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Papers by Hans-Ulrich Demuth
Journal of Biological Chemistry, 2021
The development of a targeted therapy would significantly improve the treatment of periodontitis ... more The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded β-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases. This article contains supporting information.
Journal of Biological Chemistry, Feb 1, 2000
The abbreviations used are: DPIV, dipeptidyl peptidase IV; pGlu, pyroglutamyl; Fmoc, N-9-fluoreny... more The abbreviations used are: DPIV, dipeptidyl peptidase IV; pGlu, pyroglutamyl; Fmoc, N-9-fluorenylmethyloxycarbonyl; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; EC 50 , half-maximal effective concentration; (I/A) 50 , ratio of inhibitor to agonist resulting in reduction of agonist alone 2-fold; CHO, Chinese hamster ovary; hGlucR cell, cell stably expressing the human glucagon receptor.
Alzheimers & Dementia, Jul 1, 2013
FEBS Journal, Oct 5, 2009
Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate residues at the N-terminus of s... more Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate residues at the N-terminus of several peptides and proteins from plants and animals. Recently, isoenzymes of mammalian QCs have been identified. In order to gain further insight into the biochemical characteristics of iso-QCs, the human and murine enzymes were expressed in the secretory pathway of Pichia pastoris. Replacement of the N-terminal signal anchor by an a-factor prepropeptide from Saccharomyces cerevisiae resulted in poor secretion of the protein. Insertion of an N-terminal glycosylation site and shortening of the N-terminus improved isoQC secretion 100-fold. A comparison of different recombinant isoQC proteins did not reveal an influence of mutagenic changes on catalytic activity. An initial characterization showed identical modes of substrate conversion of human isoQC and murine isoQC. Both proteins displayed a broad substrate specificity and preference for hydrophobic substrates, similar to the related QC. Likewise, a determination of the zinc content and reactivation of the apo-isoQC revealed equimolar zinc present in QC and isoQC. Far-UV CD spectroscopic analysis of murine QC and isoQC indicated virtually identical structural components. The present investigation provides the first enzymatic characterization of mammalian isoQCs. QC and isoQC represent very similar proteins, which are both present in the secretory pathway of cells. The functions of QCs and isoQC probably complement each other, suggesting a pivotal role of pyroglutamate modification for protein and peptide maturation.
Biochemistry, 1994
The mechanism of inactivation of serine proteases by N-peptidyl-0-aroylhydroxylamines was studied... more The mechanism of inactivation of serine proteases by N-peptidyl-0-aroylhydroxylamines was studied by X-ray crystallography. Cocrystals of subtilisin Carlsberg inactivated with N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-nitrobenzoylhydroxylamine were grown, and diffraction data to 1 . 8 4 resolution were obtained. The resulting electron density maps clearly reveal that the y-oxygen of the catalytic serine forms a carbamate derivative with the inhibitor. The peptide part of the inhibitor does not form the usual antiparallel P-sheet in the P binding cleft but protrudes out of the active site and is stabilized by a network of water molecules. These results, combined with kinetic characterization reported previously Eds.) ESCOM Science Publishers B.V., Amsterdam] support the existence of a t least one intermediate between the formation of the Michaelis complex and the final product. We suggest a mechanism for the inactivation of subtilisin Carlsberg by
Neuropeptides, Jan 27, 2016
Dipeptidyl peptidase 4 (DPP4; EC 3.4.14.5; CD26) is a membrane-bound or shedded serine protease t... more Dipeptidyl peptidase 4 (DPP4; EC 3.4.14.5; CD26) is a membrane-bound or shedded serine protease that hydrolyzes dipeptides from the N-terminus of peptides with either proline or alanine at the penultimate position. Substrates of DPP4 include several stress-related neuropeptides implicated in anxiety, depression and schizophrenia. A decline of DPP4-like activity has been reported in sera from depressed patient, but not fully characterized regarding DPP4-like enzymes, therapeutic interventions and protein. Sera from 16 melancholic- and 16 non-melancholic-depressed patients were evaluated for DPP4-like activities and the concentration of soluble DPP4 protein before and after treatment by anti-depressive therapies. Post-translational modification of DPP4-isoforms and degradation of NPY, Peptide YY (PYY), Galanin-like peptide (GALP), Orexin B (OrxB), OrxA, pituitary adenylate cyclase-activating polypeptide (PACAP) and substance P (SP) were studied in serum and in ex vivo human blood. N-t...
Journal of neurochemistry, Jan 26, 2016
Huntington's disease (HD) is an inherited and fatal polyglutamine neurodegenerative disorder ... more Huntington's disease (HD) is an inherited and fatal polyglutamine neurodegenerative disorder caused by an expansion of the CAG triplet repeat coding region within the HD gene. Progressive dysfunction and loss of striatal GABAergic medium spiny neurons (MSNs) may account for some of the characteristic symptoms in HD patients. Interestingly, in HD, MSNs expressing neuropeptide Y (NPY) are spared and their numbers is even up-regulated in HD patients. In line with this, we report here on increased immuno-linked NPY (IL-NPY) levels in human cerebrospinal fluid (hCSF) from HD patients. As this antibody-based detection of NPY may provide false positive differences due to the antibody-based detections of only fragments of NPY, the initial finding was validated by investigating the proteolytic stability of NPY in hCSF using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective inhibitors. A comparison between resulting NPY-fragments and...
Journal of Alzheimer's disease : JAD, Jan 27, 2015
Current treatment in Alzheimer's disease (AD) is initiated at a stage where the brain already... more Current treatment in Alzheimer's disease (AD) is initiated at a stage where the brain already has irreversible structural deteriorations. Therefore, the concept of treatment prior to obvious cognitive deficits has become widely accepted, and simple biochemical tests to discriminate normal aging from prodromal or demented stages are now common practice. The objective of the study was the differentiation of controls, mild cognitive impairment (MCI) and AD patients by novel blood-based assays in combination with neuropsychological tests. In a cross-sectional study, 143 subjects aged 18 to 85 years were recruited. All participants were classified by a comprehensive neuropsychological assessment. Blood samples were analyzed for several amyloid-β (Aβ) species, pro-inflammatory markers, anti-Aβ autoantibodies, and ApoE allele status, respectively. Plasma Aβ1-42 was significantly decreased in MCI and AD compared to age-matched controls, whereas Aβ1-40 did not differ, but increases with ...
PloS one, 2015
A hallmark of Alzheimer's disease (AD) is the accumulation of extracellular amyloid-β (Aβ) pl... more A hallmark of Alzheimer's disease (AD) is the accumulation of extracellular amyloid-β (Aβ) plaques in the brains of patients. N-terminally truncated pyroglutamate-modified Aβ (pEAβ) has been described as a major compound of Aβ species in senile plaques. pEAβ is more resistant to degradation, shows higher toxicity and has increased aggregation propensity and β-sheet stabilization compared to non-modified Aβ. Here we characterized recombinant pEAβ(3-40) in aqueous trifluoroethanol (TFE) solution regarding its aggregation propensity and structural changes in comparison to its non-pyroglutamate-modified variant Aβ(1-40). Secondary structure analysis by circular dichroism spectroscopy suggests that pEAβ(3-40) shows an increased tendency to form β-sheet-rich structures in 20% TFE containing solutions where Aβ(1-40) forms α-helices. Aggregation kinetics of pEAβ(3-40) in the presence of 20% TFE monitored by thioflavin-T (ThT) assay showed a typical sigmoidal aggregation in contrast to A...
PloS one, 2015
Alzheimer's disease (AD) is the leading cause of dementia in the elderly and is characterized... more Alzheimer's disease (AD) is the leading cause of dementia in the elderly and is characterized by memory loss and cognitive decline. Pathological hallmark of AD brains are intracellular neurofibrillary tangles and extracellular amyloid plaques. The major component of these plaques is the highly heterogeneous amyloid-β (Aβ) peptide, varying in length and modification. In recent years pyroglutamate-modified amyloid-β (pEAβ) peptides have increasingly moved into the focus since they have been described to be the predominant species of all N-terminally truncated Aβ. Compared to unmodified Aβ, pEAβ is known to show increased hydrophobicity, higher toxicity, faster aggregation and β-sheet stabilization and is more resistant to degradation. Nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful method to investigate the conformations of pEAβ isoforms in solution and to study peptide/ligand interactions for drug development. However, biophysical characterization of pEAβ...
Journal of Biological Chemistry, 2021
The development of a targeted therapy would significantly improve the treatment of periodontitis ... more The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer's disease, rheumatoid arthritis, and cardiovascular diseases. Glutaminyl cyclases (QCs) from the oral pathogens Porphyromonas gingivalis, Tannerella forsythia, and Prevotella intermedia represent attractive target enzymes for small-molecule inhibitor development, as their action is likely to stabilize essential periplasmic and outer membrane proteins by N-terminal pyroglutamination. In contrast to other microbial QCs that utilize the so-called type I enzymes, these oral pathogens possess sequences corresponding to type II QCs, observed hitherto only in animals. However, whether differences between these bacteroidal QCs and animal QCs are sufficient to enable development of selective inhibitors is not clear. To learn more, we recombinantly expressed all three QCs. They exhibit comparable catalytic efficiencies and are inhibited by metal chelators. Crystal structures of the enzymes from P. gingivalis (PgQC) and T. forsythia (TfQC) reveal a tertiary structure composed of an eight-stranded β-sheet surrounded by seven α-helices, typical of animal type II QCs. In each case, an active site Zn ion is tetrahedrally coordinated by conserved residues. Nevertheless, significant differences to mammalian enzymes are found around the active site of the bacteroidal enzymes. Application of a PgQC-selective inhibitor described here for the first time results in growth inhibition of two P. gingivalis clinical isolates in a dose-dependent manner. The insights gained by these studies will assist in the development of highly specific small-molecule bacteroidal QC inhibitors, paving the way for alternative therapies against periodontitis and associated diseases. This article contains supporting information.
Journal of Biological Chemistry, Feb 1, 2000
The abbreviations used are: DPIV, dipeptidyl peptidase IV; pGlu, pyroglutamyl; Fmoc, N-9-fluoreny... more The abbreviations used are: DPIV, dipeptidyl peptidase IV; pGlu, pyroglutamyl; Fmoc, N-9-fluorenylmethyloxycarbonyl; HPLC, high pressure liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/ionization-time of flight; EC 50 , half-maximal effective concentration; (I/A) 50 , ratio of inhibitor to agonist resulting in reduction of agonist alone 2-fold; CHO, Chinese hamster ovary; hGlucR cell, cell stably expressing the human glucagon receptor.
Alzheimers & Dementia, Jul 1, 2013
FEBS Journal, Oct 5, 2009
Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate residues at the N-terminus of s... more Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate residues at the N-terminus of several peptides and proteins from plants and animals. Recently, isoenzymes of mammalian QCs have been identified. In order to gain further insight into the biochemical characteristics of iso-QCs, the human and murine enzymes were expressed in the secretory pathway of Pichia pastoris. Replacement of the N-terminal signal anchor by an a-factor prepropeptide from Saccharomyces cerevisiae resulted in poor secretion of the protein. Insertion of an N-terminal glycosylation site and shortening of the N-terminus improved isoQC secretion 100-fold. A comparison of different recombinant isoQC proteins did not reveal an influence of mutagenic changes on catalytic activity. An initial characterization showed identical modes of substrate conversion of human isoQC and murine isoQC. Both proteins displayed a broad substrate specificity and preference for hydrophobic substrates, similar to the related QC. Likewise, a determination of the zinc content and reactivation of the apo-isoQC revealed equimolar zinc present in QC and isoQC. Far-UV CD spectroscopic analysis of murine QC and isoQC indicated virtually identical structural components. The present investigation provides the first enzymatic characterization of mammalian isoQCs. QC and isoQC represent very similar proteins, which are both present in the secretory pathway of cells. The functions of QCs and isoQC probably complement each other, suggesting a pivotal role of pyroglutamate modification for protein and peptide maturation.
Biochemistry, 1994
The mechanism of inactivation of serine proteases by N-peptidyl-0-aroylhydroxylamines was studied... more The mechanism of inactivation of serine proteases by N-peptidyl-0-aroylhydroxylamines was studied by X-ray crystallography. Cocrystals of subtilisin Carlsberg inactivated with N-((tert-butoxycarbonyl)alanylprolylphenylalanyl)-O-nitrobenzoylhydroxylamine were grown, and diffraction data to 1 . 8 4 resolution were obtained. The resulting electron density maps clearly reveal that the y-oxygen of the catalytic serine forms a carbamate derivative with the inhibitor. The peptide part of the inhibitor does not form the usual antiparallel P-sheet in the P binding cleft but protrudes out of the active site and is stabilized by a network of water molecules. These results, combined with kinetic characterization reported previously Eds.) ESCOM Science Publishers B.V., Amsterdam] support the existence of a t least one intermediate between the formation of the Michaelis complex and the final product. We suggest a mechanism for the inactivation of subtilisin Carlsberg by
Neuropeptides, Jan 27, 2016
Dipeptidyl peptidase 4 (DPP4; EC 3.4.14.5; CD26) is a membrane-bound or shedded serine protease t... more Dipeptidyl peptidase 4 (DPP4; EC 3.4.14.5; CD26) is a membrane-bound or shedded serine protease that hydrolyzes dipeptides from the N-terminus of peptides with either proline or alanine at the penultimate position. Substrates of DPP4 include several stress-related neuropeptides implicated in anxiety, depression and schizophrenia. A decline of DPP4-like activity has been reported in sera from depressed patient, but not fully characterized regarding DPP4-like enzymes, therapeutic interventions and protein. Sera from 16 melancholic- and 16 non-melancholic-depressed patients were evaluated for DPP4-like activities and the concentration of soluble DPP4 protein before and after treatment by anti-depressive therapies. Post-translational modification of DPP4-isoforms and degradation of NPY, Peptide YY (PYY), Galanin-like peptide (GALP), Orexin B (OrxB), OrxA, pituitary adenylate cyclase-activating polypeptide (PACAP) and substance P (SP) were studied in serum and in ex vivo human blood. N-t...
Journal of neurochemistry, Jan 26, 2016
Huntington's disease (HD) is an inherited and fatal polyglutamine neurodegenerative disorder ... more Huntington's disease (HD) is an inherited and fatal polyglutamine neurodegenerative disorder caused by an expansion of the CAG triplet repeat coding region within the HD gene. Progressive dysfunction and loss of striatal GABAergic medium spiny neurons (MSNs) may account for some of the characteristic symptoms in HD patients. Interestingly, in HD, MSNs expressing neuropeptide Y (NPY) are spared and their numbers is even up-regulated in HD patients. In line with this, we report here on increased immuno-linked NPY (IL-NPY) levels in human cerebrospinal fluid (hCSF) from HD patients. As this antibody-based detection of NPY may provide false positive differences due to the antibody-based detections of only fragments of NPY, the initial finding was validated by investigating the proteolytic stability of NPY in hCSF using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and selective inhibitors. A comparison between resulting NPY-fragments and...
Journal of Alzheimer's disease : JAD, Jan 27, 2015
Current treatment in Alzheimer's disease (AD) is initiated at a stage where the brain already... more Current treatment in Alzheimer's disease (AD) is initiated at a stage where the brain already has irreversible structural deteriorations. Therefore, the concept of treatment prior to obvious cognitive deficits has become widely accepted, and simple biochemical tests to discriminate normal aging from prodromal or demented stages are now common practice. The objective of the study was the differentiation of controls, mild cognitive impairment (MCI) and AD patients by novel blood-based assays in combination with neuropsychological tests. In a cross-sectional study, 143 subjects aged 18 to 85 years were recruited. All participants were classified by a comprehensive neuropsychological assessment. Blood samples were analyzed for several amyloid-β (Aβ) species, pro-inflammatory markers, anti-Aβ autoantibodies, and ApoE allele status, respectively. Plasma Aβ1-42 was significantly decreased in MCI and AD compared to age-matched controls, whereas Aβ1-40 did not differ, but increases with ...
PloS one, 2015
A hallmark of Alzheimer's disease (AD) is the accumulation of extracellular amyloid-β (Aβ) pl... more A hallmark of Alzheimer's disease (AD) is the accumulation of extracellular amyloid-β (Aβ) plaques in the brains of patients. N-terminally truncated pyroglutamate-modified Aβ (pEAβ) has been described as a major compound of Aβ species in senile plaques. pEAβ is more resistant to degradation, shows higher toxicity and has increased aggregation propensity and β-sheet stabilization compared to non-modified Aβ. Here we characterized recombinant pEAβ(3-40) in aqueous trifluoroethanol (TFE) solution regarding its aggregation propensity and structural changes in comparison to its non-pyroglutamate-modified variant Aβ(1-40). Secondary structure analysis by circular dichroism spectroscopy suggests that pEAβ(3-40) shows an increased tendency to form β-sheet-rich structures in 20% TFE containing solutions where Aβ(1-40) forms α-helices. Aggregation kinetics of pEAβ(3-40) in the presence of 20% TFE monitored by thioflavin-T (ThT) assay showed a typical sigmoidal aggregation in contrast to A...
PloS one, 2015
Alzheimer's disease (AD) is the leading cause of dementia in the elderly and is characterized... more Alzheimer's disease (AD) is the leading cause of dementia in the elderly and is characterized by memory loss and cognitive decline. Pathological hallmark of AD brains are intracellular neurofibrillary tangles and extracellular amyloid plaques. The major component of these plaques is the highly heterogeneous amyloid-β (Aβ) peptide, varying in length and modification. In recent years pyroglutamate-modified amyloid-β (pEAβ) peptides have increasingly moved into the focus since they have been described to be the predominant species of all N-terminally truncated Aβ. Compared to unmodified Aβ, pEAβ is known to show increased hydrophobicity, higher toxicity, faster aggregation and β-sheet stabilization and is more resistant to degradation. Nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful method to investigate the conformations of pEAβ isoforms in solution and to study peptide/ligand interactions for drug development. However, biophysical characterization of pEAβ...