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Dieter H Wolf - One of the best experts on this subject based on the ideXlab platform.
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previously unknown role for the ubiquitin ligase ubr1 in endoplasmic reticulum associated Protein Degradation
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Alexandra Stolz, Stefanie Besser, Heike Hottmann, Dieter H WolfAbstract:Quality control and Degradation of misfolded Proteins are essential processes of all cells. The endoplasmic reticulum (ER) is the entry site of Proteins into the secretory pathway in which Protein folding occurs and terminally misfolded Proteins are recognized and retrotranslocated across the ER membrane into the cytosol. Here, Proteins undergo polyubiquitination by one of the membrane-embedded ubiquitin ligases, in yeast Hrd1/Der3 (HMG-CoA reductase Degradation/Degradation of the ER) and Doa10 (Degradation of alpha), and are degraded by the proteasome. In this study, we identify cytosolic Ubr1 (E3 ubiquitin ligase, N-recognin) as an additional ubiquitin ligase that can participate in ER-associated Protein Degradation (ERAD) in yeast. We show that two polytopic ERAD substrates, mutated transporter of the mating type a pheromone, Ste6* (sterile), and cystic fibrosis transmembrane conductance regulator, undergo Ubr1-dependent Degradation in the presence and absence of the canonical ER ubiquitin ligases. Whereas in the case of Ste6* Ubr1 is specifically required under stress conditions such as heat or ethanol or in the absence of the canonical ER ligases, efficient Degradation of human cystic fibrosis transmembrane conductance regulator requires function of Ubr1 already in wild-type cells under standard growth conditions. Together with the Hsp70 (heat shock Protein) chaperone Ssa1 (stress-seventy subfamily A) and the AAA-type ATPase Cdc48 (cell division cycle), Ubr1 directs the substrate to proteasomal Degradation. These data unravel another layer of complexity in ERAD.
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membrane topology and function of der3 hrd1p as a ubiquitin Protein ligase e3 involved in endoplasmic reticulum Degradation
Journal of Biological Chemistry, 2001Co-Authors: Peter M Deak, Dieter H WolfAbstract:Abstract The endoplasmic reticulum contains a Protein quality control system that discovers malfolded or unassembled secretory Proteins and subjects them to Degradation in the cytosol. This requires retrograde transport of the respective Proteins from the endoplasmic reticulum back to the cytosol via the Sec61 translocon. In addition, a fully competent ubiquitination machinery and the 26 S proteasome are necessary for retrotranslocation and Degradation. Ubiquitination of mutated and malfolded Proteins of the endoplasmic reticulum is dependent mainly on the ubiquitin-conjugating enzyme Ubc7p. In addition, several new membrane components of the endoplasmic reticulum are required for Degradation. Here we present the topology of the previously discovered RING-H2 finger Protein Der3/Hrd1p, one of the new components of the endoplasmic reticulum membrane. The Protein spans the membrane six times. The amino terminus and the carboxyl terminus containing the RING finger domain face the cytoplasm. Altogether, RING finger-dependent ubiquitination of malfolded carboxypeptidase yscY in vivo, as well as of Der3/Hrd1p itself in vitro and RING finger-dependent binding of Ubc7p, uncovers Der3/Hrd1p as the ubiquitin-Protein ligase (E3) of the endoplasmic reticulum-associated Protein Degradation process.
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ricin a chain utilises the endoplasmic reticulum associated Protein Degradation pathway to enter the cytosol of yeast
FEBS Letters, 1999Co-Authors: Jeremy C Simpson, Lynne M. Roberts, Karin Romisch, John Davey, Dieter H Wolf, Michael J LordAbstract:Cytotoxic Proteins such as ricin A chain (RTA) have target substrates in the cytosol and therefore have to reach this cellular compartment in order to act. RTA is thought to translocate into the cytosol from the lumen of the endoplasmic reticulum (ER), although how it traverses the ER membrane has not been established. Using yeast mutants defective in various aspects of the ER-associated Protein Degradation (ERAD) pathway, we show that RTA introduced into the yeast ER subverts this pathway to enter the cytosol via the Sec61p translocon. A significant proportion of the exported RTA avoided proteasomal Degradation. These data are consistent with the contention that the RTA component from ricin endocytosed by mammalian cells may likewise exploit ERAD to translocate into the cytosol.
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a ring h2 finger motif is essential for the function of der3 hrd1 in endoplasmic reticulum associated Protein Degradation in the yeast saccharomyces cerevisiae
FEBS Letters, 1999Co-Authors: Javier Bordallo, Dieter H WolfAbstract:Der3/Hrd1p is a Protein required for proper Degradation of misfolded soluble and integral membrane Proteins in the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae. It is located to the ER membrane and consists of a N-terminal hydrophobic region with several transmembrane domains and a large hydrophilic tail oriented to the ER lumen containing a RING finger motif of the H2 class. We had previously reported that a truncated version of Der3p, Der3ΔRp, lacking 111 residues of the lumenal domain including the RING finger motif is not functional, suggesting the involvement of this domain in the function of the Protein in ER Degradation. We substantiated this hypothesis by constructing a mutated form of Der3/Hrd1p replacing the last cysteine of the motif with a serine. This mutated Der3C399S Protein maintains the correct localization and topology of the wild-type Protein, however, is not able to support the Degradation of soluble and integral membrane Proteins. This point mutation altering the RING-H2 motif behaves as a dominant allele especially when overexpressed from a 2μ plasmid by this increasing the half-life of CPY* more than 6-fold when compared with a wild-type strain. Furthermore co-expression of der3C399S with the wild-type allele is also able to partially suppress the temperature sensitive growth phenotype of a sec61-2 strain. Finally we have shown that overexpression of Hrd3p suppresses the dominant effect of the der3C399S mutation. These results could be explained by a competition between wild-type and mutant Der3 Protein for the interaction with some other component of the ER Degradation pathway, probably Hrd3p.
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the medial golgi ion pump pmr1 supplies the yeast secretory pathway with ca2 and mn2 required for glycosylation sorting and endoplasmic reticulum associated Protein Degradation
Molecular Biology of the Cell, 1998Co-Authors: Gabriele Durr, Dieter H Wolf, Jochen Strayle, Richard K Plemper, Saskia Elbs, Saskia K Klee, Patrice Catty, Hans K RudolphAbstract:The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects of pmr1 mutants in N-linked and O-linked Protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum Protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum Proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal Proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.
Eduard Paschke - One of the best experts on this subject based on the ideXlab platform.
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dlhex dgj a novel derivative of 1 deoxygalactonojirimycin with pharmacological chaperone activity in human gm1 gangliosidosis fibroblasts
Molecular Genetics and Metabolism, 2010Co-Authors: Katrin Fantur, Doris Hofer, Georg Schitter, Andreas J Steiner, Bettina M Pabst, Tanja M Wrodnigg, Arnold E Stutz, Eduard PaschkeAbstract:Abstract G M1 -gangliosidosis (GM1) and Morquio B disease (MBD) are rare lysosomal storage disorders caused by mutations in the gene GLB1. Its main gene product, human acid β-galactosidase (β-Gal) degrades two functionally important molecules, G M1 -ganglioside and keratan sulfate in brain and connective tissues, respectively. While GM1 is a severe, phenotypically heterogenous neurodegenerative disorder, MBD is a systemic bone disease without effects on the central nervous system. A MBD-specific mutation, p.W273L, was shown to produce stable β-Gal precursors, normally transported and processed to mature, intralysosomal β-Gal. In accordance with the MBD phenotype, elevated residual activity against G M1 -ganglioside, but strongly reduced affinity towards keratan sulfate was found. Most GM1 alleles, in contrast, were shown to affect precursor stability and intracellular transport. Specific alleles, p.R201C and p.R201H result in misfolded, unstable precursor Proteins rapidly degraded by endoplasmic reticulum-associated Protein Degradation (ERAD). They may therefore be sensitive to stabilization by small molecules which bind at the active site and provide proper conformation. Thus the stabilized Protein may escape from ERAD processes, and reach the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET). This paper demonstrates that a novel iminosugar, DLHex-DGJ, has potent effects as competitive inhibitor of human acid β-galactosidase in vitro , and describes its effects on activity, Protein expression, maturation and intracellular transport in vivo in 13 fibroblasts lines with GLB1 mutations. Beside p.R201C and p.R201H, two further alleles, p.C230R and p.G438E, displayed significant sensitivity against DLHex-DGJ, with an increase of catalytic activity, and a normalization of transport and lysosomal processing of β-Gal precursors.
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dlhex dgj a novel derivative of 1 deoxygalactonojirimycin with pharmacological chaperone activity in human gm1 gangliosidosis fibroblasts
Molecular Genetics and Metabolism, 2010Co-Authors: Katrin Fantur, Doris Hofer, Georg Schitter, Bettina M Pabst, Tanja M Wrodnigg, Arnold E Stutz, Andreas Steiner, Eduard PaschkeAbstract:G(M1)-gangliosidosis (GM1) and Morquio B disease (MBD) are rare lysosomal storage disorders caused by mutations in the gene GLB1. Its main gene product, human acid beta-galactosidase (beta-Gal) degrades two functionally important molecules, G(M1)-ganglioside and keratan sulfate in brain and connective tissues, respectively. While GM1 is a severe, phenotypically heterogenous neurodegenerative disorder, MBD is a systemic bone disease without effects on the central nervous system. A MBD-specific mutation, p.W273L, was shown to produce stable beta-Gal precursors, normally transported and processed to mature, intralysosomal beta-Gal. In accordance with the MBD phenotype, elevated residual activity against G(M1)-ganglioside, but strongly reduced affinity towards keratan sulfate was found. Most GM1 alleles, in contrast, were shown to affect precursor stability and intracellular transport. Specific alleles, p.R201C and p.R201H result in misfolded, unstable precursor Proteins rapidly degraded by endoplasmic reticulum-associated Protein Degradation (ERAD). They may therefore be sensitive to stabilization by small molecules which bind at the active site and provide proper conformation. Thus the stabilized Protein may escape from ERAD processes, and reach the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET). This paper demonstrates that a novel iminosugar, DLHex-DGJ, has potent effects as competitive inhibitor of human acid beta-galactosidase in vitro, and describes its effects on activity, Protein expression, maturation and intracellular transport in vivo in 13 fibroblasts lines with GLB1 mutations. Beside p.R201C and p.R201H, two further alleles, p.C230R and p.G438E, displayed significant sensitivity against DLHex-DGJ, with an increase of catalytic activity, and a normalization of transport and lysosomal processing of beta-Gal precursors.
Katrin Fantur - One of the best experts on this subject based on the ideXlab platform.
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dlhex dgj a novel derivative of 1 deoxygalactonojirimycin with pharmacological chaperone activity in human gm1 gangliosidosis fibroblasts
Molecular Genetics and Metabolism, 2010Co-Authors: Katrin Fantur, Doris Hofer, Georg Schitter, Andreas J Steiner, Bettina M Pabst, Tanja M Wrodnigg, Arnold E Stutz, Eduard PaschkeAbstract:Abstract G M1 -gangliosidosis (GM1) and Morquio B disease (MBD) are rare lysosomal storage disorders caused by mutations in the gene GLB1. Its main gene product, human acid β-galactosidase (β-Gal) degrades two functionally important molecules, G M1 -ganglioside and keratan sulfate in brain and connective tissues, respectively. While GM1 is a severe, phenotypically heterogenous neurodegenerative disorder, MBD is a systemic bone disease without effects on the central nervous system. A MBD-specific mutation, p.W273L, was shown to produce stable β-Gal precursors, normally transported and processed to mature, intralysosomal β-Gal. In accordance with the MBD phenotype, elevated residual activity against G M1 -ganglioside, but strongly reduced affinity towards keratan sulfate was found. Most GM1 alleles, in contrast, were shown to affect precursor stability and intracellular transport. Specific alleles, p.R201C and p.R201H result in misfolded, unstable precursor Proteins rapidly degraded by endoplasmic reticulum-associated Protein Degradation (ERAD). They may therefore be sensitive to stabilization by small molecules which bind at the active site and provide proper conformation. Thus the stabilized Protein may escape from ERAD processes, and reach the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET). This paper demonstrates that a novel iminosugar, DLHex-DGJ, has potent effects as competitive inhibitor of human acid β-galactosidase in vitro , and describes its effects on activity, Protein expression, maturation and intracellular transport in vivo in 13 fibroblasts lines with GLB1 mutations. Beside p.R201C and p.R201H, two further alleles, p.C230R and p.G438E, displayed significant sensitivity against DLHex-DGJ, with an increase of catalytic activity, and a normalization of transport and lysosomal processing of β-Gal precursors.
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dlhex dgj a novel derivative of 1 deoxygalactonojirimycin with pharmacological chaperone activity in human gm1 gangliosidosis fibroblasts
Molecular Genetics and Metabolism, 2010Co-Authors: Katrin Fantur, Doris Hofer, Georg Schitter, Bettina M Pabst, Tanja M Wrodnigg, Arnold E Stutz, Andreas Steiner, Eduard PaschkeAbstract:G(M1)-gangliosidosis (GM1) and Morquio B disease (MBD) are rare lysosomal storage disorders caused by mutations in the gene GLB1. Its main gene product, human acid beta-galactosidase (beta-Gal) degrades two functionally important molecules, G(M1)-ganglioside and keratan sulfate in brain and connective tissues, respectively. While GM1 is a severe, phenotypically heterogenous neurodegenerative disorder, MBD is a systemic bone disease without effects on the central nervous system. A MBD-specific mutation, p.W273L, was shown to produce stable beta-Gal precursors, normally transported and processed to mature, intralysosomal beta-Gal. In accordance with the MBD phenotype, elevated residual activity against G(M1)-ganglioside, but strongly reduced affinity towards keratan sulfate was found. Most GM1 alleles, in contrast, were shown to affect precursor stability and intracellular transport. Specific alleles, p.R201C and p.R201H result in misfolded, unstable precursor Proteins rapidly degraded by endoplasmic reticulum-associated Protein Degradation (ERAD). They may therefore be sensitive to stabilization by small molecules which bind at the active site and provide proper conformation. Thus the stabilized Protein may escape from ERAD processes, and reach the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET). This paper demonstrates that a novel iminosugar, DLHex-DGJ, has potent effects as competitive inhibitor of human acid beta-galactosidase in vitro, and describes its effects on activity, Protein expression, maturation and intracellular transport in vivo in 13 fibroblasts lines with GLB1 mutations. Beside p.R201C and p.R201H, two further alleles, p.C230R and p.G438E, displayed significant sensitivity against DLHex-DGJ, with an increase of catalytic activity, and a normalization of transport and lysosomal processing of beta-Gal precursors.
Hans K Rudolph - One of the best experts on this subject based on the ideXlab platform.
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deficiency of atp2c1 a golgi ion pump induces secretory pathway defects in endoplasmic reticulum er associated Degradation and sensitivity to er stress
Journal of Biological Chemistry, 2005Co-Authors: Jose Ramoscastaneda, Young Park, Ming Liu, Karin Hauser, Hans K Rudolph, Gary E Shull, Marcel F Jonkman, Kazutoshi Mori, Shigaku Ikeda, Hideoki OgawaAbstract:Abstract Relatively few clues have been uncovered to elucidate the cell biological role(s) of mammalian ATP2C1 encoding an inwardly directed secretory pathway Ca2+/Mn2+ pump that is ubiquitously expressed. Deficiency of ATP2C1 results in a human disease (Hailey-Hailey), which primarily affects keratinocytes. ATP2C1-encoded Protein is detected in the Golgi complex in a calcium-dependent manner. A small interfering RNA causes knockdown of ATP2C1 expression, resulting in defects in both post-translational processing of wild-type thyroglobulin (a secretory glycoProtein) as well as endoplasmic reticulum-associated Protein Degradation of mutant thyroglobulin, whereas Degradation of a nonglycosylated misfolded secretory Protein substrate appears unaffected. Knockdown of ATP2C1 is not associated with elevated steady state levels of ER chaperone Proteins, nor does it block cellular activation of either the PERK, ATF6, or Ire1/XBP1 portions of the ER stress response. However, deficiency of ATP2C1 renders cells hypersensitive to ER stress. These data point to the important contributions of the Golgi-localized ATP2C1 Protein in homeostatic maintenance throughout the secretory pathway.
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the medial golgi ion pump pmr1 supplies the yeast secretory pathway with ca2 and mn2 required for glycosylation sorting and endoplasmic reticulum associated Protein Degradation
Molecular Biology of the Cell, 1998Co-Authors: Gabriele Durr, Dieter H Wolf, Jochen Strayle, Richard K Plemper, Saskia Elbs, Saskia K Klee, Patrice Catty, Hans K RudolphAbstract:The yeast Ca2+ adenosine triphosphatase Pmr1, located in medial-Golgi, has been implicated in intracellular transport of Ca2+ and Mn2+ ions. We show here that addition of Mn2+ greatly alleviates defects of pmr1 mutants in N-linked and O-linked Protein glycosylation. In contrast, accurate sorting of carboxypeptidase Y (CpY) to the vacuole requires a sufficient supply of intralumenal Ca2+. Most remarkably, pmr1 mutants are also unable to degrade CpY*, a misfolded soluble endoplasmic reticulum Protein, and display phenotypes similar to mutants defective in the stress response to malfolded endoplasmic reticulum Proteins. Growth inhibition of pmr1 mutants on Ca2+-deficient media is overcome by expression of other Ca2+ pumps, including a SERCA-type Ca2+ adenosine triphosphatase from rabbit, or by Vps10, a sorting receptor guiding non-native luminal Proteins to the vacuole. Our analysis corroborates the dual function of Pmr1 in Ca2+ and Mn2+ transport and establishes a novel role of this secretory pathway pump in endoplasmic reticulum-associated processes.
Bettina M Pabst - One of the best experts on this subject based on the ideXlab platform.
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dlhex dgj a novel derivative of 1 deoxygalactonojirimycin with pharmacological chaperone activity in human gm1 gangliosidosis fibroblasts
Molecular Genetics and Metabolism, 2010Co-Authors: Katrin Fantur, Doris Hofer, Georg Schitter, Andreas J Steiner, Bettina M Pabst, Tanja M Wrodnigg, Arnold E Stutz, Eduard PaschkeAbstract:Abstract G M1 -gangliosidosis (GM1) and Morquio B disease (MBD) are rare lysosomal storage disorders caused by mutations in the gene GLB1. Its main gene product, human acid β-galactosidase (β-Gal) degrades two functionally important molecules, G M1 -ganglioside and keratan sulfate in brain and connective tissues, respectively. While GM1 is a severe, phenotypically heterogenous neurodegenerative disorder, MBD is a systemic bone disease without effects on the central nervous system. A MBD-specific mutation, p.W273L, was shown to produce stable β-Gal precursors, normally transported and processed to mature, intralysosomal β-Gal. In accordance with the MBD phenotype, elevated residual activity against G M1 -ganglioside, but strongly reduced affinity towards keratan sulfate was found. Most GM1 alleles, in contrast, were shown to affect precursor stability and intracellular transport. Specific alleles, p.R201C and p.R201H result in misfolded, unstable precursor Proteins rapidly degraded by endoplasmic reticulum-associated Protein Degradation (ERAD). They may therefore be sensitive to stabilization by small molecules which bind at the active site and provide proper conformation. Thus the stabilized Protein may escape from ERAD processes, and reach the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET). This paper demonstrates that a novel iminosugar, DLHex-DGJ, has potent effects as competitive inhibitor of human acid β-galactosidase in vitro , and describes its effects on activity, Protein expression, maturation and intracellular transport in vivo in 13 fibroblasts lines with GLB1 mutations. Beside p.R201C and p.R201H, two further alleles, p.C230R and p.G438E, displayed significant sensitivity against DLHex-DGJ, with an increase of catalytic activity, and a normalization of transport and lysosomal processing of β-Gal precursors.
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dlhex dgj a novel derivative of 1 deoxygalactonojirimycin with pharmacological chaperone activity in human gm1 gangliosidosis fibroblasts
Molecular Genetics and Metabolism, 2010Co-Authors: Katrin Fantur, Doris Hofer, Georg Schitter, Bettina M Pabst, Tanja M Wrodnigg, Arnold E Stutz, Andreas Steiner, Eduard PaschkeAbstract:G(M1)-gangliosidosis (GM1) and Morquio B disease (MBD) are rare lysosomal storage disorders caused by mutations in the gene GLB1. Its main gene product, human acid beta-galactosidase (beta-Gal) degrades two functionally important molecules, G(M1)-ganglioside and keratan sulfate in brain and connective tissues, respectively. While GM1 is a severe, phenotypically heterogenous neurodegenerative disorder, MBD is a systemic bone disease without effects on the central nervous system. A MBD-specific mutation, p.W273L, was shown to produce stable beta-Gal precursors, normally transported and processed to mature, intralysosomal beta-Gal. In accordance with the MBD phenotype, elevated residual activity against G(M1)-ganglioside, but strongly reduced affinity towards keratan sulfate was found. Most GM1 alleles, in contrast, were shown to affect precursor stability and intracellular transport. Specific alleles, p.R201C and p.R201H result in misfolded, unstable precursor Proteins rapidly degraded by endoplasmic reticulum-associated Protein Degradation (ERAD). They may therefore be sensitive to stabilization by small molecules which bind at the active site and provide proper conformation. Thus the stabilized Protein may escape from ERAD processes, and reach the lysosomes in an active state, as proposed for enzyme enhancement therapy (EET). This paper demonstrates that a novel iminosugar, DLHex-DGJ, has potent effects as competitive inhibitor of human acid beta-galactosidase in vitro, and describes its effects on activity, Protein expression, maturation and intracellular transport in vivo in 13 fibroblasts lines with GLB1 mutations. Beside p.R201C and p.R201H, two further alleles, p.C230R and p.G438E, displayed significant sensitivity against DLHex-DGJ, with an increase of catalytic activity, and a normalization of transport and lysosomal processing of beta-Gal precursors.