Schlussbericht zum Förderprogramm

Datei-Upload durch TI

Untersuchungen zur Rolle der Glutaminyl-Cyclase bei der pathologischen Bildung von Pyroglutamyl-Peptiden - [kumulative Dissertation]

Posttranslationale Modifikationen spielen eine wichtige Rolle bei der Reifung von Proteinen und beeinflussen deren Funktion und Stabilität im Organismus. Die Bildung von Pyroglutamat (pGlu) ist für eine Reihe von Peptidhormonen, wie z.B. Thyreoliberin (TRH) und Gonadoliberin (GnRH) sowie sezernierten Proteinen beschrieben worden. Die pGlu-Modifikation entsteht durch intramolekulare Zyklisierung eines Glutaminylrestes. Die Reaktion wird in Tieren und Pflanzen durch Glutaminyl-Cyclasen (QC; EC 2.3.2.5) katalysiert. Im menschlichen Organismus geht offensichtlich eine Reihe von pathophysiologischen Veränderungen mit einer Bildung von Pyroglutamylpeptiden einher. Dies betrifft u.a. neurodegenerative Erkrankungen wie die Alzheimersche Krankheit oder entzündliche Prozesse. In der vorliegenden Arbeit wurde die QC-vermittelte Bildung von pGlu am N-Terminus der Peptide Aβ und CCL2 untersucht, welchen offenbar Schlüsselfunktionen in diesen Erkrankungen zukommen. Es konnte gezeigt werden, dass QC auch die Zyklisierung von Glutamat am N-Terminus von Aβ-Peptiden nach amyloidogener und Prohormonkonvertase-vermittelter Prozessierung in Säugerzellkultur katalysiert. Die Applikation eines spezifischen QC-Inhibitors verringerte sowohl die Glutaminyl- als auch die Glutamylzyklisierung. Die QC-katalysierte Bildung von pGlu-Aβ verläuft in den zellulären Modellsystemen vorwiegend intrazellulär. Der N-terminale pGlu-Rest von CCL2 beeinflusst die Stabilität und somit die chemotaktische Potenz dieses Chemokins. Durch eine Sezernierung von nicht vollständig gereiftem CCL2 kommt es zu einem Abbau durch Aminopeptidasen, wie z.B. DP4. Die Applikation eines QCInhibitors beeinflusste die arteriosklerotischen Veränderungen im Modell der „cuff“-induzierten Arteriosklerose. In einem weiteren Teil der Arbeit konnte ein Isoenzym der QC isoliert und charakterisiert werden. Humane QC und isoQC weisen eine Sequenzidentität von ca. 50% auf. Im Unterschied zur QC ist die isoQC ein Typ II-Transmembranprotein, welches im Golgi-Apparat zurückgehalten wird. Diesbezüglich ähnelt das Protein Glykosyltransferasen. Aufgrund der stabilisierenden Wirkung des pGlu-Restes für den N-Terminus verschiedener Proteine hat die Inhibierung von QC eine proteolysefördernde und dadurch aktivitätsmodulierende Wirkung. Dies könnte zur Behandlung verschiedener Krankheitssymptome dienen, welche durch das Auftreten von stabilen pGlu-Peptiden verursacht werden. Insofern sind die im Rahmen dieser Arbeit gewonnenen Erkenntnisse für die Entwicklung spezifischer QC-Inhibitoren zur Behandlung einer Reihe von pGlu-Proteinabhängigen Erkrankungen bedeutsam.von Holger Cyni

Orally bioavailable RORγ/DHODH dual host-targeting small molecules with broad-spectrum antiviral activity

Host-directed antivirals (HDAs) represent an attractive treatment option and a strategy for pandemic preparedness, especially due to their potential broad-spectrum antiviral activity and high barrier to resistance development. Particularly, dual-targeting HDAs offer a promising approach for antiviral therapy by simultaneously disrupting multiple pathways essential for viral replication. Izumerogant (IMU-935) targets two host proteins, (i) the retinoic acid receptor-related orphan receptor γ isoform 1 (RORγ1), which modulates cellular cholesterol metabolism, and (ii) the enzyme dihydroorotate dehydrogenase (DHODH), which is involved in de novo pyrimidine synthesis. Here, we synthesized optimized derivatives of izumerogant and characterized their antiviral activity in comparison to a recently described structurally distinct RORγ/DHODH dual inhibitor. Cell culture-based infection models for enveloped and non-enveloped DNA and RNA viruses, as well as a retrovirus, demonstrated high potency and broad-spectrum activity against human viral pathogens for RORγ/DHODH dual inhibitors at nanomolar concentrations. Comparative analyses with equipotent single-target inhibitors in metabolite supplementation approaches revealed that the dual-targeting mode represents the mechanistic basis for the potent antiviral activity. For SARS-CoV-2, an optimized dual inhibitor completely blocked viral replication in human airway epithelial cells at 5 nM and displayed a synergistic drug interaction with the nucleoside analog molnupiravir. In a SARS-CoV-2 mouse model, treatment with a dual inhibitor alone, or in combination with molnupiravir, reduced the viral load by 7- and 58-fold, respectively. Considering the clinical safety, oral bioavailability, and tolerability of izumerogant in a recent Phase I study, izumerogant-like drugs represent potent dual-targeting antiviral HDAs with pronounced broad-spectrum activity for further clinical development

Immunogenicity of the Envelope Surface Unit of Human Endogenous Retrovirus K18 in Mice

The triggers for the development of multiple sclerosis (MS) have not been fully understood to date. One hypothesis proposes a viral etiology. Interestingly, viral proteins from human endogenous retroviruses (HERVs) may play a role in the pathogenesis of MS. Allelic variants of the HERV-K18 env gene represent a genetic risk factor for MS, and the envelope protein is considered to be an Epstein–Barr virus-trans-activated superantigen. To further specify a possible role for HERV-K18 in MS, the present study examined the immunogenicity of the purified surface unit (SU). HERV-K18(SU) induced envelope-specific plasma IgG in immunized mice and triggered proliferation of T cells isolated from these mice. It did not trigger phenotypic changes in a mouse model of experimental autoimmune encephalomyelitis. Further studies are needed to investigate the underlying mechanisms of HERV-K18 interaction with immune system regulators in more detail

Identification of differentially expressed human endogenous retrovirus families in human leukemia and lymphoma cell lines and stem cells

Endogenous retroviruses (ERVs) are becoming more and more relevant in cancer research and might be potential targets. The oncogenic potential of human ERVs (HERVs) has been recognized and includes immunosuppression, cell fusion, antigenicity of viral proteins, and regulation of neighboring genes. To decipher the role of HERVs in human cancers, we used a bioinformatics approach and analyzed RNA sequencing data from the LL-100 panel, covering 22 entities of hematopoietic neoplasias including T cell, B cell and myeloid malignancies. We compared HERV expression in this panel with hematopoietic stem cells (HSCs), embryonic stem cells (ESCs) and normal blood cells. RNA sequencing data were mapped against a comprehensive synthetic viral metagenome with 116 HERV sequences from 14 different HERV families. Of these, 13 HERV families and elements were differently expressed in malignant hematopoietic cells and stem cells. We found transcriptional upregulation of HERVE family in acute megakaryocytic and erythroid leukemia and of HERVFc family in multiple myeloma/plasma cell leukemia (PCL). The HERVFc member HERVFc-1 was found transcriptionally active in the multiple myeloma cell line OPM-2 and also in the Hodgkin lymphoma cell line L-428. The expression of HERVFc-1 in L-428 cells was validated by qRT-PCR. We also confirm transcriptional downregulation of ERV3 in acute megakaryocytic and erythroid leukemia, and HERVK in acute monocytic and myelocytic leukemia and a depression of HERVF in all malignant entities. Most of the higher expressed HERV families could be detected in stem cells including HERVK (HML-2), HERV-like, HERVV, HERVT, ERV9, HERVW, HERVF, HERVMER, ERV3, HERVH and HERVPABLB.Publikationsfonds ML

Dipeptidyl peptidase 4 deficiency improves survival after focal cerebral ischemia in mice and ameliorates microglia activation and specific inflammatory markers

Dipeptidyl peptidase 4 (DPP4; CD26) is involved in the regulation of various metabolic, immunological, and neurobiological processes in healthy individuals. Observations based on epidemiological data indicate that DPP4 inhibition by gliptins, typically used in patients with diabetes, may reduce the risk for cerebral ischemia and may also improve related outcomes. However, as DPP4 inhibitor application is neither complete nor specific for suppression of DPP4 enzymatic activity and DPP4 has non-enzymatic functions as well, the variety of consequences is a matter of debate. Therefore, we here used DPP4 knock-out (KO) mice to analyze the specific contribution of DPP4 to cellular, immunological, and functional consequences of experimental focal cerebral ischemia. We observed a significantly higher survival rate of DPP4 KO mice after ischemia, which was accompanied by a lower abundance of the pro-inflammatory chemokine CCL2 and reduced activation of Iba1-positive microglia cells in brain tissue of DPP4 KO mice. In addition, after ischemia for 24 h to 72 h, decreased concentrations of CCL5 and CCL12 in plasma and of CCL17 in brain tissue of DPP4 KO mice were observed when compared to wild type mice. Other aspects analyzed, such as the functional Menzies score, astrocyte activation and chemokine levels in plasma and brain tissue were affected by ischemia but appeared to be unaffected by the DPP4 KO genotype. Taken together, experimental ablation of DPP4 functions in mice improves survival and ameliorates aspects of cellular and molecular inflammation after focal cerebral ischemia

Glutaminyl cyclase contributes to the formation of focal and diffuse pyroglutamate (pGlu)-Aβ deposits in hippocampus via distinct cellular mechanisms

In the hippocampal formation of Alzheimer’s disease (AD) patients, both focal and diffuse deposits of Aβ peptides appear in a subregion- and layer-specific manner. Recently, pyroglutamate (pGlu or pE)-modified Aβ peptides were identified as a highly pathogenic and seeding Aβ peptide species. Since the pE modification is catalyzed by glutaminyl cyclase (QC) this enzyme emerged as a novel pharmacological target for AD therapy. Here, we reveal the role of QC in the formation of different types of hippocampal pE-Aβ aggregates. First, we demonstrate that both, focal and diffuse pE-Aβ deposits are present in defined layers of the AD hippocampus. While the focal type of pE-Aβ aggregates was found to be associated with the somata of QC-expressing interneurons, the diffuse type was not. To address this discrepancy, the hippocampus of amyloid precursor protein transgenic mice was analysed. Similar to observations made in AD, focal (i.e. core-containing) pE-Aβ deposits originating from QC-positive neurons and diffuse pE-Aβ deposits not associated with QC were detected in Tg2576 mouse hippocampus. The hippocampal layers harbouring diffuse pE-Aβ deposits receive multiple afferents from QC-rich neuronal populations of the entorhinal cortex and locus coeruleus. This might point towards a mechanism in which pE-Aβ and/or QC are being released from projection neurons at hippocampal synapses. Indeed, there are a number of reports demonstrating the reduction of diffuse, but not of focal, Aβ deposits in hippocampus after deafferentation experiments. Moreover, we demonstrate in neurons by live cell imaging and by enzymatic activity assays that QC is secreted in a constitutive and regulated manner. Thus, it is concluded that hippocampal pE-Aβ plaques may develop through at least two different mechanisms: intracellularly at sites of somatic QC activity as well as extracellularly through seeding at terminal fields of QC expressing projection neurons

3D-printed microstructured alginate scaffolds for neural tissue engineering

Alginate (Alg) is a versatile biopolymer for scaffold engineering and a bioink component widely used for direct cell printing. However, due to a lack of intrinsic cell-binding sites, Alg must be functionalized for cellular adhesion when used as a scaffold. Moreover, direct cell-laden ink 3D printing requires tedious disinfection procedures and cell viability is compromised by shear stress. Here, we demonstrate proof-of-concept, bioactive additive-free, microstructured Alg (M-Alg) scaffolds for neuron culture. The M-Alg scaffold was formed by introducing tetrapod-shaped ZnO (t-ZnO) microparticles into the ink as structural templates for interconnected channels and textured surfaces in the 3D-printed Alg scaffold, which were subsequently removed. Neurons exhibited significantly improved adhesion and growth on these M-Alg scaffolds compared with pristine Alg (P-Alg) scaffolds, with extensive neurite outgrowth and spontaneous neural activity, indicating the maturation of neuronal networks. These transparent, porous, additive-free Alg-based scaffolds with neuron affinity are promising for neuroregenerative and organoid-related research

Dipeptidyl-Peptidase Activity of Meprin β Links N-truncation of Aβ with Glutaminyl Cyclase-Catalyzed pGlu-Aβ Formation

The formation of amyloid-β (Aβ) peptides is causally involved in the development of Alzheimer’s disease (AD). A significant proportion of deposited Aβ is N-terminally truncated and modified at the N-terminus by a pGlu-residue (pGlu-Aβ). These forms show enhanced neurotoxicity compared to full-length Aβ. Although the truncation may occur by aminopeptidases after formation of Aβ, recently discovered processing pathways of amyloid-β protein precursor (AβPP) by proteases such as meprin β may also be involved. Here, we assessed a role of meprin β in forming Aβ3 -40/42 , which is the precursor of pGlu-Aβ3 -40/42 generated by glutaminyl cyclase (QC). Similar to QC, meprin β mRNA is significantly upregulated in postmortem brain from AD patients. A histochemical analysis supports the presence of meprin β in neurons and astrocytes in the vicinity of pGlu-Aβ containing deposits. Cleavage of AβPP-derived peptides by meprin β in vitro results in peptides Aβ1 -x , Aβ2 -x , and Aβ3 -x . The formation of N-truncated Aβ by meprin β was also corroborated in cell culture. A subset of the generated peptides was converted into pGlu-Aβ3 -40 by an addition of glutaminyl cyclase, supporting the preceding formation of Aβ3 -40 . Further analysis of the meprin β cleavage revealed a yet unknown dipeptidyl-peptidase–like activity specific for the N-terminus of Aβ1 -x . Thus, our data suggest that meprin β contributes to the formation of N-truncated Aβ by endopeptidase and exopeptidase activity to generate the substrate for QC-catalyzed pGlu-Aβ formation

Glutaminyl cyclase-mediated toxicity of pyroglutamate-beta amyloid induces striatal neurodegeneration

Background Posttranslational modifications of beta amyloid (Aβ) have been shown to affect its biophysical and neurophysiological properties. One of these modifications is N-terminal pyroglutamate (pE) formation. Enzymatic glutaminyl cyclase (QC) activity catalyzes cyclization of truncated Aβ(3-x), generating pE3-Aβ. Compared to unmodified Aβ, pE3-Aβ is more hydrophobic and neurotoxic. In addition, it accelerates aggregation of other Aβ species. To directly investigate pE3-Aβ formation and toxicity in vivo, transgenic (tg) ETNA (E at the truncated N-terminus of Aβ) mice expressing truncated human Aβ(3–42) were generated and comprehensively characterized. To further investigate the role of QC in pE3-Aβ formation in vivo, ETNA mice were intercrossed with tg mice overexpressing human QC (hQC) to generate double tg ETNA-hQC mice. Results Expression of truncated Aβ(3–42) was detected mainly in the lateral striatum of ETNA mice, leading to progressive accumulation of pE3-Aβ. This ultimately resulted in astrocytosis, loss of DARPP-32 immunoreactivity, and neuronal loss at the sites of pE3-Aβ formation. Neuropathology in ETNA mice was associated with behavioral alterations. In particular, hyperactivity and impaired acoustic sensorimotor gating were detected. Double tg ETNA-hQC mice showed similar Aβ levels and expression sites, while pE3-Aβ were significantly increased, entailing increased astrocytosis and neuronal loss. Conclusions ETNA and ETNA-hQC mice represent novel mouse models for QC-mediated toxicity of truncated and pE-modified Aβ. Due to their significant striatal neurodegeneration these mice can also be used for analysis of striatal regulation of basal locomotor activity and sensorimotor gating, and possibly for DARPP-32-dependent neurophysiology and neuropathology. The spatio-temporal correlation of pE3-Aβ and neuropathology strongly argues for an important role of this Aβ species in neurodegenerative processes in these models