Biology subjects

Thiede, B.

Publications and source records attributed to Thiede, B..

6 recordsLinked to original sources

bioRxiv2026

Proteome-wide multi-omics profiling of osteosarcoma transcription factor networks

Osteosarcoma (OS) is an aggressive bone cancer that most commonly affects children and young adults. OS exhibits a high degree of genomic complexity, as well as cellular plasticity, and dynamic transcriptional regulation is suggested to contribute to treatment resistance and metastasis. Cell lines are well characterized as models to advance our knowledge on OS biology. HOS and U2OS cells have increased invasiveness and higher migratory ability compared with MG63. In this study, we employed a tandem array of consensus transcription factor response elements (catTFREs) proteomic approach to characterize transcription factor (TF) regulatory networks related to OS aggressiveness. We mapped 7,594 proteins and enriched 352 transcription factors and coregulators. When we integrated proteomics with cell line specific gene expression and chromatin accessibility we classified the proteins into different OS cell line dependent sub-clusters and identified TFs and coregulators common for all cell lines and specific for individual cell lines. We demonstrate that RUNX2, MYBL2 and HMGA2 are specifically enriched in HOS and U2OS and may be linked to the cell aggressiveness. ETV5, JUNB, NFIX and ZEB1 were among TFs specific to MG63. Our analysis provides a more comprehensive understanding of the transcriptional drivers that shape OS regulatory landscapes and may have future therapeutic implications.

biochemistry↗

bioRxiv2022

ERK3/MAPK6 dictates Cdc42/Rac1 activity and ARP2/3-dependent actin polymerization

The actin cytoskeleton is tightly controlled by RhoGTPases, actin binding proteins and nucleation-promoting factors to perform fundamental cellular functions. Here, we show that ERK3, an atypical MAPK, directly acts as a guanine nucleotide exchange factor for Cdc42 and phosphorylates the ARP3 subunit of the ARP2/3 complex at S418 to promote filopodia formation and actin polymerization, respectively. Consistently, depletion of ERK3 prevented both basal and EGF-dependent Rac1 and Cdc42 activation, maintenance of F-actin content, filopodia formation and epithelial cell migration. Further, ERK3 protein binds directly to the purified ARP2/3 complex and augments polymerization of actin in vitro. ERK3 kinase activity is required for the formation of actin-rich protrusions in mammalian cells. These findings unveil a fundamentally unique pathway employed by cells to control actin-dependent cellular functions.

cell biology↗

bioRxiv2022

Identification of a phage display-derived peptide interacting with the N-terminal region of Factor VII activating protease (FSAP) enables characterization of zymogen activation.

Increased Factor VII activating protease (FSAP) activity has a protective effect in diverse disease conditions as inferred from studies in FSAP-/- mice and humans deficient in FSAP activity due to a single nucleotide polymorphism. The activation of FSAP zymogen in plasma is mediated by extracellular histones that are released during tissue injury or inflammation or by positively charged surfaces. However, it is not clear if this activation mechanism is specific and amenable to manipulation. Using a phage display approach we have identified a peptide, NNKC9/41, that activates pro-FSAP in plasma. Other commonly found zymogens in the plasma were not activated. Binding studies with FSAP domain deletion mutants indicate that the N-terminus of FSAP is the key interaction site of this peptide. Blocking the contact pathway of coagulation did not influence pro-FSAP activation by the peptide. In a monoclonal antibody screen, we identified MA-FSAP-38C7 that prevented the activation of pro-FSAP by the peptide. This antibody bound to the LESLDP sequence (amino acids 30-35) in the N-terminus of FSAP. The plasma clotting time was shortened by NNKC9/41 and this was reversed by MA-FSAP-38C7 demonstrating the utility of this peptide. Identification of this peptide, and the corresponding interaction site, provides proof of principle that it is possible to activate a single protease zymogen in blood in a specific manner. Peptide NNKC/41 will be useful as a tool to delineate the molecular mechanism of activation of pro-FSAP in more detail, elucidate its biological role. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/475526v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@112eea6org.highwire.dtl.DTLVardef@1f1d7eaorg.highwire.dtl.DTLVardef@967b03org.highwire.dtl.DTLVardef@1662c51_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2021

Public T-cell epitopes shared among SARS-CoV-2 variants are presented on prevalent HLA class I alleles

T-cell epitopes with broad population coverage may form the basis for a new generation of SARS-CoV-2 vaccines. However, published studies on immunoprevalence are limited by small test cohorts, low frequencies of antigen-specific cells and lack of data correlating eluted HLA ligands with T-cell responsiveness. As the protective role of pre-existing cross-reactivity to homologous peptides is unclear, we aimed to identify SARS-CoV-2-specific minimal epitopes recognized by CD8 T-cells among 48 peptides eluted from prevalent HLA alleles, and an additional 84 predicted binders, in a large cohort of convalescents (n=83) and pre-pandemic control samples (n=19). We identified nine conserved SARS-CoV-2-specific epitopes restricted by four of the most prevalent HLA class I alleles in the Norwegian study cohort, to which responding CD8 T cells were detected in 70-100% of convalescents expressing the relevant HLA allele. Only two of these were derived from the Spike protein, included in current vaccines. We found a strong correlation between immunoprevalence and immunodominance. Thus, the CD8 T-cell response to SARS-CoV-2 is more focused than previously believed. Using a new algorithm, we predict that a vaccine including these epitopes could induce a T-cell response in 83% of Caucasians.

immunology↗

bioRxiv2019

Armadillo repeat-containing protein 1 is a dual localization protein associated with mitochondrial intermembrane space bridging complex

Cristae architecture is important for the function of mitochondria, the organelles that play the central role in many cellular processes. The mitochondrial contact site and cristae organizing system (MICOS) together with the sorting and assembly machinery (SAM) forms the mitochondrial intermembrane space bridging complex (MIB), a large protein complex present in mammalian mitochondria that partakes in the formation and maintenance of cristae. We report here a new subunit of the mammalian MICOS/MIB complex, an armadillo repeat-containing protein 1 (ArmC1). ArmC1 localizes both to cytosol and mitochondria, where it associates with the outer mitochondrial membrane through its carboxy-terminus. ArmC1 interacts with other constituents of the MICOS/MIB complex and its amounts are reduced upon MICOS/MIB complex depletion. Mitochondria lacking ArmC1 do not show defects in cristae structure, respiration or protein content, but appear fragmented and with reduced motility. ArmC1 represents therefore a peripheral MICOS/MIB component that appears to play a role in mitochondrial distribution in the cell.

cell biology↗

bioRxiv2018

Critical comparison of ultracentrifugation and a commercial kit for isolation of exosomes derived from glioblastoma and breast cancer cell lines

Exosomes are small extracellular vesicles around 30-100 nm in diameter that are secreted from cells and can be found in most body fluids. Exosomes can be a vital source of biomarkers as they contain various substances (e.g. lipids, RNAs, metabolites and proteins) that can reflect the cell of origin (e.g. cancer cells). For isolation of exosomes present in biological matrices, ultracentrifugation (UC)-based procedures are most common. Other approaches exist, including commercial kits developed for easy and low sample volume isolation. In this study, differential UC and an isolation kit from a major vendor (Total Exosome Isolation Reagent from Thermo Fisher Scientific) were compared. Exosomes were isolated from cell culture media of two different cell sources (patient derived cells from glioblastoma multiforme and the breast cancer cell line MDA-MB-231). For both isolation methods, transmission electron microscopy, dynamic light scattering and western blotting indicated the presence of exosomes. The kit- and UC isolates contained similar amounts of protein measured by the bicinchoninic acid (BCA) assay with absorbance at 562 nm. Using western blot, positive exosome markers were identified in all isolates, and additional exosome markers were identified using MS-based proteomics. For the glioblastoma exosome isolates, the number of proteins identified with liquid chromatography tandem MS (LC-MS/MS) was higher for the UC isolates than the kit isolates when injecting equal protein amounts, contrary to that for the breast cancer exosome isolates. However, negative exosome markers were also found in glioblastoma isolates using LC-MS/MS. Thus, we would not use the term \"exosome isolation\" as impurities may be present with both isolation methods. Notably, potential biomarkers for both diseases were identified in the isolates using LS-MS/MS. In our opinion, the two isolation methods had rather similar performance, although with some minor differences based on cell of origin.

biochemistry↗