Inhibition of glutaminyl cyclase alters pyroglutamate formation in mammalian cells

Laboratory for Proteolytic Neuroscience, RIKEN, Вако, Saitama, Japan
Biochimica et Biophysica Acta (Impact Factor: 4.66). 11/2006; 1764(10):1618-25. DOI: 10.1016/j.bbapap.2006.08.003
Source: PubMed

ABSTRACT Mammalian cell lines were examined concerning their Glutaminyl Cyclase (QC) activity using a HPLC method. The enzyme activity was suppressed by a QC specific inhibitor in all homogenates. Aim of the study was to prove whether inhibition of QC modifies the posttranslational maturation of N-glutamine and N-glutamate peptide substrates. Therefore, the impact of QC-inhibition on amino-terminal pyroglutamate (pGlu) formation of the modified amyloid peptides Abeta(N3E-42) and Abeta(N3Q-42) was investigated. These amyloid-beta peptides were expressed as fusion proteins with either the pre-pro sequence of TRH, to be released by a prohormone convertase, or as engineered amyloid precursor protein for subsequent liberation of Abeta(N3Q-42) after beta- and gamma-secretase cleavage during posttranslational processing. Inhibition of QC leads in both expression systems to significantly reduced pGlu-formation of differently processed Abeta-peptides. This reveals the importance of QC-activity during cellular maturation of pGlu-containing peptides. Thus, QC-inhibition should impact bioactivity, stability or even toxicity of pyroglutamyl peptides preventing glutamine and glutamate cyclization.

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Available from: Holger Cynis, Sep 25, 2014
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    • "Previous studies have demonstrated that biological or chemical inhibition of QC activity leads to amelioration of AD pathology, including improvement of the memory impairment [21] [29] [34]. However, the mechanism by which this phenomenon occurs remains unclear [29] [34] [35]. In this study, we attempted to confirm the improvement of amyloid pathogenesis both in vitro and in vivo by Fig. 4. QCI treatment induces ERK activation. "
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    ABSTRACT: Alzheimer's disease is the most prevalent neurodegenerative disorder, characterized by neurofibrillary tangles, senile plaques, and neuron loss. Amyloid-β peptides (Aβ) are generated from amyloid-β precursor protein by consecutive catalysis by β- and γ-secretases. Diversely modified forms of Aβ have been discovered, including pyroglutamate Aβ (N3pE-42 Aβ). N3pE-42 Aβ has received considerable attention as one of the major constituents of the senile plaques of AD brains due to its higher aggregation velocity, stability, and hydrophobicity compared to the full-length Aβ. A previous study suggested that N3pE-42 Aβ formation is catalyzed by glutaminyl cyclase (QC) following limited proteolysis of Aβ at the N-terminus. Here, we reveal that decreasing the QC activity via application of a QC inhibitor modulates γ-secretase activity, resulting in diminished plaque formation as well as reduced N3pE-42 Aβ aggregates in the subiculum of the 5XFAD mouse model of AD. This study suggests a possible novel mechanism by which QC regulates Aβ formation, namely modulation of γ-secretase activity.
    Journal of Alzheimer's disease: JAD 01/2015; 43(3):797-807. DOI:10.3233/JAD-141356 · 4.15 Impact Factor
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    • "The QC-mediated conversion of N-terminal glutamate resulting in the formation of pathogenic pGlu-A␤ peptides has been shown to proceed optimally at mildly acidic conditions (Schilling et al., 2004). It has been shown that the inhibition of QC activity reduces the generation of pGlu-A␤ peptides in vitro (Buchholz et al., 2006; Cynis et al., 2006) and in experimental animals in vivo (Schilling et al., 2008). Based on these observations pGlu-A␤ emerged as a novel target for treatment of A␤ pathology and the inhibition of QC enzymatic activity is regarded as a rational therapeutic approach in AD (Saido, 1998; He and Barrow, 1999; Russo et al., 2002; Saido and Iwata, 2006; Schilling et al., 2006). "
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    ABSTRACT: Glutaminyl cyclases (QCs) catalyze the formation of pyroglutamate (pGlu) from glutamine precursors at the N-terminus of a number of peptide hormones, neuropeptides and chemokines. This post-translational modification stabilizes these peptides, protects them from proteolytical degradation or is important for their biological activity. However, QC is also involved in a pathogenic pGlu modification of peptides accumulating in protein aggregation disorders such as Alzheimer's disease and familial Danish and familial British dementia. Its isoenzyme (isoQC) was shown to contribute to aspects of inflammation by pGlu-modifying and thereby stabilizing the monocyte chemoattractant protein CCL2. For the generation of respective animal models and for pharmacological treatment studies the characterization of the mouse strain and brain region-specific expression of QC and isoQC is indispensible. In order to address this issue, we used enzymatic activity assays and specific antibodies to detect both QC variants by immunohistochemistry in nine different mouse strains. Comparing different brain regions, the highest enzymatic QC/isoQC activity was detected in ventral brain, followed by cortex and hippocampus. Immunohistochemical stainings revealed that QC/isoQC activity in cortex mostly arises from isoQC expression. For most brain regions, the highest QC/isoQC activity was detected in C3H and FVB mice, whereas low QC/isoQC activity was present in CD1, SJL and C57 mice. Quantification of QC- and isoQC-immunoreactive cells by unbiased stereology revealed a higher abundance of isoQC- than of QC-immunoreactive neurons in Edinger-Westphal nucleus and in substantia nigra. In the locus coeruleus, however, there were comparable densities of QC- and of isoQC-immunoreactive neurons. These observations are of considerable importance with regard to the selection of appropriate mouse strains for the study of QC/isoQC relevance in mouse models of neurodegeneration and neuroinflammation and for the testing of therapeutical interventions in these models.
    International Journal of Developmental Neuroscience 05/2014; DOI:10.1016/j.ijdevneu.2014.05.008 · 2.92 Impact Factor
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    • "QC activity was measured by a discontinuous HPLC-method using the substrate H-Gln-␤NA as described previously [27]. Briefly, QC-containing tissue lysate was incubated with substrate H-Gln-␤NA. "
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    ABSTRACT: Brains of Alzheimer's disease (AD) patients are characterized in part by the formation of high molecular weight aggregates of amyloid-β (Aβ) peptides, which interfere with neuronal function and provoke neuronal cell death. The pyroglutamate (pGlu) modification of Aβ was demonstrated to be catalyzed by the enzyme glutaminyl cyclase (QC) and to enhance pathogenicity and neurotoxicity. Here, we addressed the role of QC in AD pathogenesis in human cortex. Two sets of human postmortem brain tissue from a total of 13 non-demented controls and 11 AD cases were analyzed by immunohistochemistry and unbiased stereology, quantitative RT-PCR, and enzymatic activity assays for the expression level of QC in temporal and entorhinal cortex. Additionally, cortical Aβ and pGlu-Aβ concentrations were quantified by ELISA. Data on QC expression and Aβ peptide concentrations were correlated with each other and with the Mini-Mental State Examination (MMSE) of individual cases. In control cases, QC expression was higher in the more vulnerable entorhinal cortex than in temporal cortex. In AD brains, QC mRNA expression and the immunoreactivity of QC were increased in both cortical regions and frequently associated with pGlu-Aβ deposits. The analyses of individual cases revealed significant correlations between QC mRNA levels and the concentration of insoluble pGlu-Aβ aggregates, but not of unmodified Aβ peptides. Elevated pGlu-Aβ load showed a better correlation with the decline in MMSE than elevated concentration of unmodified Aβ. Our observations provide evidence for an involvement of QC in AD pathogenesis and cognitive decline by QC-catalyzed pGlu-Aβ formation.
    Journal of Alzheimer's disease: JAD 10/2013; 39(2). DOI:10.3233/JAD-131535 · 4.15 Impact Factor
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