelial na channel involves Proteolytic Processing of the α and γ subunits
Journal of Biological Chemistry, 2003Co-Authors: Rebecca P Hughey, Gunhild M Mueller, James B Bruns, Carol L Kinlough, Paul A Poland, Keri L Harkleroad, Marcelo D Carattino, Thomas R KleymanAbstract:Abstract The epithelial Na+ channel (ENaC) is a tetramer of two α-, one β-, and one γ-subunit, but little is known about its assembly and Processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone α-(95 kDa), β-(96 kDa), and γ-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of αβγENaC subunits revealed a second band for each subunit (65 kDa for α, 110 kDa for β, and 75 kDa for γ) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed α- and γ-subunits is also consistent with Proteolytic cleavage. By using α- and γ-subunits with epitope tags at both the amino and carboxyl termini, Proteolytic Processing of the α- and γ-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for α and 18 kDa for γ) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that Proteolytic cleavage represents maturation rather than degradation of the channel.
