Browsing by Affiliation "Biochemistry"
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- Research DataMechanism of the glycan-driven MHC I quality control cycle mediated by a dedicated chaperone network2025-04-24Trowitzsch, SimonTampé, RobertHeinke, Tim Julius (DataCollector)Fahim, Amin (DataCollector)Trowitzsch, Simon (ContactPerson)Tampé, Robert (ContactPerson)Protein folding in the endoplasmic reticulum (ER) is crucial for about one third of the mammalian proteome. N-linked glycosylation and subsequent restructuring of glycans barcodes glycoproteins during their maturation. UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) and the chaperones calnexin and calreticulin together with glucosidase I play a vital role in this process. MHC I molecules, key for adaptive immunity, additionally rely on the specialized chaperones tapasin and TAPBPR (TAP-binding protein-related) for their maturation and loading of antigenic peptides. Here, we delineate the functional interplay between tapasin, TAPBPR, UGGT1, and calreticulin, during recycling of MHC I molecules via purified components. The transfer of peptide-receptive MHC I from TAPBPR back to tapasin relies on the recognition of the mono-glucosylated glycan by calreticulin. Our findings unveil a finetuned dynamic network of glycan-dependent and MHC I-specific chaperones that guarantee maturation of MHC I molecules and highlight the fundamental processes driving ER protein quality control.
37 6 - Research DataNMR screening of low molecular weight inhibitors targeting the papain-like protease (PLPro) of SARS-CoV-22025-03-10Pyper, Dennis JoshuaSreeramulu, SridharLanham, Benjamin T.Engle, Elizabeth M.Fushman, DavidThe Papain-like protease (PLPro) from SARS-CoV-2 plays an important role in the cleavage of the polyproteins Pp1a and Pp1ab as well as in the suppression of the immune response by deISG15ylation. Considerable effort is therefore devoted to developing low molecular weight inhibitors as starting points for antiviral drugs. Here, we present the results of an NMR screening study of PLPro for binding to the DSI-poised fragment library containing 607 compounds. Based on Saturation-Transfer Difference (STD)- and WaterLOGSY-NMR experiments, we identified 86 binding compounds. We prioritized five candidates for further in-depth analysis. For three of those, we determined dissociation constants and two distinct binding sites on PLPro.
32 6 - Research DataStructural Insights into Spare-Tire DNA G-Quadruplex from the Human VEGF Promoter2025-03-10The vascular endothelial growth factor (VEGF) promoter region, which is involved in cancer progression, con-tains guanine-rich sequences capable of forming G-quadruplex (G4) structures. G4s play a critical role in transcriptional regulation and genomic stability and exhibit high structural polymorphism. The major VEGF G4 adopts a parallel topology involving the first four of five G-tracts (VEGF1234), while a potential "spare-tire" mechanism suggests the formation of VEGF1245 in response to oxidative damage. Here, we characterize this alternative G4 (VEGF1245), formed by excluding the third G-tract, using circular dichroism (CD) and nu-clear magnetic resonance (NMR) spectroscopy. Structural analysis reveals that VEGF1245 folds in a hybrid conformation. Different to other five tract containing G4s, for which various stand topologies can rapidly interconvert, VEGF1245 remains thermodynamically metastable and does not refold spontaneously into VEGF1234 at physiological temperatures. Further trapping of the VEGF1245 conformation by a photolabile protecting group and its in-situ release documents that the transition to VEGF1234 requires elevated tem-peratures, implicating kinetic barriers in the refolding process and the delineation of VEGF1245 as prominent metastable conformation. Our findings provide new insights into transcriptional regulation and DNA repair for the cancer-related VEGF-G4.
20 3 - Research DataTargeting the SARS-CoV-2 RNA translation initiation element SL1 by molecules of low molecular weight2025-03-25We present the development of low molecular weight inhibitors that target the 5’-terminal RNA stem-loop 1 (SL1) of the SARS-CoV-2 genome. SL1 is crucial for allowing viral protein synthesis in the context of global translation repression in infected cells. We applied compound- and RNA-detected nuclear magnetic resonance spectroscopy (NMR) experiments to guide a fragment-growth strategy based on two primary NMR screening hits from a diverse fragment library poised for follow-up chemistry. These primary hits with molecular weights of around 200 Da were derivatized with the aim to improve solubility, binding affinity, and target specificity. We used NMR to monitor solubility changes, binding affinity, and specific binding to the SL1 binding pocket along the fragment derivatization campaign. The six compounds scoring best in all three categories were tested for their inhibitory effect on SL1 in a cell-free translation assay, where the best two compounds, A.2 and A.13, showed both significant and selective inhibition. Our results demonstrate that small molecules targeting translation initiation of SARS-CoV-2 can be rapidly obtained using NMR-guided medicinal chemistry, and that the correlation between affinity, selectivity, and in situ function of the derived compounds is still to be explored.
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