TY - JOUR A1 - Jost, Priska A1 - Klein, Franziska A1 - Brand, Benjamin A1 - Wahl, Vanessa A1 - Wyatt, Amanda A1 - Yildiz, Daniela A1 - Boehm, Ulrich A1 - Niemeyer, Barbara A. A1 - Vaeth, Martin A1 - Alansary, Dalia T1 - Acute downregulation but not genetic ablation of murine MCU impairs suppressive capacity of regulatory CD4 T cells JF - International Journal of Molecular Sciences N2 - By virtue of mitochondrial control of energy production, reactive oxygen species (ROS) generation, and maintenance of Ca\(^{2+}\) homeostasis, mitochondria play an essential role in modulating T cell function. The mitochondrial Ca\(^{2+}\) uniporter (MCU) is the pore-forming unit in the main protein complex mediating mitochondrial Ca\(^{2+}\) uptake. Recently, MCU has been shown to modulate Ca\(^{2+}\) signals at subcellular organellar interfaces, thus fine-tuning NFAT translocation and T cell activation. The mechanisms underlying this modulation and whether MCU has additional T cell subpopulation-specific effects remain elusive. However, mice with germline or tissue-specific ablation of Mcu did not show impaired T cell responses in vitro or in vivo, indicating that ‘chronic’ loss of MCU can be functionally compensated in lymphocytes. The current work aimed to specifically investigate whether and how MCU influences the suppressive potential of regulatory CD4 T cells (Treg). We show that, in contrast to genetic ablation, acute siRNA-mediated downregulation of Mcu in murine Tregs results in a significant reduction both in mitochondrial Ca\(^{2+}\) uptake and in the suppressive capacity of Tregs, while the ratios of Treg subpopulations and the expression of hallmark transcription factors were not affected. These findings suggest that permanent genetic inactivation of MCU may result in compensatory adaptive mechanisms, masking the effects on the suppressive capacity of Tregs. KW - mitochondrial calcium uniporter KW - regulatory T cells KW - suppressive capacity Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313621 SN - 1422-0067 VL - 24 IS - 9 ER - TY - JOUR A1 - Tejero, Rocio A1 - Alsakkal, Mohammad A1 - Hennlein, Luisa A1 - Lopez-Cabello, Ana M. A1 - Jablonka, Sibylle A1 - Tabares, Lucia T1 - Nifedipine ameliorates cellular differentiation defects of Smn-deficient motor neurons and enhances neuromuscular transmission in SMA mice JF - International Journal of Molecular Sciences N2 - In spinal muscular atrophy (SMA), mutations in or loss of the Survival Motor Neuron 1 (SMN1) gene reduce full-length SMN protein levels, which leads to the degeneration of a percentage of motor neurons. In mouse models of SMA, the development and maintenance of spinal motor neurons and the neuromuscular junction (NMJ) function are altered. Since nifedipine is known to be neuroprotective and increases neurotransmission in nerve terminals, we investigated its effects on cultured spinal cord motor neurons and motor nerve terminals of control and SMA mice. We found that application of nifedipine increased the frequency of spontaneous Ca\(^{2+}\) transients, growth cone size, cluster-like formations of Cav2.2 channels, and it normalized axon extension in SMA neurons in culture. At the NMJ, nifedipine significantly increased evoked and spontaneous release at low-frequency stimulation in both genotypes. High-strength stimulation revealed that nifedipine increased the size of the readily releasable pool (RRP) of vesicles in control but not SMA mice. These findings provide experimental evidence about the ability of nifedipine to prevent the appearance of developmental defects in SMA embryonic motor neurons in culture and reveal to which extent nifedipine could still increase neurotransmission at the NMJ in SMA mice under different functional demands. KW - spinal muscular atrophy KW - motor neurons KW - synaptic transmission KW - neuromuscular junction KW - calcium channels KW - nifedipine KW - growth cone KW - axons KW - synaptic vesicles KW - postsynaptic potentials Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313636 SN - 1422-0067 VL - 24 IS - 8 ER - TY - JOUR A1 - Brenner, Daniela A1 - Geiger, Nina A1 - Schlegel, Jan A1 - Diesendorf, Viktoria A1 - Kersting, Louise A1 - Fink, Julian A1 - Stelz, Linda A1 - Schneider-Schaulies, Sibylle A1 - Sauer, Markus A1 - Bodem, Jochen A1 - Seibel, Jürgen T1 - Azido-ceramides, a tool to analyse SARS-CoV-2 replication and inhibition — SARS-CoV-2 is inhibited by ceramides JF - International Journal of Molecular Sciences N2 - Recently, we have shown that C6-ceramides efficiently suppress viral replication by trapping the virus in lysosomes. Here, we use antiviral assays to evaluate a synthetic ceramide derivative α-NH2-ω-N3-C6-ceramide (AKS461) and to confirm the biological activity of C6-ceramides inhibiting SARS-CoV-2. Click-labeling with a fluorophore demonstrated that AKS461 accumulates in lysosomes. Previously, it has been shown that suppression of SARS-CoV-2 replication can be cell-type specific. Thus, AKS461 inhibited SARS-CoV-2 replication in Huh-7, Vero, and Calu-3 cells up to 2.5 orders of magnitude. The results were confirmed by CoronaFISH, indicating that AKS461 acts comparable to the unmodified C6-ceramide. Thus, AKS461 serves as a tool to study ceramide-associated cellular and viral pathways, such as SARS-CoV-2 infections, and it helped to identify lysosomes as the central organelle of C6-ceramides to inhibit viral replication. KW - ceramides KW - SARS-CoV-2 KW - azido-ceramides KW - sphingolipids Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313581 SN - 1422-0067 VL - 24 IS - 8 ER - TY - JOUR A1 - Müller, Patrick A1 - Meta, Mergim A1 - Meidner, Jan Laurenz A1 - Schwickert, Marvin A1 - Meyr, Jessica A1 - Schwickert, Kevin A1 - Kersten, Christian A1 - Zimmer, Collin A1 - Hammerschmidt, Stefan Josef A1 - Frey, Ariane A1 - Lahu, Albin A1 - de la Hoz-Rodríguez, Sergio A1 - Agost-Beltrán, Laura A1 - Rodríguez, Santiago A1 - Diemer, Kira A1 - Neumann, Wilhelm A1 - Gonzàlez, Florenci V. A1 - Engels, Bernd A1 - Schirmeister, Tanja T1 - Investigation of the compatibility between warheads and peptidomimetic sequences of protease inhibitors — a comprehensive reactivity and selectivity study JF - International Journal of Molecular Sciences N2 - Covalent peptidomimetic protease inhibitors have gained a lot of attention in drug development in recent years. They are designed to covalently bind the catalytically active amino acids through electrophilic groups called warheads. Covalent inhibition has an advantage in terms of pharmacodynamic properties but can also bear toxicity risks due to non-selective off-target protein binding. Therefore, the right combination of a reactive warhead with a well-suited peptidomimetic sequence is of great importance. Herein, the selectivities of well-known warheads combined with peptidomimetic sequences suited for five different proteases were investigated, highlighting the impact of both structure parts (warhead and peptidomimetic sequence) for affinity and selectivity. Molecular docking gave insights into the predicted binding modes of the inhibitors inside the binding pockets of the different enzymes. Moreover, the warheads were investigated by NMR and LC-MS reactivity assays against serine/threonine and cysteine nucleophile models, as well as by quantum mechanics simulations. KW - covalent inhibitors KW - in vitro study KW - protease inhibitors KW - peptidomimetic sequence KW - warhead KW - reactivity and selectivity study Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-313596 SN - 1422-0067 VL - 24 IS - 8 ER - TY - JOUR A1 - Glaser, Kirsten A1 - Kern, David A1 - Speer, Christian P. A1 - Schlegel, Nicolas A1 - Schwab, Michael A1 - Thome, Ulrich H. A1 - Härtel, Christoph A1 - Wright, Clyde J. T1 - Imbalanced inflammatory responses in preterm and term cord blood monocytes and expansion of the CD14\(^+\)CD16\(^+\) subset upon toll-like receptor stimulation JF - International Journal of Molecular Sciences N2 - Developmentally regulated features of innate immunity are thought to place preterm and term infants at risk of infection and inflammation-related morbidity. Underlying mechanisms are incompletely understood. Differences in monocyte function including toll-like receptor (TLR) expression and signaling have been discussed. Some studies point to generally impaired TLR signaling, others to differences in individual pathways. In the present study, we assessed mRNA and protein expression of pro- and anti-inflammatory cytokines in preterm and term cord blood (CB) monocytes compared with adult controls stimulated ex vivo with Pam3CSK4, zymosan, polyinosinic:polycytidylic acid, lipopolysaccharide, flagellin, and CpG oligonucleotide, which activate the TLR1/2, TLR2/6, TLR3, TLR4, TLR5, and TLR9 pathways, respectively. In parallel, frequencies of monocyte subsets, stimulus-driven TLR expression, and phosphorylation of TLR-associated signaling molecules were analyzed. Independent of stimulus, pro-inflammatory responses of term CB monocytes equaled adult controls. The same held true for preterm CB monocytes—except for lower IL-1β levels. In contrast, CB monocytes released lower amounts of anti-inflammatory IL-10 and IL-1ra, resulting in higher ratios of pro-inflammatory to anti-inflammatory cytokines. Phosphorylation of p65, p38, and ERK1/2 correlated with adult controls. However, stimulated CB samples stood out with higher frequencies of intermediate monocytes (CD14\(^+\)CD16\(^+\)). Both pro-inflammatory net effect and expansion of the intermediate subset were most pronounced upon stimulation with Pam3CSK4 (TLR1/2), zymosan (TR2/6), and lipopolysaccharide (TLR4). Our data demonstrate robust pro-inflammatory and yet attenuated anti-inflammatory responses in preterm and term CB monocytes, along with imbalanced cytokine ratios. Intermediate monocytes, a subset ascribed pro-inflammatory features, might participate in this inflammatory state. KW - neonatal immunology KW - inflammation KW - preterm infants KW - monocytes KW - cord blood KW - monocyte subsets KW - cytokines KW - Toll-like receptor signaling Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311056 SN - 1422-0067 VL - 24 IS - 5 ER - TY - JOUR A1 - Michalke, Bernhard A1 - Venkataramani, Vivek T1 - Editorial to the special issue “Homeostasis: metals and cellular redox and immunity status” JF - International Journal of Molecular Sciences N2 - No abstract available KW - homeostasis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311061 SN - 1422-0067 VL - 24 IS - 5 ER - TY - JOUR A1 - Watermann, Christoph A1 - Meyer, Malin Tordis A1 - Wagner, Steffen A1 - Wittekindt, Claus A1 - Klussmann, Jens Peter A1 - Erguen, Sueleyman A1 - Baumgart-Vogt, Eveline A1 - Karnati, Srikanth T1 - Peroxisomes are highly abundant and heterogeneous in human parotid glands JF - International Journal of Molecular Sciences N2 - The parotid gland is one of the major salivary glands producing a serous secretion, and it plays an essential role in the digestive and immune systems. Knowledge of peroxisomes in the human parotid gland is minimal; furthermore, the peroxisomal compartment and its enzyme composition in the different cell types of the human parotid gland have never been subjected to a detailed investigation. Therefore, we performed a comprehensive analysis of peroxisomes in the human parotid gland’s striated duct and acinar cells. We combined biochemical techniques with various light and electron microscopy techniques to determine the localization of parotid secretory proteins and different peroxisomal marker proteins in parotid gland tissue. Moreover, we analyzed the mRNA of numerous gene encoding proteins localized in peroxisomes using real-time quantitative PCR. The results confirm the presence of peroxisomes in all striated duct and acinar cells of the human parotid gland. Immunofluorescence analyses for various peroxisomal proteins showed a higher abundance and more intense staining in striated duct cells compared to acinar cells. Moreover, human parotid glands comprise high quantities of catalase and other antioxidative enzymes in discrete subcellular regions, suggesting their role in protection against oxidative stress. This study provides the first thorough description of parotid peroxisomes in different parotid cell types of healthy human tissue. KW - peroxisomes KW - parotid gland KW - human KW - catalase KW - differential expression KW - PSP KW - mRNA KW - immunofluorescence Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-311079 SN - 1422-0067 VL - 24 IS - 5 ER - TY - JOUR A1 - Geiger, Nina A1 - Diesendorf, Viktoria A1 - Roll, Valeria A1 - König, Eva-Maria A1 - Obernolte, Helena A1 - Sewald, Katherina A1 - Breidenbach, Julian A1 - Pillaiyar, Thanigaimalai A1 - Gütschow, Michael A1 - Müller, Christa E. A1 - Bodem, Jochen T1 - Cell type-specific anti-viral effects of novel SARS-CoV-2 main protease inhibitors JF - International Journal of Molecular Sciences N2 - Recently, we have described novel pyridyl indole esters and peptidomimetics as potent inhibitors of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) main protease. Here, we analysed the impact of these compounds on viral replication. It has been shown that some antivirals against SARS-CoV-2 act in a cell line-specific way. Thus, the compounds were tested in Vero, Huh-7, and Calu-3 cells. We showed that the protease inhibitors at 30 µM suppress viral replication by up to 5 orders of magnitude in Huh-7 cells, while in Calu-3 cells, suppression by 2 orders of magnitude was achieved. Three pyridin-3-yl indole-carboxylates inhibited viral replication in all cell lines, indicating that they might repress viral replication in human tissue as well. Thus, we investigated three compounds in human precision-cut lung slices and observed donor-dependent antiviral activity in this patient-near system. Our results provide evidence that even direct-acting antivirals may act in a cell line-specific manner. KW - SARS-CoV-2 KW - protease inhibitors KW - cell line specificity pyridyl indole carboxylates KW - azapeptide nitriles KW - peptidomimetics Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304034 SN - 1422-0067 VL - 24 IS - 4 ER - TY - JOUR A1 - Feigl, Frederik Fabian A1 - Stahringer, Anika A1 - Peindl, Matthias A1 - Dandekar, Gudrun A1 - Koehl, Ulrike A1 - Fricke, Stephan A1 - Schmiedel, Dominik T1 - Efficient redirection of NK cells by genetic modification with chemokine receptors CCR4 and CCR2B JF - International Journal of Molecular Sciences N2 - Natural killer (NK) cells are a subset of lymphocytes that offer great potential for cancer immunotherapy due to their natural anti-tumor activity and the possibility to safely transplant cells from healthy donors to patients in a clinical setting. However, the efficacy of cell-based immunotherapies using both T and NK cells is often limited by a poor infiltration of immune cells into solid tumors. Importantly, regulatory immune cell subsets are frequently recruited to tumor sites. In this study, we overexpressed two chemokine receptors, CCR4 and CCR2B, that are naturally found on T regulatory cells and tumor-resident monocytes, respectively, on NK cells. Using the NK cell line NK-92 as well as primary NK cells from peripheral blood, we show that genetically engineered NK cells can be efficiently redirected using chemokine receptors from different immune cell lineages and migrate towards chemokines such as CCL22 or CCL2, without impairing the natural effector functions. This approach has the potential to enhance the therapeutic effect of immunotherapies in solid tumors by directing genetically engineered donor NK cells to tumor sites. As a future therapeutic option, the natural anti-tumor activity of NK cells at the tumor sites can be increased by co-expression of chemokine receptors with chimeric antigen receptors (CAR) or T cell receptors (TCR) on NK cells can be performed in the future. KW - chemokine receptor KW - migration KW - immune cell infiltration KW - trafficking KW - NK cells KW - immunotherapy KW - CCR2 KW - CCR4 KW - genetic engineering Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304049 SN - 1422-0067 VL - 24 IS - 4 ER - TY - JOUR A1 - Goeritzer, Madeleine A1 - Kuentzel, Katharina B. A1 - Beck, Sarah A1 - Korbelius, Melanie A1 - Rainer, Silvia A1 - Bradić, Ivan A1 - Kolb, Dagmar A1 - Mussbacher, Marion A1 - Schrottmaier, Waltraud C. A1 - Assinger, Alice A1 - Schlagenhauf, Axel A1 - Rost, René A1 - Gottschalk, Benjamin A1 - Eichmann, Thomas O. A1 - Züllig, Thomas A1 - Graier, Wolfgang F. A1 - Vujić, Nemanja A1 - Kratky, Dagmar T1 - Monoglyceride lipase deficiency is associated with altered thrombogenesis in mice JF - International Journal of Molecular Sciences N2 - Monoglyceride lipase (MGL) hydrolyzes monoacylglycerols (MG) to glycerol and one fatty acid. Among the various MG species, MGL also degrades 2-arachidonoylglycerol, the most abundant endocannabinoid and potent activator of the cannabinoid receptors 1 and 2. We investigated the consequences of MGL deficiency on platelet function using systemic (Mgl\(^{−/−}\)) and platelet-specific Mgl-deficient (platMgl\(^{−/−}\)) mice. Despite comparable platelet morphology, loss of MGL was associated with decreased platelet aggregation and reduced response to collagen activation. This was reflected by reduced thrombus formation in vitro, accompanied by a longer bleeding time and a higher blood volume loss. Occlusion time after FeCl\(_3\)-induced injury was markedly reduced in Mgl\(^{−/−}\) mice, which is consistent with contraction of large aggregates and fewer small aggregates in vitro. The absence of any functional changes in platelets from platMgl\(^{−/−}\) mice is in accordance with lipid degradation products or other molecules in the circulation, rather than platelet-specific effects, being responsible for the observed alterations in Mgl\(^{−/−}\) mice. We conclude that genetic deletion of MGL is associated with altered thrombogenesis. KW - platelets KW - MGL KW - in vitro and in vivo thrombus formation KW - platelet activation KW - platelet aggregation Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-304052 SN - 1422-0067 VL - 24 IS - 4 ER -