Project description:The biguanide drug metformin is a safe and widely prescribed drug for type 2 diabetes. Interestingly, hundreds of clinical trials were set to evaluate the potential role of metformin in the prevention and treatment of cancer including colorectal cancer (CRC). However, the metformin-induced cell signaling remains controversial. To interrogate cell signaling events and networks in CRC and explore the druggability of the metformin-rewired phosphorylation network, we performed a proteomic and phosphoproteomic analysis on a panel of 12 molecularly heterogeneous CRC cell lines. Using in-depth data-independent analysis mass spectrometry (DIA-MS), we profiled a total of 10,142 proteins and 56,080 phosphosites (P-sites) in CRC cells treated with metformin for 30 minutes and 24 hours. Our results indicate that metformin tends to not trigger or inhibit significant immediate phosphorylation events. Instead, it primarily remodels cell signaling in the long-term. Strikingly, the phosphorylation response to metformin was highly heterogeneous in the CRC panel. We further performed a network analysis to systematically estimate kinase/phosphatase activities and reconstruct signaling cascades in each cell line. We created a “MetScore” which catalogs the most consistently perturbed P-sites among CRC cells for future studies. Finally, we leveraged the metformin P-site signature to identify pharmacodynamic interactions, revealing and confirming a number of candidate metformin-interacting drugs, including navitoclax, a BCL-2/BCL-xL inhibitor. Together, we provide a state-of-the-art phosphoproteomic resource to explore the metformin-induced cell signaling for potential cancer therapeutics.
Project description:Nociceptors play an essential role in both acute pain and chronic pain conditions. In this study, we examined the proteome of mouse dorsal root ganglia and compared NaV1.8Cre+/-; ROSA26-flox-stop-flox-DTA (Diphtheria toxin fragment A) mutant mice (NaV1.8Cre-DTA), in which NaV1.8-positive neurons (mainly nociceptors) in dorsal root ganglia (DRG) were ablated, with respective littermate wildtype controls.
Project description:Recent work in immunometabolism has emphasised the role of mitochondria in both the innate and adaptive immune system. Mitochondria are important in T cell development and differentiation, but less is known about their role in CD8+ effector T cells (CTLs). We found that CTLs that lack the mitochondrial deubiquitinase USP30 undergo promiscuous mitophagy, destroying most of the cellular mitochondria. Surprisingly, this results in a markedly diminished killing capacity, while motility, signalling and secretion remain intact. This study uses quantitative DIA proteomics to measure the impact of USP30 deficiency on the global proteome of IL-2 maintained, 4.5 h TCR retriggered and 4.5 h TCR retriggered + cycloheximide CTL. Unexpectedly, inhibition of mitochondrial translation, through genetic or pharmacologic methods, was the mechanism by which CTL killing was impaired. Reduced mitochondrial translation triggered attenuated cytosolic translation which precluded replenishment of secreted effector molecules thereby limiting the capacity of CTLs to serially kill multiple targets. Thus, mitochondria emerge as a previously unappreciated homeostatic regulator of protein translation required for serial CTL killing.
Project description:Effector cytotoxic T lymphocytes (CTLs) are critical for ridding the body of infected or cancerous cells. Recognition of an antigenic ligand by the CTL’s T cell receptor (TCR) triggers a signalling cascade that ultimately results in the targeted release of cytolytic granules and/or secretion of cytokines and chemokines to alert and recruit additional immune cells. These signalling cascades are capable of initiating transcription, translation, and cytoskeletal rearrangements. While previous work has demonstrated how translation and intracellular reorganisation contribute to CTL effector responses, the role of transcription is less well studied. To address this, we examined the impact of blocking transcription on the CTL proteome during TCR stimulation. These data demonstrated a strong transcriptional requirement for expression of cytokines and chemokines but not cytolytic molecules. Together with functional studies, these data reveal differential molecular control of the cell-cell communication and cytolytic functions of effector CTLs. CTLs exhibit complete and persistent priming for cytolytic activity prior to target cell encounter, but they require de novo transcription to recruit additional immune cells that amplify the response.
Project description:Polycythemia vera (PV) is a myeloproliferative neoplasm (MPN) characterized by hyper-proliferation of the erythroid, megakaryocytic and granulocytic lineages and the presence of an activating mutation in JAK2. To elucidate mechanisms that regulate PV stem cells, we applied a newly developed data-independent acquisition (DIA) mass spectrometry (MS) technology to purified hematopoietic stem and progenitor cell (HSPC) subpopulations of patients with chronic and progressed PV. Proteomic analyses were supplemented by RNA-sequencing (RNA-seq) and identified targets validated by flow cytometry and functional in vitro assays.
Project description:Genomic DNA from 187 wild type and 169 asy1 Col-0 x Ws-4 F2 individuals was extracted using CTAB and used to generate sequencing libraries as described (Lawrence et al, 2019 Current Biology). Sequencing data was analysed to identify crossovers using the TIGER pipeline as previously described (Rowan et al, 2015 G3 (Bethesda); Yelina et al, 2015 Genes & Dev; Lawrence et al, 2019 Current Biology).
Project description:Genomic DNA from 191 asy1/+ Col x Ler F2 individuals was extracted using CTAB and used to generate sequencing libraries as described (Lawrence et al, 2019 Current Biology). Sequencing data was analysed to identify crossovers using the TIGER pipeline as previously described (Rowan et al, 2015 G3 (Bethesda); Yelina et al, 2015 Genes & Dev; Lawrence et al, 2019 Current Biology).
Project description:Identification of specific chromatin interactions of 49 selected genes with the Capture-C technique in DG75 iBZLF1 cells prior to and 15 h after expression of EBV's protein BZLF1. The experiments were performed as triplicates.
Project description:Acute postsurgerical pain and it's management represent a major clinical challenge. In particular, severe and prolonged pain impairs immediate recovery and might lead to long-term consequences like chronic pain, opioid abuse, and reduced quality of life. Rodent incision models greatly contributed to the understanding of cutaneous wound repair, but the underlying mechanisms' knowledge remains incomplete, yet translational data are urgently needed to develop novel treatment options and preventive measures. We combined extensive sensory phenotyping with unbiased quantitative proteomics in human and mouse skin after an incision to assess interspecies protein signatures to overcome this gap. Additionally, stratification of human volunteers based on the hyperalgesic area after surgery in correlation with the corresponding proteomic fingerprint revealed novel insides into human-specific mechanisms.Unbiased skin proteome analysis of both species unveiled comparable protein signatures after incision, notably for inflammatory activity and actin polymerization. Upon incision, we could discern 50 commonly regulated proteins, amounting to 61% of all regulated human and 10% regulated mouse proteins. Notably, the direction of regulation upon incision, i.e. up-or downregulated, was conserved in humans and mice to a great extent. Integrative analysis of pain-related phenotyping with quantitative mass spectrometry identified hitherto unknown skin protein signatures, providing a tool for stratification on the protein level. Protein-protein interaction (PPI)-networks differed between volunteers with small incision-related hyperalgesic areas (termed"low responder") versus those with large areas ("high responder"). In particular, PPIs of volunteers exhibiting a large hyperalgesic area were characterized by a predominant dysregulated proteolytic environment associated with persistent inflammation reponse. In contrast, PPI- networks of low responders point to anti-inflammatory processes. Here, we present a framework for specific-specific alterations in human and mice upon incision, highlighting similarities and differences on a protein network level. Moreover, the detection of developing hyperalgesia and pain after surgery in volunteers and it's correlation with corresponding molecular fingerprints indicates the need for tailored treatments to prevent chronification processes in humans after surgery.