Project description:The intermediate filament protein Nestin serves as a biomarker for stem cells and has been used to identify subsets of cancer stem-like cells. However, the mechanistic contributions of Nestin to cancer pathogenesis are not understood. Here we report that Nestin binds the hedgehog pathway transcription factor Gli3 to mediate the development of medulloblastomas of the hedgehog subtype. In a mouse model system, Nestin levels increased progressively during medulloblastoma formation resulting in enhanced tumor growth. Conversely, loss of Nestin dramatically inhibited proliferation and promoted differentiation. Mechanistic investigations revealed that the tumor-promoting effects of Nestin were mediated by binding to Gli3, a zinc finger transcription factor that negatively regulates hedgehog signaling. Nestin binding to Gli3 blocked Gli3 phosphorylation and its subsequent proteolytic processing, thereby abrogating its ability to negatively regulate the hedgehog pathway. Our findings show how Nestin drives hedgehog pathway-driven cancers and uncover in Gli3 a therapeutic target to treat these malignancies.
Project description:The intermediate filament protein Nestin serves as a biomarker for stem cells and has been used to identify subsets of cancer stem-like cells. However, the mechanistic contributions of Nestin to cancer pathogenesis are not understood. Here we report that Nestin binds the hedgehog pathway transcription factor Gli3 to mediate the development of medulloblastomas of the hedgehog subtype. In a mouse model system, Nestin levels increased progressively during medulloblastoma formation resulting in enhanced tumor growth. Conversely, loss of Nestin dramatically inhibited proliferation and promoted differentiation. Mechanistic investigations revealed that the tumor-promoting effects of Nestin were mediated by binding to Gli3, a zinc finger transcription factor that negatively regulates hedgehog signaling. Nestin binding to Gli3 blocked Gli3 phosphorylation and its subsequent proteolytic processing, thereby abrogating its ability to negatively regulate the hedgehog pathway. Our findings show how Nestin drives hedgehog pathway-driven cancers and uncover in Gli3 a therapeutic target to treat these malignancies. Nestin+ and Nestin- GNPs (granule neuron precursors) were purified from Nestin-CFP/Math1-Cre/Ptch1-loxp cerebella at postnatal day 4 by FACs, and total RNA from these two cell populations were extracted, and then labeled and hybridized to Affymetrix Mouse Genome 430 2.0 arrays.
Project description:To evaluate the emergence of RAS mutation in patients with metastatic colorectal cancer, circulating free DNA will be analyzed using mass spectrometric genotyping in subjects during cetuximab treatment. The hypothesis of this study is that acquired RAS mutation is responsible for the resistance to cetuximab treatment in wild-type colorectal cancer. The usefulness of liquid biopsy to monitor dynamic genetic alterations in colorectal cancer during treatment will also be investigated in this study.
Project description:In order to identify novel stress-induced signalling peptides, we searched for Arabidopsis thaliana transcripts encoding short proteins (<150 amino acids) with a predicted signal peptide, which were induced upon biotic elicitor treatment (Bjornson et al., 2021). Through this analysis, we identified an uncharacterised family of peptides with 5 predicted members, which we named CTNIP1 to 5 (pronounced catnip) based on relatively conserved residues within the peptides. CTNIP4 is perceived by Arabidopsis, inducing some hallmark outputs of defence (Pattern-Triggered Immunity) signalling. Mass spectrometric analyses indicated that HSL3 may be the receptor mediating CTNIP recognition. Through assaying transcriptomic responses in wild type and hsl3-1 mutants, we were able to confirm similarity of CTNIP and defence responses at the transcriptional level, and the dependence of these responses on HSL3.
Project description:Current proteomic methods are not well suited to detect protein isoforms. On the one hand, standard shotgun (that is, bottom-up) proteomics involves digestion of proteins into peptides. While this approach identifies many proteins, it results in a loss of isoform information. On the other hand, mass spectrometric analysis of intact proteins (that is, top-down proteomics) distinguishes protein isoforms but only covers a small subset of the proteome. We developed peptide correlation profiling (PepCP) as a method to obtain protein-level information from peptide-centric (that is, bottom-up proteomic) data: First, proteins are fractionated by SDS-PAGE to polypeptides of different length. Second, individual protein fractions are digested into peptides. Third, peptides are identified and quantified in all fractions using quantitative mass spectrometry-based proteomics. Finally, peptide abundance profiles across fractions are analysed to obtain protein-level information.
Project description:Current proteomic methods are not well suited to detect protein isoforms. On the one hand, standard shotgun (that is, bottom-up) proteomics involves digestion of proteins into peptides. While this approach identifies many proteins, it results in a loss of isoform information. On the other hand, mass spectrometric analysis of intact proteins (that is, top-down proteomics) distinguishes protein isoforms but only covers a small subset of the proteome. We developed peptide correlation profiling (PepCP) as a method to obtain protein-level information from peptide-centric (that is, bottom-up proteomic) data: First, proteins are fractionated by SDS-PAGE to polypeptides of different length. Second, individual protein fractions are digested into peptides. Third, peptides are identified and quantified in all fractions using quantitative mass spectrometry-based proteomics. Finally, peptide abundance profiles across fractions are analysed to obtain protein-level information.