27 results on '"Wolfgang Heinemeyer"'
Search Results
2. Structure-Based Design of Inhibitors Selective for Human Proteasome β2c or β2i Subunits
- Author
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Bogdan I. Florea, Emily S. Weyburne, Bo-Tao Xin, Marissa Janssens, Wolfgang Heinemeyer, Eva M. Huber, Gerjan de Bruin, Michael Groll, Christoph Driessen, Gijsbert A. van der Marel, Alexei F. Kisselev, Christofer Espinal, Yimeng Du, Elmer Maurits, and Herman S. Overkleeft
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Proteasome Endopeptidase Complex ,Saccharomyces cerevisiae Proteins ,Structural similarity ,Protein subunit ,Recombinant Fusion Proteins ,Mutagenesis (molecular biology technique) ,Plasma protein binding ,Saccharomyces cerevisiae ,Crystallography, X-Ray ,Protein Engineering ,01 natural sciences ,Article ,Small Molecule Libraries ,03 medical and health sciences ,Mice ,Peptide Library ,Catalytic Domain ,Cell Line, Tumor ,Drug Discovery ,Animals ,Humans ,Peptide library ,030304 developmental biology ,0303 health sciences ,Chemistry ,Stereoisomerism ,Protein engineering ,ddc ,0104 chemical sciences ,010404 medicinal & biomolecular chemistry ,Protein Subunits ,Proteasome ,Structural biology ,Biochemistry ,Drug Design ,Mutation ,Molecular Medicine ,Oligopeptides ,Proteasome Inhibitors ,Protein Binding - Abstract
Subunit-selective proteasome inhibitors are valuable tools to assess the biological and medicinal relevance of individual proteasome active sites. Whereas the inhibitors for the beta 1c, beta 1i, beta 5c, and beta 5i subunits exploit the differences in the substrate-binding channels identified by X-ray crystallography, compounds selectively targeting beta 2c or beta 2i could not yet be rationally designed because of the high structural similarity of these two subunits. Here, we report the development, chemical synthesis, and biological screening of a compound library that led to the identification of the beta 2c- and beta 2i-selective-compounds LU-002c (4; IC50 beta 2c: 8 nM, IC50 beta 2i/beta 2c: 40-fold) and LU-002i (5; IC50 beta 2i: 220 nM, IC50 beta 2c/beta 2i: 45-fold), respectively. Co-crystal structures with beta 2 humanized yeast proteasomes visualize protein-ligand interactions crucial for subunit specificity. Altogether, organic syntheses, activity-based protein profiling, yeast mutagenesis, and structural biology allowed us to decipher significant differences of beta 2 substrate-binding channels and to complete the set of subunit-selective proteasome inhibitors.
- Published
- 2019
3. Structural Elucidation of a Nonpeptidic Inhibitor Specific for the Human Immunoproteasome
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Eva M. Huber, Michael Groll, Haissi Cui, Regina Baur, Camille Le Chapelain, Christian Dubiella, and Wolfgang Heinemeyer
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Models, Molecular ,0301 basic medicine ,Proteasome Endopeptidase Complex ,Molecular Structure ,Yeast Model ,Stereochemistry ,Organic Chemistry ,Selective inhibition ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,chemistry ,Electrophile ,Humans ,Immunologic Factors ,Molecular Medicine ,Peptides ,Thiazole ,Oligopeptides ,Proteasome Inhibitors ,Molecular Biology ,Lead compound - Abstract
Selective inhibition of the immunoproteasome is a promising approach towards the development of immunomodulatory drugs. Recently, a class of substituted thiazole compounds that combine a non-peptidic scaffold with the absence of an electrophile has been reported in a patent. Herein, we describe the mode of action of the lead compound using a sophisticated chimeric yeast model of the human immunoproteasome for structural studies. The inhibitor adopts a unique orientation perpendicular to the β5i substrate binding channel. Distinctive interactions between the inhibitor and the subpockets of the human immunoproteasome account for its isotype selectivity.
- Published
- 2017
4. Defective immuno- and thymoproteasome assembly causes severe immunodeficiency
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Thomas Wieland, Tim M. Strom, Kathy D. McCoy, Christian Andres, Eckhard Wolf, Birgit Rathkolb, Eva M. Huber, Simon Grassmann, Christina Landbrecht, Laura Helming, Valerie Gailus-Durner, Frauke Neff, Thure Adler, Michael Basler, Marcus Groettrup, Andrew J. Macpherson, Irina Treise, Martin Hrabě de Angelis, Wolfgang Heinemeyer, Markus Ollert, Michael Groll, Tanja Klein-Rodewald, Matthias Klaften, Helmut Fuchs, and Dirk H. Busch
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Male ,0301 basic medicine ,Proteasome Endopeptidase Complex ,Cell Survival ,Mutant ,lcsh:Medicine ,Mutagenesis (molecular biology technique) ,610 Medicine & health ,Mice, SCID ,Biology ,Article ,Mice ,03 medical and health sciences ,0302 clinical medicine ,ddc:570 ,medicine ,Animals ,Humans ,Missense mutation ,lcsh:Science ,Immunodeficiency ,Mice, Knockout ,Mice, Inbred BALB C ,Severe combined immunodeficiency ,Multidisciplinary ,Point mutation ,lcsh:R ,medicine.disease ,Molecular biology ,ddc ,3. Good health ,Mice, Inbred C57BL ,Protein Subunits ,030104 developmental biology ,Proteasome assembly ,Knockout mouse ,lcsh:Q ,Female ,Cell death and immune response, Mechanisms of disease, Primary immunodeficiency disorders, Proteasome ,030217 neurology & neurosurgery - Abstract
By N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated the mutant mouse line TUB6 that is characterised by severe combined immunodeficiency (SCID) and systemic sterile autoinflammation in homozygotes, and a selective T cell defect in heterozygotes. The causative missense point mutation results in the single amino acid exchange G170W in multicatalytic endopeptidase complex subunit-1 (MECL-1), the β2i-subunit of the immuno- and thymoproteasome. Yeast mutagenesis and crystallographic data suggest that the severe TUB6-phenotype compared to the MECL-1 knockout mouse is caused by structural changes in the C-terminal appendage of β2i that prevent the biogenesis of immuno- and thymoproteasomes. Proteasomes are essential for cell survival, and defective proteasome assembly causes selective death of cells expressing the mutant MECL-1, leading to the severe immunological phenotype. In contrast to the immunosubunits β1i (LMP2) and β5i (LMP7), mutations in the gene encoding MECL-1 have not yet been assigned to human disorders. The TUB6 mutant mouse line exemplifies the involvement of MECL-1 in immunopathogenesis and provides the first mouse model for primary immuno- and thymoproteasome-associated immunodeficiency that may also be relevant in humans.
- Published
- 2018
5. Interactions of the natural product kendomycin and the 20S proteasome
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Michael Groll, Anna-Lena Späth, Rolf Müller, Yasser A. Elnakady, Philipp Beck, and Wolfgang Heinemeyer
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Models, Molecular ,Cytoplasm ,Proteasome Endopeptidase Complex ,Cell Survival ,Mutant ,Saccharomyces cerevisiae ,Crystallography, X-Ray ,Mass Spectrometry ,chemistry.chemical_compound ,Structural Biology ,In vivo ,Humans ,Mode of action ,Molecular Biology ,Chromatography, High Pressure Liquid ,Biological Products ,Natural product ,Ubiquitin ,Drug discovery ,Wild type ,Kinetics ,Rifabutin ,chemistry ,Proteasome ,Biochemistry ,Mutation ,Kendomycin ,Female ,Proteasome Inhibitors ,HeLa Cells ,Protein Binding - Abstract
Natural products are a valuable source for novel lead structures in drug discovery, but for the majority of isolated bioactive compounds, the cellular targets are unknown. The structurally unique ansa-polyketide kendomycin (KM) was reported to exert its potent cytotoxic effects via impairment of the ubiquitin proteasome system, but the exact mode of action remained unclear. Here, we present a systematic biochemical characterization of KM-proteasome interactions in vitro and in vivo, including complex structures of wild type and mutant yeast 20S proteasome with KM. Our results provide evidence for a polypharmacological mode of action for KM's cytotoxic effect on cancer cells.
- Published
- 2014
6. A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit β5i
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Michael Groll, Wolfgang Heinemeyer, Gerjan de Bruin, Herman S. Overkleeft, and Eva M. Huber
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0301 basic medicine ,Resource ,Proteasome Endopeptidase Complex ,Protein subunit ,Saccharomyces cerevisiae ,Biology ,Crystallography, X-Ray ,01 natural sciences ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Mice ,Animals ,Humans ,Enzyme Inhibitors ,Mode of action ,Molecular Biology ,General Immunology and Microbiology ,010405 organic chemistry ,General Neuroscience ,Ligand (biochemistry) ,Yeast ,Recombinant Proteins ,0104 chemical sciences ,030104 developmental biology ,Biochemistry ,Proteasome ,Protein Binding - Abstract
Inhibition of the immunoproteasome subunit β5i alleviates autoimmune diseases in preclinical studies and represents a promising new anti-inflammatory therapy. However, the lack of structural data on the human immunoproteasome still hampers drug design. Here, we systematically determined the potency of seven α' β' epoxyketone inhibitors with varying N-caps and P3-stereochemistry for mouse/human β5c/β5i and found pronounced differences in their subunit and species selectivity. Using X-ray crystallography, the compounds were analyzed for their modes of binding to chimeric yeast proteasomes that incorporate key parts of human β5c, human β5i or mouse β5i and the neighboring β6 subunit. The structural data reveal exceptional conformations for the most selective human β5i inhibitors and highlight subtle structural differences as the major reason for the observed species selectivity. Altogether, the presented results validate the humanized yeast proteasome as a powerful tool for structure-based development of β5i inhibitors with potential clinical applications.
- Published
- 2016
7. A unified mechanism for proteolysis and autocatalytic activation in the 20S proteasome
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Xia Li, Michael Groll, Cassandra S. Arendt, Wolfgang Heinemeyer, Eva M. Huber, and Mark Hochstrasser
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0301 basic medicine ,Threonine ,Proteasome Endopeptidase Complex ,Saccharomyces cerevisiae Proteins ,Science ,Proteolysis ,General Physics and Astronomy ,Threonine protease ,Saccharomyces cerevisiae ,Biology ,Crystallography, X-Ray ,General Biochemistry, Genetics and Molecular Biology ,Article ,Catalysis ,03 medical and health sciences ,Catalytic Domain ,Catalytic triad ,medicine ,Serine ,Cysteine ,Protein Precursors ,Aspartic Acid ,Multidisciplinary ,medicine.diagnostic_test ,Lysine ,Wild type ,Active site ,General Chemistry ,ddc ,030104 developmental biology ,Proteasome ,Biochemistry ,biology.protein ,Mutagenesis, Site-Directed ,Autolysis ,Biogenesis - Abstract
Biogenesis of the 20S proteasome is tightly regulated. The N-terminal propeptides protecting the active-site threonines are autocatalytically released only on completion of assembly. However, the trigger for the self-activation and the reason for the strict conservation of threonine as the active site nucleophile remain enigmatic. Here we use mutagenesis, X-ray crystallography and biochemical assays to suggest that Lys33 initiates nucleophilic attack of the propeptide by deprotonating the Thr1 hydroxyl group and that both residues together with Asp17 are part of a catalytic triad. Substitution of Thr1 by Cys disrupts the interaction with Lys33 and inactivates the proteasome. Although a Thr1Ser mutant is active, it is less efficient compared with wild type because of the unfavourable orientation of Ser1 towards incoming substrates. This work provides insights into the basic mechanism of proteolysis and propeptide autolysis, as well as the evolutionary pressures that drove the proteasome to become a threonine protease., The proteasome, an essential molecular machine, is a threonine protease, but the evolution and the components of its proteolytic centre are unclear. Here, the authors use structural biology and biochemistry to investigate the role of proteasome active site residues on maturation and activity.
- Published
- 2016
8. Immuno- and constitutive proteasome crystal structures reveal differences in substrate and inhibitor specificity
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Ricarda Schwab, Marcus Groettrup, Christopher J. Kirk, Wolfgang Heinemeyer, Michael Groll, Michael Basler, and Eva M. Huber
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Models, Molecular ,Proteasome Endopeptidase Complex ,Protein subunit ,Molecular Sequence Data ,Peptide ,Saccharomyces cerevisiae ,Crystallography, X-Ray ,Major histocompatibility complex ,General Biochemistry, Genetics and Molecular Biology ,Mice ,03 medical and health sciences ,0302 clinical medicine ,ddc:570 ,Hydrolase ,Animals ,Amino Acid Sequence ,030304 developmental biology ,chemistry.chemical_classification ,Antigen Presentation ,0303 health sciences ,biology ,Biochemistry, Genetics and Molecular Biology(all) ,Histocompatibility Antigens Class I ,Active site ,Yeast ,3. Good health ,Cell biology ,Proteasome ,Biochemistry ,chemistry ,Docking (molecular) ,030220 oncology & carcinogenesis ,biology.protein ,Oligopeptides ,Proteasome Inhibitors ,Sequence Alignment - Abstract
Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 Å resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit Beta5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of Beta5c/X but not Beta5i, thereby explaining the selectivity of PR-957 for Beta5i. Time-resolved structures of yeast proteasome: PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders.
- Published
- 2012
9. Systematic Analyses of Substrate Preferences of 20S Proteasomes Using Peptidic Epoxyketone Inhibitors
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Eva M. Huber, Gerjan de Bruin, Guillem Paniagua Soriano, Michael Groll, Wolfgang Heinemeyer, and Herman S. Overkleeft
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Models, Molecular ,Substrate Specificities ,Proteasome Endopeptidase Complex ,Stereochemistry ,Cleavage (embryo) ,Biochemistry ,Catalysis ,Cell Line ,Substrate Specificity ,Colloid and Surface Chemistry ,Catalytic Domain ,Yeasts ,Humans ,Amino Acid Sequence ,Peptide sequence ,Chemistry ,Rational design ,General Chemistry ,Ketones ,Protein Subunits ,Proteasome ,Caspases ,Substrate specificity ,Peptides ,Proteasome Inhibitors - Abstract
Cleavage analyses of 20S proteasomes with natural or synthetic substrates allowed to infer the substrate specificities of the active sites and paved the way for the rational design of high-affinity proteasome inhibitors. However, details of cleavage preferences remained enigmatic due to the lack of appropriate structural data. In a unique approach, we here systematically examined substrate specificities of yeast and human proteasomes using irreversibly acting α',β'epoxyketone (ep) inhibitors. Biochemical and structural analyses provide unique insights into the substrate preferences of the distinct active sites and highlight differences between proteasome types that may be considered in future inhibitor design efforts. (1) For steric reasons, epoxyketones with Val or Ile at the P1 position are weak inhibitors of all active sites. (2) Identification of the β2c selective compound Ac-LAE-ep represents a promising starting point for the development of compounds that discriminate between β2c and β2i. (3) The compound Ac-LAA-ep was found to favor subunit β5c over β5i by three orders of magnitude. (4) Yeast β1 and human β1c subunits preferentially bind Asp and Leu in their S1 pockets, while Glu and large hydrophobic residues are not accepted. (5) Exceptional structural features in the β1/2 substrate binding channel give rise to the β1 selectivity of compounds featuring Pro at the P3 site. Altogether, 23 different epoxyketone inhibitors, five proteasome mutants, and 43 crystal structures served to delineate a detailed picture of the substrate and ligand specificities of proteasomes and will further guide drug development efforts toward subunit-specific proteasome inhibitors for applications as diverse as cancer and autoimmune disorders.
- Published
- 2015
10. Cic1, an adaptor protein specifically linking the 26S proteasome to its substrate, the SCF component Cdc4
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Dieter H. Wolf, Jochen Strayle, Wolfgang Heinemeyer, and Sibylle Jäger
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Proteasome Endopeptidase Complex ,F-Box-WD Repeat-Containing Protein 7 ,Saccharomyces cerevisiae Proteins ,Ubiquitin-Protein Ligases ,Molecular Sequence Data ,Cell Cycle Proteins ,Protein degradation ,Biology ,F-box protein ,Article ,General Biochemistry, Genetics and Molecular Biology ,Substrate Specificity ,Fungal Proteins ,SCF complex ,Humans ,Amino Acid Sequence ,Cell division control protein 4 ,Peptide Synthases ,Cell Cycle Protein ,Ubiquitins ,Molecular Biology ,Fungal protein ,SKP Cullin F-Box Protein Ligases ,General Immunology and Microbiology ,F-Box Proteins ,General Neuroscience ,Signal transducing adaptor protein ,Cell biology ,Proteasome ,biology.protein ,Carrier Proteins ,Peptide Hydrolases - Abstract
In eukaryotes, the ubiquitin–proteasome system plays a major role in selective protein breakdown for cellular regulation. Here we report the discovery of a new essential component of this degradation machinery. We found the Saccharomyces cerevisiae protein Cic1 attached to 26S proteasomes playing a crucial role in substrate specificity for proteasomal destruction. Whereas degradation of short-lived test proteins is not affected, cic1 mutants stabilize the F-box proteins Cdc4 and Grr1, substrate recognition subunits of the SCF complex. Cic1 interacts in vitro and in vivo with Cdc4, suggesting a function as a new kind of substrate recruiting factor or adaptor associated with the proteasome.
- Published
- 2001
11. Contribution of Proteasomal β-Subunits to the Cleavage of Peptide Substrates Analyzed with Yeast Mutants
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Robert Huber, Dieter H. Wolf, Wieland Keilholz, Martin Deeg, Wolfgang Heinemeyer, Michael Groll, Hansjörg Schild, Stefan Stevanovic, Tobias P. Dick, Markus Schirle, Alexander K. Nussbaum, and Hans-Georg Rammensee
- Subjects
Proteasome Endopeptidase Complex ,Leupeptins ,Protein subunit ,Molecular Sequence Data ,Peptide ,Saccharomyces cerevisiae ,Biology ,Major histocompatibility complex ,Cleavage (embryo) ,Biochemistry ,Fluorescence ,Substrate Specificity ,Fungal Proteins ,Coumarins ,Multienzyme Complexes ,MHC class I ,Animals ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,Histocompatibility Antigens Class I ,Cell Biology ,Yeast ,Acetylcysteine ,Cysteine Endopeptidases ,Isocoumarins ,chemistry ,Proteasome ,Vertebrates ,biology.protein ,Peptides - Abstract
Proteasomes generate peptides that can be presented by major histocompatibility complex (MHC) class I molecules in vertebrate cells. Using yeast 20 S proteasomes carrying different inactivated beta-subunits, we investigated the specificities and contributions of the different beta-subunits to the degradation of polypeptide substrates containing MHC class I ligands and addressed the question of additional proteolytically active sites apart from the active beta-subunits. We found a clear correlation between the contribution of the different subunits to the cleavage of fluorogenic and long peptide substrates, with beta5/Pre2 cleaving after hydrophobic, beta2/Pup1 after basic, and beta1/Pre3 after acidic residues, but with the exception that beta2/Pup1 and beta1/Pre3 can also cleave after some hydrophobic residues. All proteolytic activities including the "branched chain amino acid-preferring" component are associated with beta5/Pre2, beta1/Pre3, or beta2/Pup1, arguing against additional proteolytic sites. Because of the high homology between yeast and mammalian 20 S proteasomes in sequence and subunit topology and the conservation of cleavage specificity between mammalian and yeast proteasomes, our results can be expected to also describe most of the proteolytic activity of mammalian 20 S proteasomes leading to the generation of MHC class I ligands.
- Published
- 1998
12. The Active Sites of the Eukaryotic 20 S Proteasome and Their Involvement in Subunit Precursor Processing
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Michael Fischer, Ulrike Stachon, Thomas Krimmer, Dieter H. Wolf, and Wolfgang Heinemeyer
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Threonine ,Proteasome Endopeptidase Complex ,Genotype ,Macromolecular Substances ,medicine.medical_treatment ,Protein subunit ,DNA Mutational Analysis ,Molecular Sequence Data ,Restriction Mapping ,Saccharomyces cerevisiae ,Biology ,Protein degradation ,Polymerase Chain Reaction ,Biochemistry ,Multienzyme Complexes ,medicine ,Animals ,Chymotrypsin ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Peptide sequence ,DNA Primers ,Mammals ,Enzyme Precursors ,Binding Sites ,Protease ,Sequence Homology, Amino Acid ,Active site ,Cell Biology ,Archaea ,Recombinant Proteins ,Models, Structural ,Cysteine Endopeptidases ,Proteasome ,Mutagenesis, Site-Directed ,biology.protein ,Protein Processing, Post-Translational ,Sequence Alignment ,Gene Deletion - Abstract
The 26 S proteasome is the central protease involved in ubiquitin-mediated protein degradation and fulfills vital regulatory functions in eukaryotes. The proteolytic core of the complex is the 20 S proteasome, a cylindrical particle with two outer rings each made of 7 different alpha-type subunits and two inner rings made of 7 different beta-type subunits. In the archaebacterial 20 S proteasome ancestor proteolytically active sites reside in the 14 uniform beta-subunits. Their N-terminal threonine residues, released by precursor processing, perform the nucleophilic attack for peptide bond hydrolysis. By directed mutational analysis of 20 S proteasomal beta-type proteins of Saccharomyces cerevisiae, we identified three active site-carrying subunits responsible for different peptidolytic activities as follows: Pre3 for post-glutamyl hydrolyzing, Pup1 for trypsin-like, and Pre2 for chymotrypsin-like activity. Double mutants harboring only trypsin-like or chymotrypsin-like activity were viable. Mutation of two potentially active site threonine residues in the Pre4 subunit excluded its catalytic involvement in any of the three peptidase activities. The generation of different, incompletely processed forms of the Pre4 precursor in active site mutants suggested that maturation of non-active proteasomal beta-type subunits is exerted by active subunits and occurs in the fully assembled particle. This trans-acting proteolytic activity might also account for processing intermediates of the active site mutated Pre2 subunit, which was unable to undergo autocatalytic maturation.
- Published
- 1997
13. PRE2, highly homologous to the human major histocompatibility complex-linked RING10 gene, codes for a yeast proteasome subunit necessary for chrymotryptic activity and degradation of ubiquitinated proteins
- Author
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V Möhrle, Y Mahé, A. Gruhler, Wolfgang Heinemeyer, and Dieter H. Wolf
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biology ,Molecular mass ,Protein subunit ,Mutant ,Antigen presentation ,Cell Biology ,Major histocompatibility complex ,Biochemistry ,Homology (biology) ,Proteasome ,biology.protein ,Gene family ,Molecular Biology - Abstract
We have cloned the yeast PRE2 gene by complementation of pre2 mutants, which are defective in the chymotrypsin-like activity of the 20 S proteasome (multicatalytic-multifunctional proteinase complex). The PRE2 gene, a beta-type member of the proteasomal gene family, is essential for life and codes for a 287-amino acid proteasomal subunit with a predicted molecular mass of 31.6 kDa. Missense mutations in two pre2 mutant alleles were identified. They led to enhanced sensitivity of yeast cells against stress. At the same time, pre2 mutants accumulated ubiquitinated proteins. The Pre2 protein shows striking homology to the human Ring10 protein (60% identity excluding the 70 amino-terminal residues), which is encoded in the major histocompatibility complex class II region. It represents a component of the low molecular mass polypeptide complex, previously shown to be a special type of the 20 S proteasome. The low molecular mass polypeptide complex is assumed to be involved in antigen presentation, generating peptides from cytosolic protein antigens, which are subsequently presented to cytotoxic T-lymphocytes on the cell surface. The high homology of Pre2 to Ring10 implies the hypothesis that Ring10 is a subunit of the low molecular mass polypeptide complex central in its chymotryptic activity. One might further suggest that replacement of constitutive proteasomal components by functionally related major histocompatibility complex-linked low molecular mass polypeptides, as is Ring10, adapts mammalian proteasomes for functions in the immune response.
- Published
- 1993
14. Studies on the Yeast Proteasome Uncover Its Basic Structural Features and Multiple in vivo Functions
- Author
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Wolfgang Heinemeyer, Wolfgang Hilt, and Dieter H. Wolf
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Proteasome Endopeptidase Complex ,Protein Conformation ,Cellular differentiation ,medicine.medical_treatment ,ATPase ,Genes, Fungal ,Saccharomyces cerevisiae ,Biology ,Biochemistry ,Multienzyme Complexes ,Stress, Physiological ,medicine ,Animals ,Humans ,Ubiquitins ,Gene ,Protease ,Molecular Structure ,Cell Cycle ,Yeast ,Fructose-Bisphosphatase ,Cell biology ,Repressor Proteins ,Cysteine Endopeptidases ,Proteasome ,Cytoplasm ,biology.protein ,Fatty Acid Synthases ,Function (biology) - Abstract
Proteasomes are large multicatalytic protease complexes found in the cytoplasm and nucleus of all eukaryotic cells. 20S proteasomes are cylindrically shaped particles composed of a set of different subunits arranged in a stack of 4 rings with 7-fold symmetry. In yeast 14 different genes are known, which are proposed to code for the complete set of 20S proteasomal subunits. They can be divided in 7 alpha- and 7 beta-type subunits. 26S proteasomes are even larger proteinase complexes which contain the 20S proteasome as the functional proteolytic core. They degrade ubiquitinylated proteins in vitro. Several yeast 26S proteasome subunits have been characterized as members of a novel ATPase family. Studies with yeast 20S and 26S proteasome mutants uncovered the function of proteasomes in stress-dependent and ubiquitin-mediated proteolytic pathways. Proteasomes are important for cellular regulation, cell differentiation, adaptation to environmental changes and are involved in cell cycle control.
- Published
- 1993
15. The proteasome/multicatalytic-multifunctional proteinase In vivo function in the ubiquitin-dependent N-end rule pathway of protein degradation in eukaryotes
- Author
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Dieter H. Wolf, Birgit Richter-Ruoff, and Wolfgang Heinemeyer
- Subjects
Proteasome Endopeptidase Complex ,Recombinant Fusion Proteins ,Proteolysis ,Mutant ,Biophysics ,N-end rule ,Saccharomyces cerevisiae ,Proteinase yscE ,Biology ,Protein degradation ,Biochemistry ,Catalysis ,Substrate Specificity ,Ubiquitin ,Multienzyme Complexes ,Structural Biology ,Genetics ,medicine ,Chymotrypsin ,Ubiquitins ,Molecular Biology ,Ribonucleoprotein ,chemistry.chemical_classification ,medicine.diagnostic_test ,Proteins ,Proteasome/multicatalytic—multifunctional proteinase ,Cell Biology ,beta-Galactosidase ,Yeast ,Cysteine Endopeptidases ,Enzyme ,Proteasome ,chemistry ,Mutation ,biology.protein - Abstract
Proteinase yscE, the proteasome/multicatalytic—multifunctional proteinase of yeast had been shown to function in stress response and in the degradation of ubiquitinated proteins [(1991) EMBO J. 10, 555–562]. A well-defined set of proteins degraded via ubiquitin-mediated proteolysis are the substrates of the N-end rule pathway [(1986) Science 234, 179–186; (1989) Science 243, 1576–1583]. We show that mutants defective in the chymotryptic activity of proteinase yscE fail to degrade substrates of the N-end rule pathway. This gives further proof of the proteasome being a central catalyst in ubiquitin-mediated proteolysis.
- Published
- 1992
16. Proteinase yscE, the yeast proteasome/multicatalytic-multifunctional proteinase: mutants unravel its function in stress induced proteolysis and uncover its necessity for cell survival
- Author
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Dieter H. Wolf, J A Kleinschmidt, J Saidowsky, C Escher, and Wolfgang Heinemeyer
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Transcription, Genetic ,Macromolecular Substances ,Proteolysis ,Genes, Fungal ,Molecular Sequence Data ,Restriction Mapping ,Saccharomyces cerevisiae ,Biology ,Protein degradation ,General Biochemistry, Genetics and Molecular Biology ,chemistry.chemical_compound ,Proteinase 3 ,medicine ,Amino Acid Sequence ,Cloning, Molecular ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,Base Sequence ,General Immunology and Microbiology ,medicine.diagnostic_test ,General Neuroscience ,Genetic Complementation Test ,biology.organism_classification ,Amino acid ,Cysteine Endopeptidases ,Kinetics ,Biochemistry ,Proteasome ,chemistry ,Protein Biosynthesis ,Mutation ,Chromosome Deletion ,Chromosomes, Fungal ,Canavanine ,Research Article - Abstract
Proteinase yscE is the yeast equivalent of the proteasome, a multicatalytic-multifunctional proteinase found in higher eukaryotic cells. We have isolated three mutants affecting the proteolytic activity of proteinase yscE. The mutants show a specific reduction in the activity of the complex against peptide substrates with hydrophobic amino acids at the cleavage site and define two complementation groups, PRE1 and PRE2. The PRE1 gene was cloned and shown to be essential. The deduced amino acid sequence encoded by the PRE1 gene reveals weak, but significant similarities to proteasome subunits of other organisms. Two-dimensional gel electrophoresis identified the yeast proteasome to be composed of 14 different subunits. Comparison of these 14 subunits with the translation product obtained from PRE1 mRNA synthesized in vitro demonstrated that PRE1 encodes the 22.6 kd subunit (numbered 11) of the yeast proteasome. Diploids homozygous for pre1-1 are defective in sporulation. Strains carrying the pre1-1 mutation show enhanced sensitivity to stresses such as incorporation of the amino acid analogue canavanine into proteins or a combination of poor growth medium and elevated temperature. Under these stress conditions pre1-1 mutant cells exhibit decreased protein degradation and accumulate ubiquitin-protein conjugates.
- Published
- 1991
17. The virD genes from the vir region of the Ti plasmid: T-region border dependent processing steps in different rec mutants of Escherichia coli
- Author
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Juliane Alt-Mörbe, Joachim Schröder, and Wolfgang Heinemeyer
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DNA, Bacterial ,biology ,Mutant ,Genetic transfer ,Context (language use) ,General Medicine ,Agrobacterium tumefaciens ,medicine.disease_cause ,biology.organism_classification ,Molecular biology ,Substrate Specificity ,Blotting, Southern ,Ti plasmid ,Plasmid ,Genes, Bacterial ,Mutation ,Escherichia coli ,Genetics ,medicine ,Gene ,Plasmids - Abstract
We evaluated the substrate requirements for virD-mediated T-circle formation in an in vivo binary test system in Escherichia coli. Two copies of the 25-bp sequence which defines the right border of the T-DNA (transferred DNA) are sufficient, and the right and the left copy of the border are equivalent in function in this system. Experiments with different rec mutants show that the occurrence and frequency of circular double-stranded and single-stranded T-DNA equivalents strongly depend on rec functions of the host. These results are discussed in the context of processing of the tumor-inducing Ti plasmid preceding the T-DNA transfer from agrobacteria to plants.
- Published
- 1990
18. Constitutive and immunoproteasome crystal structures reveal differences in substrate and inhibitor specificity
- Author
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Ricarda Schwab, Michael Groll, Wolfgang Heinemeyer, Michael Basler, Eva M. Huber, Christopher J. Kirk, and Marcus Groettrup
- Subjects
Crystallography ,Materials science ,Immunology ,Substrate (chemistry) ,Crystal structure ,Molecular Biology - Published
- 2012
19. The catalytic sites of 20S proteasomes and their role in subunit maturation: a mutational and crystallographic study
- Author
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Michael Groll, Tobias C. Ullrich, Wolfgang Heinemeyer, Sibylle Jäger, Matthias Bochtler, Dieter H. Wolf, and Robert Huber
- Subjects
Autolysis (biology) ,Proteasome Endopeptidase Complex ,Multidisciplinary ,Crystallography ,biology ,Protein subunit ,Mutant ,Active site ,Saccharomyces cerevisiae ,Cleavage (embryo) ,Substrate Specificity ,Cysteine Endopeptidases ,Structure-Activity Relationship ,Biochemistry ,Proteasome ,Acetylation ,Multienzyme Complexes ,Catalytic Domain ,Colloquium Paper ,Mutation ,biology.protein ,Protein precursor - Abstract
We present a biochemical and crystallographic characterization of active site mutants of the yeast 20S proteasome with the aim to characterize substrate cleavage specificity, subunit intermediate processing, and maturation. beta1(Pre3), beta2(Pup1), and beta5(Pre2) are responsible for the postacidic, tryptic, and chymotryptic activity, respectively. The maturation of active subunits is independent of the presence of other active subunits and occurs by intrasubunit autolysis. The propeptides of beta6(Pre7) and beta7(Pre4) are intermediately processed to their final forms by beta2(Pup1) in the wild-type enzyme and by beta5(Pre2) and beta1(Pre3) in the beta2(Pup1) inactive mutants. A role of the propeptide of beta1(Pre3) is to prevent acetylation and thereby inactivation. A gallery of proteasome mutants that contain active site residues in the context of the inactive subunits beta3(Pup3), beta6(Pre7), and beta7(Pre4) show that the presence of Gly-1, Thr1, Asp17, Lys33, Ser129, Asp166, and Ser169 is not sufficient to generate activity.
- Published
- 1999
20. Cleavage motifs of the yeast 20S proteasome beta subunits deduced from digests of enolase 1
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Michael Groll, Hansjörg Schild, Wieland Keilholz, Klaus Dietz, Dieter H. Wolf, Tobias P. Dick, Alexander K. Nussbaum, Stefan Stevanovic, Hans-Georg Rammensee, Markus Schirle, Robert Huber, and Wolfgang Heinemeyer
- Subjects
chemistry.chemical_classification ,Proteasome Endopeptidase Complex ,Multidisciplinary ,Hydrolysis ,Enolase ,Saccharomyces cerevisiae ,Mutant ,Molecular Sequence Data ,Biology ,Biological Sciences ,Cleavage (embryo) ,biology.organism_classification ,Yeast ,Amino acid ,Substrate Specificity ,Cysteine Endopeptidases ,chemistry ,Biochemistry ,Proteasome ,Multienzyme Complexes ,Phosphopyruvate Hydratase ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Amino Acid Sequence ,Peptide sequence - Abstract
The 436-amino acid protein enolase 1 from yeast was degradedin vitroby purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active β subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.
- Published
- 1998
21. The Proteasome and Protein Degradation in Yeast
- Author
-
Dieter H. Wolf, Wolfgang Hilt, and Wolfgang Heinemeyer
- Subjects
Complementation ,Proteasome ,Biochemistry ,biology ,Chemistry ,Protein subunit ,Saccharomyces cerevisiae ,Protein degradation ,Endoplasmic-reticulum-associated protein degradation ,Ubiquitin-conjugating enzyme ,biology.organism_classification ,Yeast - Abstract
In 1984 a high molecular mass multisubunit protease complex was isolated from Saccharomyces cerevisiae [Achstetter et al. 1984] which proved to be the yeast homologue of the 20S proteasome complexes found in all eukaryotic cells [Kleinschmidt et al. 1988]. The yeast 20S proteasome is able to cleave chromo- and fluorogenic peptides at the carboxyterminus of hydrophobic, basic or acidic amino acids (chymotrypsin-like-, trypsin-like- and peptidyl-glutamyl-peptide hydrolyzing activity, respectively) [Heinemeyer et al. 1991]. The yeast 20S proteasome is composed of different subunits, showing a set of protein bands in the SDS-PAGE with molecular masses ranging from 20 to 35 kDa. They can be separated into 14 protein spots after two-dimensional gel electrophoresis [Heinemeyer et al. 1991]. Genes named Y7, Y13, PRS1 and PRS2 (independendly cloned as Y8 and SCL1) were cloned and sequenced on the basis of protein sequences of 20S proteasome subunits, genes named PRS3, PUP1, PUP2 and PUP3 were sequenced by chance [for summary see Hilt et al. 1993b]. We cloned the s-type genes PREI, PRE2, PRE3 and PRE4 by complementation of mutants defective in the chymotrypsin-like- (prel and pre2 mutants) or the PGPH-activity (pre3 and pre4 mutants) of the proteasome [Heinemeyer et al. 1991, Heinemeyer et al. 1993, Hilt et al. 1993a, Enenkel et al. 1994]. Additionally we cloned two α-type genes PRE5 and PRE6 using peptide sequences derived from purified proteasome subunits, extending the number of yeast 20S proteasome subunit genes to 14 [Heinemeyer et al. 1994].
- Published
- 1996
22. Die Fortschrittszahlen als logistisches Konzept in der Automobilindustrie
- Author
-
Wolfgang Heinemeyer
- Published
- 1994
23. Die Planung und Steuerung des logistischen Prozesses mit Fortschrittszahlen
- Author
-
Wolfgang Heinemeyer
- Abstract
Der Wettbewerb zwingt den Unternehmer, neben Produkten und Technologien auch die Organisationsstrukturen der Betriebe immer wieder zu uberprufen und zu verbessern. Durch struktur- und ablauforganisatorische Masnahmen wird eine zunehmende Integration der Planungs-, Steuerungs- und Uberwachungsaktivitaten angestrebt. So ist die Integration der Systeme der Produktionssteuerung mit denen der Arbeitsvorbereitung im Gedankengut der CIM-Welt1 stark ausgepragt.
- Published
- 1992
24. Lysosomal and non-lysosomal proteolysis in the eukaryotic cell: studies on yeast
- Author
-
Angela Simeon, Hans H. Hirsch, Dieter H. Wolf, Hans H. Schiffer, Wolfgang Heinemeyer, and Ulrich Teichert
- Subjects
Mannose 6-phosphate receptor ,Biochemistry ,Chemistry ,Endopeptidases ,Lysosomal proteolysis ,Saccharomyces cerevisiae ,Lysosomes ,Yeast ,Eukaryotic cell ,Cell biology - Published
- 1991
25. Nucleotide sequence of the clustered genes for apocytochrome b6 and subunit 4 of the cytochrome b/f complex in the spinach plastid chromosome
- Author
-
Juliane Alt, Wolfgang Heinemeyer, and Reinhold G. Herrmann
- Subjects
Plastid chromosome ,Biochemistry ,Cytochrome b ,Base pair ,Protein subunit ,Genetics ,Nucleic acid sequence ,General Medicine ,Biology ,Peptide sequence ,Gene ,Homology (biology) - Abstract
A 2.4 kilobase-pair segment of the spinach plastid chromosome carrying the genes for apocytochrome b6 and subunit 4 of the thylakoid membrane cytochrome b/f complex has been analysed by DNA sequencing and Northern blot analysis. The nucleotide sequence reveals two uninterrupted open reading frames of 211 and 139 triplets coding for two hydrophobic proteins of 23.7 kd (cytochrome b6) and 15.2 kd (subunit 4). The genes are located on the same strand and are separated from each other by 1018 untranslated base pairs. They map adjacent to the gene for the P680 chlorophyll α apoprotein of the photosystem II reaction center. The three genes appear to be under common transcriptional control and the transcripts post-transcriptionally modified. The deduced amino acid sequences of cytochrome b6 and subunit 4 both exhibit significant homology with published sequences from mitochondrial b cytochromes (42 kd) suggesting that these functionally equivalent polypeptides in photosynthetic and respiratory electron transport chains arose monophyletically.
- Published
- 1984
26. Two Agrobacterium tumefaciens genes for cytokinin biosynthesis: Ti plasmid-coded isopentenyltransferases adapted for function in prokaryotic or eukaryotic cells
- Author
-
Thomas Botz, Elmar W. Weiler, Joachim Schröder, Juliane Alt-Moerbe, Ilka Buchmann, John David Windass, Dave W. Tonge, and Wolfgang Heinemeyer
- Subjects
chemistry.chemical_classification ,Rhizobiaceae ,Agrobacterium ,Agrobacterium tumefaciens ,Biology ,biology.organism_classification ,medicine.disease_cause ,chemistry.chemical_compound ,Ti plasmid ,Enzyme ,Biosynthesis ,chemistry ,Biochemistry ,parasitic diseases ,Genetics ,medicine ,Molecular Biology ,Escherichia coli ,Gene - Abstract
Tzs and ipt are two Ti plasmid genes coding for proteins with isopentenyltransferase (IPT) activity in vitro. We cloned both genes for protein expression in Escherichia coli and in Agrobacterium tumefaciens, and we investigated differences between the two genes by analysing the properties of the proteins in vitro and in vivo. In vitro, extracts with tzs or ipt-coded proteins had high IPT activity, and the enzymes were identical in most properties. The most important difference was detected in vivo: the tzs-encoded protein was very active in cytokinin production, while the ipt protein required overexpression in order to obtain measurable activity in bacteria. In both cases, rans-zeatin was the major product of the gene activity. Formation of this cytokinin requires a hydroxylase function in addition to the IPT reaction. No such activity could be ascribed to tzs or ipt-encoded proteins in vitro or in vivo, but cytokinin hydroxylase activity was detected in cells and extracts of E. coli, regardless of the presence or absence of the cytokinin genes. Based on these results it is proposed that both genes code for a single enzyme activity (isopentenyltransferase), that the genes and proteins are adapted for function either in bacteria (tzs) or in transformed plant cells (ipt), and that in both prokaryotic and eukaryotic cells hydroxylation to trans-zeatin is a function contributed by host enzymes.
- Published
- 1987
27. Bortezomib-Resistant Mutant Proteasomes: Structural and Biochemical Evaluation with Carfilzomib and ONX 0914
- Author
-
Eva M. Huber, Michael Groll, and Wolfgang Heinemeyer
- Subjects
Models, Molecular ,Drug ,Proteasome Endopeptidase Complex ,Protein Conformation ,media_common.quotation_subject ,Mutant ,Mutagenesis (molecular biology technique) ,Drug resistance ,Biology ,Crystallography, X-Ray ,Bortezomib ,chemistry.chemical_compound ,X-Ray Diffraction ,Structural Biology ,Catalytic Domain ,Yeasts ,medicine ,Molecular Biology ,media_common ,Molecular Structure ,Cell growth ,Boronic Acids ,Carfilzomib ,3. Good health ,Proteasome ,chemistry ,Biochemistry ,Drug Resistance, Neoplasm ,Mutagenesis ,Pyrazines ,Mutation ,Cancer research ,Oligopeptides ,medicine.drug - Abstract
SummaryInhibition of the 20S proteasome by bortezomib (Velcade) constitutes a successfully applied therapy for blood cancer. However, emerging resistance restricts its medicinal use. For example, mutations in the proteolytically active β5-subunit of the proteasome, the main target of inhibitors, were reported to impair drug binding and thus to reduce therapeutic efficacy. Using yeast as a model system, we describe here a systematic evaluation of these mutations by cell growth analysis, proteasome inhibition assays, and X-ray crystallography. The 11 mutants examined display decreased proliferation rates, impaired proteolytic activity, and marked resistance to bortezomib as well as the α′,β′-epoxyketone inhibitors carfilzomib (Kyprolis) and ONX 0914, while the second-generation compound carfilzomib was the least affected. In total, 49 proteasome X-ray structures, including structural data on proteasome-carfilzomib complexes, reveal three distinct molecular mechanisms that hamper both drug binding and natural substrate turnover to an extent that is still compatible with cell survival.
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