Discrete cytosolic macromolecular BRAF complexes exhibit distinct activities and composition

As a central element within the RAS/ERK pathway, the serine/threonine kinase BRAF plays a key role in development and homeostasis and represents the most frequently mutated kinase in tumors. Consequently, it has emerged as an important therapeutic target in various malignancies. Nevertheless, the BRAF activation cycle still raises many mechanistic questions as illustrated by the paradoxical action and side effects of RAF inhibitors. By applying SEC‐PCP‐SILAC, we analyzed protein–protein interactions of hyperactive BRAFV600E and wild‐type BRAF (BRAFWT). We identified two macromolecular, cytosolic BRAF complexes of distinct molecular composition and phosphorylation status. Hyperactive BRAFV600E resides in large complexes of higher molecular mass and activity, while BRAFWT is confined to smaller, slightly less active complexes. However, expression of oncogenic K‐RasG12V, either by itself or in combination with RAF dimer promoting inhibitors, induces the incorporation of BRAFWT into large, active complexes, whereas pharmacological inhibition of BRAFV600E has the opposite effect. Thus, the quaternary structure of BRAF complexes is shaped by its activation status, the conformation of its kinase domain, and clinically relevant inhibitors

Degradation of protein translation machinery by amino acid starvation-induced macroautophagy

Macroautophagy is regarded as a nonspecific bulk degradation process of cytoplasmic material within the lysosome. However, the process has mainly been studied by nonspecific bulk degradation assays using radiolabeling. In the present study we monitor protein turnover and degradation by global, unbiased approaches relying on quantitative mass spectrometry-based proteomics. Macroautophagy is induced by rapamycin treatment, and by amino acid and glucose starvation in differentially, metabolically labeled cells. Protein dynamics are linked to image-based models of autophagosome turnover. Depending on the inducing stimulus, protein as well as organelle turnover differ. Amino acid starvation-induced macroautophagy leads to selective degradation of proteins important for protein translation. Thus, protein dynamics reflect cellular conditions in the respective treatment indicating stimulus-specific pathways in stress-induced macroautophagy

Studerendes skabelse af personlig teori om børneperspektiver i pædagoguddannelsens praktik

DK resuméVia analyser fra et etnografisk inspireret feltarbejde af pædagogstuderendes arbejde med børneperspektiver i deres praktik i daginstitutioner bidrager artiklen til nuancerede forståelser indenfor forskningsfeltet voksenlæring og praktik. Med afsæt i en kombination af Lave og Wengers antropologisk og sociologisk inspirerede læringsteori og Jarvis’ filosofisk eksistentielt inspirerede læringsteori viser artiklen de studerendes refleksioner over det konfliktuelle og modsætningsfulde i praksis som forudsætning for deres læring. Artiklen sætter dermed fokus på de studerendes modsætningsfulde praksiserfaringer som udgangspunkt for læring på pædagoguddannelsen og bidrager med et kritisk korrektiv til en instrumentel og kompetencemålstyret kvalifikationstænkning. Abstract – UKHow do students in social education create a personal theory about children’s perspective during an internshipBased on an ethnographic inspired fieldwork of students’ work with children’s perspective in day care institutions this article offers new understandings in the field of adult education and internship. Using Lave and Wenger’s social, anthropological inspired theory of learning in combination with Peter Jarvis’ philosophical, existential theory the article stresses the importance of reflection on conflicts and disjunctures during internship and offers a critical corrective to the current instrumental perspective on learning ruled by competence goals

The BioGRID interaction database: 2015 update

The Biological General Repository for Interaction Datasets (BioGRID: http://thebiogrid.org) is an open access database that houses genetic and protein interactions curated from the primary biomedical literature for all major model organism species and humans. As of September 2014, the BioGRID contains 749 912 interactions as drawn from 43 149 publications that represent 30 model organisms. This interaction count represents a 50% increase compared to our previous 2013 BioGRID update. BioGRID data are freely distributed through partner model organism databases and meta-databases and are directly downloadable in a variety of formats. In addition to general curation of the published literature for the major model species, BioGRID undertakes themed curation projects in areas of particular relevance for biomedical sciences, such as the ubiquitin-proteasome system and various human disease-associated interaction networks. BioGRID curation is coordinated through an Interaction Management System (IMS) that facilitates the compilation interaction records through structured evidence codes, phenotype ontologies, and gene annotation. The BioGRID architecture has been improved in order to support a broader range of interaction and post-translational modification types, to allow the representation of more complex multi-gene/protein interactions, to account for cellular phenotypes through structured ontologies, to expedite curation through semi-automated text-mining approaches, and to enhance curation quality control

Multilayered proteomics reveals molecular switches dictating ligand-dependent EGFR trafficking

A fascinating conundrum in cell signaling is how stimulation of the same receptor tyrosine kinase with distinct ligands generates specific outcomes. To decipher the functional selectivity of EGF and TGF-α, which induce epidermal growth factor receptor (EGFR) degradation and recycling, respectively, we devised an integrated multilayered proteomics approach (IMPA). We analyzed dynamic changes in the receptor interactome, ubiquitinome, phosphoproteome, and late proteome in response to both ligands in human cells by quantitative MS and identified 67 proteins regulated at multiple levels. We identified RAB7 phosphorylation and RCP recruitment to EGFR as switches for EGF and TGF-α outputs, controlling receptor trafficking, signaling duration, proliferation, and migration. By manipulating RCP levels or phosphorylation of RAB7 in EGFR-positive cancer cells, we were able to switch a TGF-α-mediated response to an EGF-like response or vice versa as EGFR trafficking was rerouted. We propose IMPA as an approach to uncover fine-tuned regulatory mechanisms in cell signaling.</p

Multi-omics characterization of a diet-induced obese model of non-alcoholic steatohepatitis

To improve the understanding of the complex biological processes underlying the development of non-alcoholic steatohepatitis (NASH), a multi-omics approach combining bulk RNA-sequencing based transcriptomics, quantitative proteomics and single-cell RNA-sequencing was used to characterize tissue biopsies from histologically validated diet-induced obese (DIO) NASH mice compared to chow-fed controls. Bulk RNA-sequencing and proteomics showed a clear distinction between phenotypes and a good correspondence between mRNA and protein level regulations, apart from specific regulatory events discovered by each technology. Transcriptomics-based gene set enrichment analysis revealed changes associated with key clinical manifestations of NASH, including impaired lipid metabolism, increased extracellular matrix formation/remodeling and pro-inflammatory responses, whereas proteomics-based gene set enrichment analysis pinpointed metabolic pathway perturbations. Integration with single-cell RNA-sequencing data identified key regulated cell types involved in development of NASH demonstrating the cellular heterogeneity and complexity of NASH pathogenesis.</p