Biology subjects

Schwarzer, C.

Publications and source records attributed to Schwarzer, C..

4 recordsLinked to original sources

bioRxiv2024

EasyAb: A High-Throughput Workflow for Antibody-Based PTM Peptide Enrichment Method Coupled to Mass Spectrometry

Reversible post-translational modification (PTMs) is a fundamental mechanism of cellular signal transduction. In vivo and ex vivo studies to profile PTMs have greatly advanced our understanding of the complexities of cellular signaling. However, apart from the most commonly studied PTMs, large-scale analysis is still very challenging, limiting our understanding of various cellular processes. PTM-bearing peptides often enriched by antibodies, followed by unbiased mass spectrometry (MS)-based readout of hundreds or thousands of sites. To extend the reach of this powerful technology to in vivo and ex vivo studies with small protein starting amounts, we here developed EasyAb, a streamlined and high throughput MS workflow for antibody-based PTM profiling. Using epidermal growth factor receptor (EGFR) signaling and acute myeloid leukemia (AML) cell systems, we demonstrate that EasyAb increases sensitivity and enables multiple large-scale systems-level studies. Furthermore, EasyAb resolves in vivo brain G protein-coupled receptor-mediated tyrosine kinase activation and reveals the long elusive hypothalamic neuron-specific leptin signaling architecture. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=125 SRC="FIGDIR/small/620939v1_ufig1.gif" ALT="Figure 1"> View larger version (62K): org.highwire.dtl.DTLVardef@259537org.highwire.dtl.DTLVardef@1675a43org.highwire.dtl.DTLVardef@570b61org.highwire.dtl.DTLVardef@ac45df_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSEasyAb, a sensitive, rapid and high-throughput method to quantify post translationally modified peptides in diverse cell systems. EasyAb reveals differential tyrosine phosphorylation of mRNA splicing proteins in AML patient samples. Activation of KOR by "G-protein-biased" non-aversive agonist 6GNTI elicits Src kinase activity in mice. Elucidation of in vivo hypothalamic Leptin induced LEPRb-Jak2 phosphotyrosine signaling architecture.

biochemistry↗

bioRxiv2024

Super-enhancer profiling reveals ThPOK/ZBTB7B, a CD4+ cell lineage commitment factor, as a master regulator that restricts breast cancer cells to a luminal non-migratory phenotype

Despite efforts to understand breast cancer biology, metastatic disease remains a clinical challenge. Identifying suppressors of breast cancer progression and mechanisms of transition to more invasive phenotypes could provide game changing therapeutic opportunities. Transcriptional deregulation is central to all malignancies, highlighted by the extensive reprogramming of regulatory elements that underlie oncogenic programs. Among these, super-enhancers (SEs) stand out due to their enrichment in genes controlling cancer hallmarks. To reveal novel breast cancer dependencies, we integrated the analysis of the SE landscape with master regulator activity inference for a series of breast cancer cell lines. As a result, we identified T-helper-inducing Poxviruses and Zinc-finger (POZ)/Kruppel-like factor (ThPOK, ZBTB7B), a CD4+ cell lineage commitment factor, as a breast cancer master regulator that is recurrently associated with a SE. ThPOK expression is highest in luminal breast cancer but is significantly reduced in the basal subtype. Manipulation of ThPOK levels in cell lines shows that its repressive function restricts breast cancer cells to an epithelial phenotype by suppressing the expression of genes involved in the epithelial-mesenchymal transition (EMT), WNT/{beta}-catenin target genes, and the pro-metastatic TGF{beta} pathway. Our study reveals ThPOK as a master transcription factor that restricts the acquisition of metastatic features in breast cancer cells.

cancer biology↗

bioRxiv2022

A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF(V600E) colorectal tumors

BRAFV600E mutation confers a poor prognosis in metastatic colorectal cancer (CRC) despite combinatorial targeted therapies based on the latest understanding of signaling circuitry. To identify parallel resistance mechanisms induced by BRAF/MEK/EGFR co-targeting, we used a high throughput kinase activity mapping platform. We found that SRC kinases are systematically activated in BRAFV600E CRC following targeted inhibition of BRAF {+/-} EGFR, and that coordinated targeting of SRC with BRAF {+/-} EGFR increases efficacy in vitro and in vivo. SRC drives resistance to BRAF {+/-} anti-EGFR therapy independently of ERK signaling by inducing transcriptional reprogramming via beta-catenin (CTNNB1). The EGFR-independent compensatory activation of SRC kinases is mediated by an autocrine prostaglandin E2-loop that can be blocked with cyclooxygenase-2 (COX2) inhibitors. Co-targeting of COX2 with BRAF+EGFR promotes durable suppression of tumor growth in patient-derived tumor xenograft (PDX) models. COX2 inhibition represents a novel drug-repurposing strategy to overcome therapeutic resistance in BRAFV600E CRC.

cancer biology↗

bioRxiv2022

Characterization of the intrahippocampal kainic acid model in female mice with a special focus on seizure suppression by antiseizure drugs and DMSO

ObjectivesAffecting around 50 million people, men and women likewise, epilepsies are among the most common neurological diseases worldwide. Despite special challenges in the medical treatment of women with epilepsy, previous research has mainly focused on males, in particular preclinical animal studies, leaving a gap that needs to be urgently addressed. The intrahippocampal kainic acid (IHKA) mouse model of temporal lobe epilepsy (TLE) as one of the most frequently studied models in males is used for screening of novel antiepileptic therapies. In this study we investigate the IHKA model of TLE in female mice, in particular drug-resistance of hippocampal paroxysmal discharges. Furthermore, we provide evidence for anti-seizure effects of dimethyl sulfoxide (DMSO) in epileptic, but not naIve mice. MethodsAfter injecting KA unilaterally into the hippocampus of female mice, we monitored the development of epileptiform activity in in-vivo EEG recordings, evaluated responsiveness to the commonly prescribed antiseizure drugs (ASDs) lamotrigine (LTG), oxcarbazepine (OXC) and levetiracetam (LEV) and assessed typical neuropathological alterations of the hippocampus. Moreover, the effect of different doses of DMSO was tested in the IHKA chronic epilepsy model as well as on the PTZ-induced acute seizure threshold in both female and male mice. ResultsIn the IHKA model, female mice replicated the key features of human TLE (EEG and neuropathological changes). Importantly, hippocampal paroxysmal discharges (HPDs) in female mice did not respond to commonly prescribed ASDs, thus representing a suitable model of drug-resistant seizures. The solvent DMSO caused a significant short-term reduction of HPDs, but did not affect the threshold of acute seizures. SignificanceBy characterizing the drug-resistance of HPDs in the IHKA model of TLE in female mice we have laid a foundation for future research addressing sex-specific aspects. Considering the special issues complicating the therapeutic management of women, inclusion of females in the quest for novel treatment strategies is imperative. The observed effect of DMSO on epileptiform activity underlines that its application in epilepsy research is problematic and that the choice of solvent and appropriate vehicle control is crucial.

pharmacology and toxicology↗