Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.

The development of a targeted therapy would significantly improve the treatment of periodontitis and its associated diseases including Alzheimer 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 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 ß-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.

Mammalian-like type II glutaminyl cyclases in Porphyromonas gingivalis and other oral pathogenic bacteria as targets for treatment of periodontitis.

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 ß-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.

CD33-directed immunotherapy with third-generation chimeric antigen receptor T cells and gemtuzumab ozogamicin in intact and CD33-edited acute myeloid leukemia and hematopoietic stem and progenitor cells.

Immunotherapies, such as chimeric antigen receptor (CAR) modified T cells and antibody-drug conjugates (ADCs), have revolutionized the treatment of cancer, especially of lymphoid malignancies. The application of targeted immunotherapy to patients with acute myeloid leukemia (AML) has been limited in particular by the lack of a tumor-specific target antigen. Gemtuzumab ozogamicin (GO), an ADC targeting CD33, is the only approved immunotherapeutic agent in AML. In our study, we introduce a CD33-directed third-generation CAR T-cell product (3G.CAR33-T) for the treatment of patients with AML. 3G.CAR33-T cells could be expanded up to the end-of-culture, that is, 17 days after transduction, and displayed significant cytokine secretion and robust cytotoxic activity when incubated with CD33-positive cells including cell lines, drug-resistant cells, primary blasts as well as normal hematopoietic stem and progenitor cells (HSPCs). When compared to second-generation CAR33-T cells, 3G.CAR33-T cells exhibited higher viability, increased proliferation and stronger cytotoxicity. Also, GO exerted strong antileukemia activity against CD33-positive AML cells. Upon genomic deletion of CD33 in HSPCs, 3G.CAR33-T cells and GO preferentially killed wildtype leukemia cells, while sparing CD33-deficient HSPCs. Our data provide evidence for the applicability of CD33-targeted immunotherapies in AML and its potential implementation in CD33 genome-edited stem cell transplantation approaches.

Combination of the Glutaminyl Cyclase Inhibitor PQ912 (Varoglutamstat) and the Murine Monoclonal Antibody PBD-C06 (m6) Shows Additive Effects on Brain Aß Pathology in Transgenic Mice.

Compelling evidence suggests that pyroglutamate-modified Aß (pGlu3-Aß; AßN3pG) peptides play a pivotal role in the development and progression of Alzheimer's disease (AD). Approaches targeting pGlu3-Aß by glutaminyl cyclase (QC) inhibition (Varoglutamstat) or monoclonal antibodies (Donanemab) are currently in clinical development. Here, we aimed at an assessment of combination therapy of Varoglutamstat (PQ912) and a pGlu3-Aß-specific antibody (m6) in transgenic mice. Whereas the single treatments at subtherapeutic doses show moderate (16-41%) but statistically insignificant reduction of Aß42 and pGlu-Aß42 in mice brain, the combination of both treatments resulted in significant reductions of Aß by 45-65%. Evaluation of these data using the Bliss independence model revealed a combination index of ≈1, which is indicative for an additive effect of the compounds. The data are interpreted in terms of different pathways, in which the two drugs act. While PQ912 prevents the formation of pGlu3-Aß in different compartments, the antibody is able to clear existing pGlu3-Aß deposits. The results suggest that combination of the small molecule Varoglutamstat and a pE3Aß-directed monoclonal antibody may allow a reduction of the individual compound doses while maintaining the therapeutic effect.

Structural and functional analyses of pyroglutamate-amyloid-ß-specific antibodies as a basis for Alzheimer immunotherapy.

Alzheimer disease is associated with deposition of the amyloidogenic peptide Aß in the brain. Passive immunization using Aß-specific antibodies has been demonstrated to reduce amyloid deposition both in vitro and in vivo Because N-terminally truncated pyroglutamate (pE)-modified Aß species (AßpE3) exhibit enhanced aggregation potential and propensity to form toxic oligomers, they represent particularly attractive targets for antibody therapy. Here we present three separate monoclonal antibodies that specifically recognize AßpE3 with affinities of 1-10 nm and inhibit AßpE3 fibril formation in vitro. In vivo application of one of these resulted in improved memory in AßpE3 oligomer-treated mice. Crystal structures of Fab-AßpE3 complexes revealed two distinct binding modes for the peptide. Juxtaposition of pyroglutamate pE3 and the F4 side chain (the "pEF head") confers a pronounced bulky hydrophobic nature to the AßpE3 N terminus that might explain the enhanced aggregation properties of the modified peptide. The deep burial of the pEF head by two of the antibodies explains their high target specificity and low cross-reactivity, making them promising candidates for the development of clinical antibodies.

Passive Aß Immunotherapy: Current Achievements and Future Perspectives.

Passive immunotherapy has emerged as a very promising approach for the treatment of Alzheimer's disease and other neurodegenerative disorders, which are characterized by the misfolding and deposition of amyloid peptides. On the basis of the amyloid hypothesis, the majority of antibodies in clinical development are directed against amyloid ß (Aß), the primary amyloid component in extracellular plaques. This review focuses on the current status of Aß antibodies in clinical development, including their characteristics and challenges that came up in clinical trials with these new biological entities (NBEs). Emphasis is placed on the current view of common side effects observed with passive immunotherapy, so-called amyloid-related imaging abnormalities (ARIAs), and potential ways to overcome this issue. Among these new ideas, a special focus is placed on molecules that are directed against post-translationally modified variants of the Aß peptide, an emerging approach for development of new antibody molecules.

Immunohistochemical Evidence from APP-Transgenic Mice for Glutaminyl Cyclase as Drug Target to Diminish pE-Abeta Formation.

Oligomeric assemblies of neurotoxic amyloid beta (Abeta) peptides generated by proteolytical processing of the amyloid precursor protein (APP) play a key role in the pathogenesis of Alzheimer's disease (AD). In recent years, a substantial heterogeneity of Abeta peptides with distinct biophysical and cell biological properties has been demonstrated. Among these, a particularly neurotoxic and disease-specific Abeta variant is N-terminally truncated and modified to pyroglutamate (pE-Abeta). Cell biological and animal experimental studies imply the catalysis of this modification by the enzyme glutaminyl cyclase (QC). However, direct histopathological evidence in transgenic animals from comparative brain region and cell type-specific expression of transgenic hAPP and QC, on the one hand, and on the formation of pE-Abeta aggregates, on the other, is lacking. Here, using single light microscopic, as well as triple immunofluorescent, labeling, we report the deposition of pE-Abeta only in the brain regions of APP-transgenic Tg2576 mice with detectable human APP and endogenous QC expression, such as the hippocampus, piriform cortex, and amygdala. Brain regions showing human APP expression without the concomitant presence of QC (the anterodorsal thalamic nucleus and perifornical nucleus) do not display pE-Abeta plaque formation. However, we also identified brain regions with substantial expression of human APP and QC in the absence of pE-Abeta deposition (the Edinger-Westphal nucleus and locus coeruleus). In these brain regions, the enzymes required to generate N-truncated Abeta peptides as substrates for QC might be lacking. Our observations provide additional evidence for an involvement of QC in AD pathogenesis via QC-catalyzed pE-Abeta formation.

Neuropeptide Y (NPY) in cerebrospinal fluid from patients with Huntington's Disease: increased NPY levels and differential degradation of the NPY1-30 fragment.

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. Consistent with this, we report here on increased immuno-linked NPY (IL-NPY) levels in human cerebrospinal fluid (hCSF) from HD patients (Control n = 10; early HD n = 9; mid HD n = 11). As this antibody-based detection of NPY may provide false positive differences as a result of 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 detailed epitope analysis verified significant differences in IL-NPY1-36/3-36 and NPY1-30 levels between HD patients and control subjects with no significant differences between early vs mid HD cases. Ex vivo degradomics analysis demonstrated that NPY is initially degraded to NPY1-30 by cathepsin D in both HD patients and control subjects. Yet, NPY1-30 is then further differentially hydrolyzed by thimet oligopeptidase (TOP) in HD patients and by neprilysin (NEP) in control subjects. Furthermore, altered hCSF TOP-inhibitor Dynorphin A1-13 (Dyn-A1-13 ) and TOP-substrate Dyn-A1-8 levels indicate an impaired Dyn-A-TOP network in HD patients. Thus, we conclude that elevated IL-NPY-levels in conjunction with TOP-/NEP-activity/protein as well as Dyn-A1-13 -peptide levels may serve as a potential biomarker in human CSF of HD. Huntington's disease (HD) patients' cerebrospinal fluid (CSF) exhibits higher neuropeptide Y (NPY) levels. Further degradomics studies show that CSF-NPY is initially degraded to NPY1-30 by Cathepsin D. The NPY1-30 fragment is then differentially degraded in HD vs control involving Neprilysin (NEP), Thimet Oligopeptidase (TOP), and TOP-Dynorphin-A network. Together, these findings may help in search for HD biomarkers.

Enhanced Fibril Fragmentation of N-Terminally Truncated and Pyroglutamyl-Modified Aß Peptides.

N-terminal truncation and pyroglutamyl (pE) formation are naturally occurring chemical modifications of the Aß peptide in Alzheimer's disease. We show herein that these two modifications significantly reduce the fibril length and the transition midpoint of thermal unfolding of the fibrils, but they do not substantially perturb the fibrillary peptide conformation. This observation implies that the N terminus of the unmodified peptide protects Aß fibrils against mechanical stress and fragmentation and explains the high propensity of pE-modified peptides to form small and particularly toxic aggregates.

A non-canonical function of eukaryotic elongation factor 1A1: regulation of interleukin-6 expression.

Interleukin-6 is one of the most prominent triggers of inflammatory processes. We have shown recently that heteroarylketones (HAKs) interfere with stimulated interleukin-6 expression in astrocytes by suppression of STAT3 phosphorylation at serine 727. Surprisingly, this effect is not based on the inhibition of STAT3-relevant kinases. Therefore, we here used the structurally modified HAK compound biotin-HAK-3 in a reverse chemical approach to identify the relevant molecular target in UV-mediated cross-linking experiments. Employing streptavidin-specific 2D-immunoblotting followed by mass spectrometry we identified nine proteins putatively interacting with biotin-HAK-3. After co-immunoprecipitation, co-immunofluorescence, surface plasmon resonance analyses and RNAi-mediated knock-down, the eukaryotic elongation factor 1A1 (eEF1A1) was verified as the relevant target of HAK bioactivity. eEF1A1 forms complexes with STAT3 and PKCδ, which are crucial for STAT3(S727) phosphorylation and for NF-κB/STAT3-enhanced interleukin-6 expression. Furthermore, the intracellular HAK accumulation is strongly dependent on eEF1A1 expression. Taken together, the results reveal a novel molecular mechanism for a non-canonical role of eEF1A1 in signal transduction via direct modulation of kinase-dependent phosphorylation events.

Proteolytic degradation of neuropeptide Y (NPY) from head to toe: Identification of novel NPY-cleaving peptidases and potential drug interactions in CNS and Periphery.

The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application. The bioactivity of neuropeptide Y (NPY) is either N-terminally modulated with respect to receptor selectivity by dipeptidyl peptidase 4 (DP4)-like enzymes or proteolytic degraded by neprilysin or meprins, thereby abrogating signal transduction. However, neither the subcellular nor the compartmental differentiation of these regulatory mechanisms is fully understood. Using mass spectrometry, selective inhibitors and histochemistry, studies across various cell types, body fluids, and tissues revealed that most frequently DP4-like enzymes, aminopeptidases P, secreted meprin-A (Mep-A), and cathepsin D (CTSD) rapidly hydrolyze NPY, depending on the cell type and tissue under study. Novel degradation of NPY by cathepsins B, D, L, G, S, and tissue kallikrein could also be identified. The expression of DP4, CTSD, and Mep-A at the median eminence indicates that the bioactivity of NPY is regulated by peptidases at the interphase between the periphery and the CNS. Detailed ex vivo studies on human sera and CSF samples recognized CTSD as the major NPY-cleaving enzyme in the CSF, whereas an additional C-terminal truncation by angiotensin-converting enzyme could be detected in serum. The latter finding hints to potential drug interaction between antidiabetic DP4 inhibitors and anti-hypertensive angiotensin-converting enzyme inhibitors, while it ablates suspected hypertensive side effects of only antidiabetic DP4-inhibitors application.

Contribution of amino acids in the active site of dipeptidyl peptidase 4 to the catalytic action of the enzyme.

Dipeptidyl peptidase 4 (DP4)/CD26 regulates the biological function of various peptide hormones by releasing dipeptides from their N-terminus. The enzyme is a prominent target for the treatment of type-2 diabetes and various DP4 inhibitors have been developed in recent years, but their efficacy and side effects are still an issue. Many available crystal structures of the enzyme give a static picture about enzyme-ligand interactions, but the influence of amino acids in the active centre on binding and single catalysis steps can only be judged by mutagenesis studies. In order to elucidate their contribution to inhibitor binding and substrate catalysis, especially in discriminating the P1 amino acid of substrates, the amino acids R125, N710, E205 and E206 were investigated by mutagenesis studies. Our studies demonstrated, that N710 is essential for the catalysis of dipeptide substrates. We found that R125 is not important for dipeptide binding but interacts in the P1`position of the peptide backbone. In contrast to dipeptide substrates both amino acids play an essential role in the binding and arrangement of long natural substrates, particularly if lacking proline in the P1 position. Thus, it can be assumed that the amino acids R125 and N710 are important in the DP4 catalysed substrate hydrolysis by interacting with the peptide backbone of substrates up- and downstream of the cleavage site. Furthermore, we confirmed the important role of the amino acids E205 and E206. However, NP Y, displaying proline in P1 position, is still processed without the participation of E205 or E206.

Identification of isoaspartate-modified transthyretin as potential target for selective immunotherapy of transthyretin amyloidosis.

BACKGROUND: Numerous studies suggest a progressive accumulation of post-translationally modified peptides within amyloid fibrils, including isoaspartate (isoD) modifications. Here, we generated and characterised novel monoclonal antibodies targeting isoD-modified transthyretin (TTR). The antibodies were used to investigate the presence of isoD-modified TTR in deposits from transthyretin amyloidosis patients and to mediate antibody-dependent phagocytosis of TTR fibrils. METHODS: Monoclonal antibodies were generated by immunisation of mice using an isoD-modified peptide and subsequent hybridoma generation. The antibodies were characterised in terms of affinity and specificity to isoD-modified TTR using surface plasmon resonance, transmission electron microscopy and immunohistochemical staining of human cardiac tissue. The potential to elicit antibody-dependent phagocytosis of TTR fibrils was assessed using THP-1 cells. RESULTS: We developed two mouse monoclonal antibodies, 2F2 and 4D4, with high nanomolar affinity for isoD-modified TTR and strong selectivity over the unmodified epitope. Both antibodies show presence of isoD-modified TTR in human cardiac tissue, but not in freshly purified recombinant TTR, suggesting isoD modification only present in aged fibrillar deposits. Likewise, the antibodies only facilitated phagocytosis of TTR fibrils and not TTR monomers by THP-1 cells. CONCLUSIONS: These antibodies label aged, non-native TTR deposits, leaving native TTR unattended and thereby potentially enabling new therapeutic approaches.

Consequences of Amyloid-ß Deficiency for the Liver.

The hepatic content of amyloid beta (Aß) decreases drastically in human and rodent cirrhosis highlighting the importance of understanding the consequences of Aß deficiency in the liver. This is especially relevant in view of recent advances in anti-Aß therapies for Alzheimer's disease (AD). Here, it is shown that partial hepatic loss of Aß in transgenic AD mice immunized with Aß antibody 3D6 and its absence in amyloid precursor protein (APP) knockout mice (APP-KO), as well as in human liver spheroids with APP knockdown upregulates classical hallmarks of fibrosis, smooth muscle alpha-actin, and collagen type I. Aß absence in APP-KO and deficiency in immunized mice lead to strong activation of transforming growth factor-ß (TGFß), alpha secretases, NOTCH pathway, inflammation, decreased permeability of liver sinusoids, and epithelial-mesenchymal transition. Inversely, increased systemic and intrahepatic levels of Aß42 in transgenic AD mice and neprilysin inhibitor LBQ657-treated wild-type mice protect the liver against carbon tetrachloride (CCl4)-induced injury. Transcriptomic analysis of CCl4-treated transgenic AD mouse livers uncovers the regulatory effects of Aß42 on mitochondrial function, lipid metabolism, and its onco-suppressive effects accompanied by reduced synthesis of extracellular matrix proteins. Combined, these data reveal Aß as an indispensable regulator of cell-cell interactions in healthy liver and a powerful protector against liver fibrosis.

Structure of glutaminyl cyclase from Drosophila melanogaster in space group I4.

The structure of ligand-free glutaminyl cyclase (QC) from Drosophila melanogaster (DmQC) has been determined in a novel crystal form. The protein crystallized in space group I4, with unit-cell parameters a = b = 122.3, c = 72.7 Å. The crystal diffracted to a resolution of 2 Å at the home source. The structure was solved by molecular replacement and was refined to an R factor of 0.169. DmQC exhibits a typical α/ß-hydrolase fold. The electron density of three monosaccharides could be localized. The accessibility of the active site will facilitate structural studies of novel inhibitor-binding modes.

Crystal structures of glutaminyl cyclases (QCs) from Drosophila melanogaster reveal active site conservation between insect and mammalian QCs.

Glutaminyl cyclases (QCs), which catalyze the formation of pyroglutamic acid (pGlu) at the N-terminus of a variety of peptides and proteins, have attracted particular attention for their potential role in Alzheimer's disease. In a transgenic Drosophila melanogaster (Dm) fruit fly model, oral application of the potent competitive QC inhibitor PBD150 was shown to reduce the burden of pGlu-modified Aß. In contrast to mammals such as humans and rodents, there are at least three DmQC species, one of which (isoDromeQC) is localized to mitochondria, whereas DromeQC and an isoDromeQC splice variant possess signal peptides for secretion. Here we present the recombinant expression, characterization, and crystal structure determination of mature DromeQC and isoDromeQC, revealing an overall fold similar to that of mammalian QCs. In the case of isoDromeQC, the putative extended substrate binding site might be affected by the proximity of the N-terminal residues. PBD150 inhibition of DromeQC is roughly 1 order of magnitude weaker than that of the human and murine QCs. The inhibitor binds to isoDromeQC in a fashion similar to that observed for human QCs, whereas it adopts alternative binding modes in a DromeQC variant lacking the conserved cysteines near the active center and shows a disordered dimethoxyphenyl moiety in wild-type DromeQC, providing an explanation for the lower affinity. Our biophysical and structural data suggest that isoDromeQC and human QC are similar with regard to functional aspects. The two Dm enzymes represent a suitable model for further in-depth analysis of the catalytic mechanism of animal QCs, and isoDromeQC might serve as a model system for the structure-based design of potential AD therapeutics.

Evidence for Enhanced Efficacy of Passive Immunotherapy against Beta-Amyloid in CD33-Negative 5xFAD Mice.

Passive immunotherapy is a very promising approach for the treatment of Alzheimer's disease (AD). Among the different antibodies under development, those targeting post-translationally modified Aß peptides might combine efficient reduction in beta-amyloid accompanied by lower sequestration in peripheral compartments and thus anticipated and reduced treatment-related side effects. In that regard, we recently demonstrated that the antibody-mediated targeting of isoD7-modified Aß peptides leads to the attenuation of AD-like amyloid pathology in 5xFAD mice. In order to assess novel strategies to enhance the efficacy of passive vaccination approaches, we investigated the role of CD33 for Aß phagocytosis in transgenic mice treated with an isoD7-Aß antibody. We crossbred 5xFAD transgenic mice with CD33 knock out (CD33KO) mice and compared the amyloid pathology in the different genotypes of the crossbreds. The knockout of CD33 in 5xFAD mice leads to a significant reduction in Aß plaques and concomitant rescue of behavioral deficits. Passive immunotherapy of 5xFAD/CD33KO showed a significant increase in plaque-surrounding microglia compared to 5xFAD treated with the antibody. Additionally, we observed a stronger lowering of Aß plaque load after passive immunotherapy in 5xFAD/CD33KO mice. The data suggest an additive effect of passive immunotherapy and CD33KO in terms of lowering Aß pathology. Hence, a combination of CD33 antagonists and monoclonal antibodies might represent a strategy to enhance efficacy of passive immunotherapy in AD.

Structures of glycosylated mammalian glutaminyl cyclases reveal conformational variability near the active center.

Formation of N-terminal pyroglutamate (pGlu or pE) from glutaminyl or glutamyl precursors is catalyzed by glutaminyl cyclases (QC). As the formation of pGlu-amyloid has been linked with Alzheimer's disease, inhibitors of QCs are currently the subject of intense development. Here, we report three crystal structures of N-glycosylated mammalian QC from humans (hQC) and mice (mQC). Whereas the overall structures of the enzymes are similar to those reported previously, two surface loops in the neighborhood of the active center exhibit conformational variability. Furthermore, two conserved cysteine residues form a disulfide bond at the base of the active center that was not present in previous reports of hQC structure. Site-directed mutagenesis suggests a structure-stabilizing role of the disulfide bond. At the entrance to the active center, the conserved tryptophan residue, W(207), which displayed multiple orientations in previous structure, shows a single conformation in both glycosylated human and murine QCs. Although mutagenesis of W(207) into leucine or glutamine altered substrate conversion significantly, the binding constants of inhibitors such as the highly potent PQ50 (PBD150) were minimally affected. The crystal structure of PQ50 bound to the active center of murine QC reveals principal binding determinants provided by the catalytic zinc ion and a hydrophobic funnel. This study presents a first comparison of two mammalian QCs containing typical, conserved post-translational modifications.

Isolation of dipeptidyl peptidase IV (DP 4) isoforms from porcine kidney by preparative isoelectric focusing to improve crystallization.

Abstract In the present studies we resolved the post-translational microheterogeneity of purified porcine dipeptidyl peptidase IV (DP 4) from kidney cortex. Applying SDS-homogeneous DP 4 onto an analytical agarose isoelectric focusing (IEF) gel, pH 4-6, activity staining resulted in at least 17 isoforms between pH 4.8-6.0. These could be separated into fractions with only two to six isoforms by means of preparative liquid-phase IEF, using a Rotofor cell. Starting off with three parallel Rotofor runs under the same conditions at pH 5-6, the fractions were pooled according to the specific activity of DP 4, pH and analytical IEF profile, and further refractionated without any additional ampholytes. Since excessive dilution of ampholytes and proteins was kept to the minimum, a second refractionation step could be introduced, resulting in pH gradients between 0.022 and 0.028 pH increments per fraction. By performing two consecutive refractionation steps, the high resolution necessary for the separation of DP 4 isoforms could be achieved. This represents an alternative method if isolation of isoforms with similar pI's results in precipitation and denaturation in presence of a narrow pH range. Furthermore, it demonstrates that preparative IEF is a powerful tool to resolve post-translational microheterogeneity of a purified protein required for crystallization processing.

Selective inhibition of dipeptidyl peptidase 4 by targeting a substrate-specific secondary binding site.

Dipeptidyl peptidase 4/CD26 (DP4) is a multifunctional serine protease liberating dipeptide from the N-terminus of (oligo)peptides which can modulate the activity of these peptides. The enzyme is involved in physiological processes such as blood glucose homeostasis and immune response. DP4 substrate specificity is characterized in detail using synthetic dipeptide derivatives. The specificity constant k(cat)/K(m) strongly depends on the amino acid in P1-position for proline, alanine, glycine and serine with 5.0 x 105 M⁻¹ s⁻¹, 1.8 x 104 M⁻¹ s⁻¹, 3.6 x 10² M⁻¹ s⁻¹, 1.1 x 10² M⁻¹ s⁻¹, respectively. By contrast, kinetic investigation of larger peptide substrates yields a different pattern. The specific activity of DP4 for neuropeptide Y (NPY) cleavage comprising a proline in P1-position is the same range as the k(cat)/K(m) values of NPY derivatives containing alanine or serine in P1-position with 4 x 105 M⁻¹ s⁻¹, 9.5 x 105 M⁻¹ s⁻¹ and 2.1 x 105 M⁻¹ s⁻¹, respectively. The proposed existence of an additional binding region outside the catalytic center is supported by measurements of peptide substrates with extended chain length. This 'secondary' binding site interaction depends on the amino acid sequence in P4'-P8'-position. Interactions with this binding site could be specifically blocked for substrates of the GRF/glucagon peptide family. By contrast, substrates not belonging to this peptide family and dipeptide derivative substrates that only bind to the catalytic center of DP4 were not inhibited. This more selective inhibition approach allows, for the first time, to distinguish between substrate families by substrate-discriminating inhibitors.