Project description:The RNA binding protein IGF2BP2/IMP2 alters the cargo of cancer cell-derived extracellular vesicles supporting tumor-associated macrophages
Project description:Background: Aggressiveness guides treatment in IDH-mutant gliomas. Objective grading of oligodendrogliomas is therefore urgently needed. Material and Methods: 211 primary and recurrent resections from 111 oligodendroglioma patients were collected, complemented with 91 samples for validation. Samples were subjected to Ki-67 staining, proteomics and DNA-methylation profiling. Data were analyzed using various multivariate differential models and algorithms. Results & conclusion: We developed CGCψ, a continuous grading coefficient independent of tumor typing, and demonstrate its prognostic value in oligodendrogliomas. Its prognostic value outperformed WHO grade at tumor recurrence. CGCψ is linked to large scale DNA de-methylation, but increased DNA methylation of polycomb transcription factors, aging, Ki-67, and losses on chr4 and chr9p. DNA de-methylation at tumor recurrence/higher grade is sequence context specific and associated with TET recognition sites. Both oligodendrogliomas and astrocytomas progress along this shared epi-genetic axis. Oligosarcomas are characterized by a high CGCψ and low tumor purity.
Project description:Lysine acetyltransferases (KATs) such as p300 and CBP regulate gene networks in healthy and malignant cells by scaffolding transcriptional machinery and by acetylating chromatin and transcription factors (TFs). In certain cancers including diffuse large B cell lymphomas, they contribute to tumor suppression and are therapeutic vulnerabilities when deregulated. Inhibitors and degraders of KATs are limited by potential toxicity associated with targeting these essential proteins. We introduce KAT-TCIPs (lysine acetyltransferase transcriptional/epigenetic chemical inducers of proximity), bivalent molecules that redirect the KAT and co-activating activity of p300 and CBP towards programmed cell death signaling repressed by the oncogenic TF BCL6. The lead KAT-TCIP molecule kills lymphoma cells at ~0.8 nM by redistributing chromatin acetylation, activating the pro-apoptotic protein PUMA, and repressing MYC. Analysis of the ternary co-crystal structure containing p300 and BCL6 reveals fortuitous interactions that drive the potency and specificity of KAT-TCIPs and sets a precedent for KAT-targeting induced proximity therapeutics.
Project description:The dynamic protein distribution within and across the plasma membrane is pivotal in regulating cell communications. Yet, methods facilitating rapid labeling, applicable across diverse cell types, and amenable to multiplexed live imaging are scarce. We discovered that live mammalian cell surface proteins can be labeled using N-hydroxysuccinimide (NHS)-ester-based amine crosslinking of fluorescent dyes. Using mouse DC2.4 dendritic cells, human Jurkat T cells, and mouse primary lymphoma T cells as model systems, we revealed the membrane topology and membrane-associated cell communications previously challenging to capture by fluorescence microscopy techniques. In DC2.4 cells, we observed transient membrane protein accumulation at cell-cell contacts and their bidirectional migration patterns guided by membrane fibers. We further visualized the involvement of membrane fibers in the transfer of membrane-derived particles and ovalbumin, as well as caveolin-dependent endocytosis and subsequent transport of membrane labels in late endosomes and lysosomes. In addition, we demonstrate caveolin-1 phosphorylation-dependent insulin receptor endocytosis in HEK 293T cells. Volumetric structured illumination microscopy revealed changes in the membrane topology of Jurkat T cells in response to the activating surface, generation of membrane-derived microvesicles, and phagocytosis of CD3/CD28 Dynabeads on the glass surface. Finally, we demonstrate that the membrane label remains stable in the in vivo environment and evidence of intercellular membrane protein transfer from splenocytes from the donor T cell lymphoma SNF5 mouse to the healthy splenocytes in the C57/BL6 mouse 24 and 48-h after cell transfer. The ultrahigh membrane labeling density, coupled with high multiplexity and rapid labeling, enables innovative fluorescence microscopy applications to dissect membrane and membrane-derived structures in intercellular and intracellular communication both in vitro and in vivo.
Project description:Many pathogenic bacteria use proteinaceous ethanolamine-utilization microcompartments (Eut BMCs) to facilitate the catabolism of ethanolamine, an abundant nutrient in the mammalian gut. The ability to metabolize ethanolamine gives pathogens a competitive edge over commensal microbiota which can drive virulence in the inflamed gut. Despite their critical functions, the molecular mechanisms underlying the synthesis of Eut BMCs in bacterial cells remain elusive. Here, we report a systematic study for dissecting the molecular basis underlying Eut BMC assembly in Salmonella. We determined the functions of individual constituent proteins in the structure and function of Eut BMCs and demonstrated that EutQ plays an essential role in both cargo encapsulation and Eut BMC formation through specific association with the shell and cargo enzymes. Furthermore, our data reveal that Eut proteins can self-assemble to form cargo and shell aggregates independently in vivo, and that the biogenesis of Eut BMCs follows a “Shell-first” pathway. Cargo enzymes exhibit dynamic liquid-like organization within the Eut BMC. These discoveries provide mechanistic insights into the structure and assembly of the Eut BMC, which serves as a paradigm for membrane-less organelles.
Project description:Phosphoproteomics analysis following unflavored e-cigarette exposed donor-derived primary lung epithelial cells from one donor and 3 technical replicates condition using a Bruker timsTOF HT mass spectrometer paired to a ThermoScientific Vaquish Neo HPLC. Conditions were room air (“Air”) or vape (“Vape”).
Project description:Cancer growth is fueled by nutrients obtained from circulation and local biosynthesis. Isolating and exploiting tumoral nutrient dependencies to potentiate current anti-cancer therapies is undergoing active research. We used unbiased metabolomics on patient samples and identified reprogramming of the arginine-proline-glutamine axis in MYCN amplified neuroblastoma. Tumoral acquisition of these non-essential amnio acids, as revealed by stable isotope tracing, was primarily by import, while extra-tumoral deamination of arginine indirectly feeds tumor ornithine via circulation. Dietary depletion of proline and arginine reduced systemic ornithine, the direct precursor of polyamine biosynthesis, and in combination with the clinically approved polyamine biosynthesis inhibitor, difluoromethylornithine, enhanced their depletion to improve survival in a MYCN-driven neuroblastoma mouse model. Mechanistically, ribosome profiling indicated specific translation defects, with ribosomal pausing at adenine-ending codons, to cause reprogramed protein biosynthesis affecting cell cycle and inducing neuronal differentiation. This work provides proof of concept for combined small molecule and nutrient depletion therapy for simultaneous targeting of translation in cancers dependent on external nutrient uptake.