Preprint
Refine
Document Type
- Preprint (34) (remove)
Language
- English (34)
Has Fulltext
- yes (34)
Is part of the Bibliography
- no (34)
Keywords
- Antigens/Peptides/Epitopes (1)
- BRAF (1)
- Buparlisib (1)
- CRISPR/Cas9 (1)
- EGFR (1)
- HER2 (1)
- HLA class I (1)
- HRAS 28 Gefitinib (1)
- Human (1)
- MET (1)
Institute
- Biochemie und Chemie (34) (remove)
The ubiquitin (Ub) code denotes the complex Ub architectures, including Ub chains of different length, linkage-type and linkage combinations, which enable ubiquitination to control a wide range of protein fates. Although many linkage-specific interactors have been described, how interactors are able to decode more complex architectures is not fully understood. We conducted a Ub interactor screen, in humans and yeast, using Ub chains of varying length, as well as, homotypic and heterotypic branched chains of the two most abundant linkage types – K48- and K63-linked Ub. We identified some of the first K48/K63 branch-specific Ub interactors, including histone ADP-ribosyltransferase PARP10/ARTD10, E3 ligase UBR4 and huntingtin-interacting protein HIP1. Furthermore, we revealed the importance of chain length by identifying interactors with a preference for Ub3 over Ub2 chains, including Ub-directed endoprotease DDI2, autophagy receptor CCDC50 and p97-adaptor FAF1. Crucially, we compared datasets collected using two common DUB inhibitors – Chloroacetamide and N-ethylmaleimide. This revealed inhibitor-dependent interactors, highlighting the importance of inhibitor consideration during pulldown studies. This dataset is a key resource for understanding how the Ub code is read.
With the emergence of immunotherapies, the understanding of functional HLA class I antigen presentation to T cells is more relevant than ever. Current knowledge on antigen presentation is based on decades of research in a wide variety of cell types with varying antigen presentation machinery (APM) expression patterns, proteomes and HLA haplotypes. This diversity complicates the establishment of individual APM contributions to antigen generation, selection and presentation. Therefore, we generated a novel Panel of APM Knockout Cell lines (PAKC) from the same genetic origin. After CRISPR/Cas9 genome-editing of ten individual APM components in a human cell line, we derived clonal cell lines and confirmed their knockout status and phenotype. We then show how PAKC will accelerate research on the functional interplay between APM components and their role in antigen generation and presentation. This will lead to improved understanding of peptide-specific T cell responses in infection, cancer and autoimmunity.
Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and to structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness is a key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned by cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen thickness for cryo-ET on lamellae has not been systematically investigated. Furthermore, the ions used to ablate material can cause damage in the lamellae, thereby reducing STA resolution. Here, we systematically benchmark the resolution depending on lamella thickness and the depth of the particles within the sample. Up to ca. 180 nm, lamella thickness does not negatively impact resolution. This shows that there is no need to generate very thin lamellae and thickness can be chosen such that it captures major cellular features. Furthermore, we show that gallium-ion-induced damage extends to depths of up to 30 nm from either lamella surface.
Nuclear pore complexes (NPCs) constitute giant channels within the nuclear envelope that mediate nucleocytoplasmic exchange. NPC diameter is thought to be regulated by nuclear envelope tension, but how such diameter changes are physiologically linked to cell differentiation, where mechanical properties of nuclei are remodeled and nuclear mechanosensing occurs, remains unstudied. Here we used cryo-electron tomography to show that NPCs dilate during differentiation of mouse embryonic stem cells into neural progenitors. In Nup133-deficient cells, which are known to display impaired neural differentiation, NPCs however fail to dilate. By analyzing the architectures of individual NPCs with template matching, we revealed that the Nup133-deficient NPCs are structurally heterogeneous and frequently disintegrate, resulting in the formation of large nuclear envelope openings. We propose that the elasticity of the NPC scaffold mechanically safeguards the nuclear envelope. Our studies provide a molecular explanation for how genetic perturbation of scaffolding components of macromolecular complexes causes tissue-specific phenotypes.
Stimulated emission depletion (STED) microscopy is a super-resolution technique that surpasses the diffraction limit and has contributed to the study of dynamic processes in living cells. However, high laser intensities induce fluorophore photobleaching and sample phototoxicity, limiting the number of fluorescence images obtainable from a living cell. Here, we address these challenges by using ultra-low irradiation intensities and a neural network for image restoration, enabling extensive imaging of single living cells. The endoplasmic reticulum (ER) was chosen as the target structure due to its dynamic nature over short and long timescales. The reduced irradiation intensity combined with denoising permitted continuous ER dynamics observation in living cells for up to 7 hours with a temporal resolution of seconds. This allowed for quantitative analysis of ER structural features over short (seconds) and long (hours) timescales within the same cell, and enabled fast 3D live-cell STED microscopy. Overall, the combination of ultra-low irradiation with image restoration enables comprehensive analysis of organelle dynamics over extended periods in living cells.
Oncogenic transformation of lung epithelial cells is a multi-step process, frequently starting with the inactivation of tumor suppressors and subsequent activating mutations in proto-oncogenes, such as members of the PI3K or MAPK family. Cells undergoing transformation have to adjust to changes, such as metabolic requirements. This is achieved, in part, by modulating the protein abundance of transcription factors, which manifest these adjustments. Here, we report that the deubiquitylase USP28 enables oncogenic reprogramming by regulating the protein abundance of proto-oncogenes, such as c-JUN, c-MYC, NOTCH and ΔNP63, at early stages of malignant transformation. USP28 is increased in cancer compared to normal cells due to a feed-forward loop, driven by increased amounts of oncogenic transcription factors, such as c-MYC and c-JUN. Irrespective of oncogenic driver, interference with USP28 abundance or activity suppresses growth and survival of transformed lung cells. Furthermore, inhibition of USP28 via a small molecule inhibitor reset the proteome of transformed cells towards a ‘pre-malignant’ state, and its inhibition cooperated with clinically established compounds used to target EGFRL858R, BRAFV600E or PI3KH1047R driven tumor cells. Targeting USP28 protein abundance already at an early stage via inhibition of its activity therefore is a feasible strategy for the treatment of early stage lung tumours and the observed synergism with current standard of care inhibitors holds the potential for improved targeting of established tumors.
Light-driven sodium pumps (NaRs) are unique ion-transporting microbial rhodopsins. The major group of NaRs is characterized by an NDQ motif and has two aspartic acid residues in the central region essential for sodium transport. Here we identified a new subgroup of the NDQ rhodopsins bearing an additional glutamic acid residue in the close vicinity to the retinal Schiff base. We thoroughly characterized a member of this subgroup, namely the protein ErNaR from Erythrobacter sp. HL-111 and showed that the additional glutamic acid results in almost complete loss of pH sensitivity for sodium-pumping activity, which is in contrast to previously studied NaRs. ErNaR is capable of transporting sodium efficiently even at acidic pH levels. X-ray crystallography and single particle cryo-electron microscopy reveal that the additional glutamic acid residue mediates the connection between the other two Schiff base counterions and strongly interacts with the aspartic acid of the characteristic NDQ motif. Hence, it reduces its pKa. Our findings shed light on a new subgroup of NaRs and might serve as a basis for their rational optimization for optogenetics.
The TOM complex is the main entry point for precursor proteins into mitochondria. Precursor proteins containing targeting sequences are recognized by the TOM complex and imported into the mitochondria. We have determined the structure of the TOM core complex from Neurospora crassa by single-particle cryoEM at 3.3 Å resolution, showing its interaction with a bound presequence at 4 Å resolution, and of the TOM holo complex including the Tom20 receptor at 6-7 Å resolution. TOM is a transmembrane complex consisting of two β-barrels, three receptor subunits and three short transmembrane subunits. Tom20 has a transmembrane helix and a receptor domain on the cytoplasmic side. We propose that Tom20 acts as a dynamic gatekeeper, guiding precursor proteins into the pores of the TOM complex. We analyze the interactions of Tom20 with other TOM subunits, present insights into the structure of the TOM holo complex, and suggest a translocation mechanism.
The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350-400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy. Using antibodies labeled with short DNA oligonucleotides, multiple targets are visualized successively by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. The structural integrity of the tissue is maintained owing to only a single labelling step during sample preparation. Multiple targets are imaged using a single laser wavelength, minimizing chromatic aberration. This method proved robust for multi-target imaging in semi-thin tissue sections, paving the way towards structural cell biology with single-molecule super-resolution microscopy.
DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a super-resolution technique with relatively easy-to-implement multi-target imaging. However, image acquisition is slow as sufficient statistical data has to be generated from spatio-temporally isolated single emitters. Here, we trained the neural network (NN) DeepSTORM to predict fluorophore positions from high emitter density DNA-PAINT data. This achieves image acquisition in one minute. We demonstrate multi-color super-resolution imaging of structure-conserved semi-thin neuronal tissue and imaging of large samples. This improvement can be integrated into any single-molecule microscope and enables fast single-molecule super-resolution microscopy.