Now showing 1 - 10 of 19
  • Publication
    Mechanochemical Stimulation of MCF7 Cells with Rod-Shaped Fe-Au Janus Particles Induces Cell Death through Paradoxical Hyperactivation of ERK
    Multifunctional nanoparticles that actively target-specific tissues are studied for cancer diagnosis and treatment. Magnetically and optically active particles are of particular interest because they enable multiple imaging modalities and physically modulated therapies, such as magnetic hyperthermia. Fe–Au nanorods are synthesized that have a long iron segment, coated with polyethylene glycol, and a short gold tip functionalized with heregulin (HRG), a known ligand of ErbB family of receptors. HRG–nanorods preferentially target MCF7 cells relative to MDA-MB-231 cells, as demonstrated in a novel microfluidics device. Targeting rates of these classical breast cancer cells correlate with their differential expression of ErbB2/3 receptors. HRG–nanorod binding stimulates the extracellular signal-regulated kinase 1/2 (ERK) phosphorylation in MCF7 cells. The increase in ERK phosphorylation is linked to 'active zones,' dynamic regions in the cell periphery, which exhibit higher rates of particle binding than the rest of the cell. Periodically stretching cells using magnetic tweezers further activates ERK, which leads to cell death in cells co-treated with B-Raf inhibitors, through ERK hyperactivation. Although to a lesser extent, cell death is also achieved through magnetic hyperthermia. These results demonstrate nanoscale targeting and localized mechanochemical treatment of specific cancer cell lines based on their receptor expression using multifunctional nanoparticles.
      624Scopus© Citations 26
  • Publication
    An Integrated Global Analysis of Compartmentalized HRAS Signaling
    Modern omics technologies allow us to obtain global information on different types of biological networks. However, integrating these different types of analyses into a coherent framework for a comprehensive biological interpretation remains challenging. Here, we present a conceptual framework that integrates protein interaction, phosphoproteomics, and transcriptomics data. Applying this method to analyze HRAS signaling from different subcellular compartments shows that spatially defined networks contribute specific functions to HRAS signaling. Changes in HRAS protein interactions at different sites lead to different kinase activation patterns that differentially regulate gene transcription. HRAS-mediated signaling is the strongest from the cell membrane, but it regulates the largest number of genes from the endoplasmic reticulum. The integrated networks provide a topologically and functionally resolved view of HRAS signaling. They reveal distinct HRAS functions including the control of cell migration from the endoplasmic reticulum and TP53-dependent cell survival when signaling from the Golgi apparatus.
      371Scopus© Citations 24
  • Publication
    Regulation of IL-1β-induced NF-κB by hydroxylases links key hypoxic and inflammatory signaling pathways
    Hypoxia is a prominent feature of chronically inflamed tissues. Oxygen-sensing hydroxylases control transcriptional adaptation to hypoxia through the regulation of hypoxia-inducible factor (HIF) and nuclear factor κB (NF-κB), both of which can regulate the inflammatory response. Furthermore, pharmacologic hydroxylase inhibitors reduce inflammation in multiple animal models. However, the underlying mechanism(s) linking hydroxylase activity to inflammatory signaling remains unclear. IL-1β, a major proinflammatory cytokine that regulates NF-κB, is associated with multiple inflammatory pathologies. We demonstrate that a combination of prolyl hydroxylase 1 and factor inhibiting HIF hydroxylase isoforms regulates IL-1β-induced NF-κB at the level of (or downstream of) the tumor necrosis factor receptor-associated factor 6 complex. Multiple proteins of the distal IL-1β-signaling pathway are subject to hydroxylation and form complexes with either prolyl hydroxylase 1 or factor inhibiting HIF. Thus, we hypothesize that hydroxylases regulate IL-1β signaling and subsequent inflammatory gene expression. Furthermore, hydroxylase inhibition represents a unique approach to the inhibition of IL-1β-dependent inflammatory signaling.
      223Scopus© Citations 121
  • Publication
    On-Beads Digestion in Conjunction with Data-Dependent Mass Spectrometry: A Shortcut to Quantitative and Dynamic Interaction Proteomics
    With the advent of the '-omics' era, biological research has shifted from functionally analyzing single proteins to understanding how entire protein networks connect and adapt to environmental cues. Frequently, pathological processes are initiated by a malfunctioning protein network rather than a single protein. It is therefore crucial to investigate the regulation of proteins in the context of a pathway first and signaling network second. In this study, we demonstrate that a quantitative interaction proteomic approach, combining immunoprecipitation, in-solution digestion and label-free quantification mass spectrometry, provides data of high accuracy and depth. This protocol is applicable, both to tagged, exogenous and untagged, endogenous proteins. Furthermore, it is fast, reliable and, due to a label-free quantitation approach, allows the comparison of multiple conditions. We further show that we are able to generate data in a medium throughput fashion and that we can quantify dynamic interaction changes in signaling pathways in response to mitogenic stimuli, making our approach a suitable method to generate data for system biology approaches.
      234Scopus© Citations 91
  • Publication
    HEATR2 Plays a Conserved Role in Assembly of the Ciliary Motile Apparatus
    Cilia are highly conserved microtubule-based structures that perform a variety of sensory and motility functions during development and adult homeostasis. In humans, defects specifically affecting motile cilia lead to chronic airway infections, infertility and laterality defects in the genetically heterogeneous disorder Primary Ciliary Dyskinesia (PCD). Using the comparatively simple Drosophila system, in which mechanosensory neurons possess modified motile cilia, we employed a recently elucidated cilia transcriptional RFX-FOX code to identify novel PCD candidate genes. Here, we report characterization of CG31320/HEATR2, which plays a conserved critical role in forming the axonemal dynein arms required for ciliary motility in both flies and humans. Inner and outer arm dyneins are absent from axonemes of CG31320 mutant flies and from PCD individuals with a novel splice-acceptor HEATR2 mutation. Functional conservation of closely arranged RFX-FOX binding sites upstream of HEATR2 orthologues may drive higher cytoplasmic expression of HEATR2 during early motile ciliogenesis. Immunoprecipitation reveals HEATR2 interacts with DNAI2, but not HSP70 or HSP90, distinguishing it from the client/chaperone functions described for other cytoplasmic proteins required for dynein arm assembly such as DNAAF1-4. These data implicate CG31320/HEATR2 in a growing intracellular pre-assembly and transport network that is necessary to deliver functional dynein machinery to the ciliary compartment for integration into the motile axoneme.
      360Scopus© Citations 51
  • Publication
    Autophosphorylation on S614 inhibits the activity and the transforming potential of BRAF
    The BRAF proto-oncogene serine/threonine-protein kinase, known as BRAF, belongs to the RAF kinase family. It regulates the MAPK/ERK signalling pathway affecting several cellular processes such as growth, survival, differentiation, and cellular transformation. BRAF is mutated in ~8% of all human cancers with the V600E mutation constituting ~90% of mutations. Here, we have used quantitative mass spectrometry to map and compare phosphorylation site patterns between BRAF and BRAF V600E. We identified sites that are shared as well as several quantitative differences in phosphorylation abundance. The highest difference is phosphorylation of S614 in the activation loop which is ~5fold enhanced in BRAF V600E. Mutation of S614 increases the kinase activity of both BRAF and BRAF V600E and the transforming ability of BRAF V600E. The phosphorylation of S614 is mitogen inducible and the result of autophosphorylation. These data suggest that phosphorylation at this site is inhibitory, and part of the physiological shut-down mechanism of BRAF signalling.
      354Scopus© Citations 4
  • Publication
    Common and Distinctive Functions of the Hippo Effectors Taz and Yap in Skeletal Muscle Stem Cell Function
    Hippo pathway downstream effectors Yap and Taz play key roles in cell proliferation and regeneration, regulating gene expression especially via Tead transcription factors. To investigate their role in skeletal muscle stem cells, we analyzed Taz in vivo and ex vivo in comparison with Yap. Small interfering RNA knockdown or retroviral-mediated expression of wild-type human or constitutively active TAZ mutants in satellite cells showed that TAZ promoted proliferation, a function shared with YAP. However, at later stages of myogenesis, TAZ also enhanced myogenic differentiation of myoblasts, whereas YAP inhibits such differentiation. Functionally, while muscle growth was mildly affected in Taz (gene Wwtr1–/–) knockout mice, there were no overt effects on regeneration. Conversely, conditional knockout of Yap in satellite cells of Pax7Cre-ERT2/+: Yapfl°x/fl°x:Rosa26Lacz mice produced a regeneration deficit. To identify potential mechanisms, microarray analysis showed many common TAZ/YAP target genes, but TAZ also regulates some genes independently of YAP, including myogenic genes such as Pax7, Myf5, and Myod1 (ArrayExpress–E-MTAB-5395). Proteomic analysis revealed many novel binding partners of TAZ/YAP in myogenic cells, but TAZ also interacts with proteins distinct from YAP that are often involved in myogenesis and aspects of cytoskeleton organization (ProteomeXchange–PXD005751). Neither TAZ nor YAP bind members of the Wnt destruction complex but both regulated expression of Wnt and Wnt-cross talking genes with known roles in myogenesis. Finally, TAZ operates through Tead4 to enhance myogenic differentiation. In summary, Taz and Yap have overlapping functions in promoting myoblast proliferation but Taz then switches to enhance myogenic differentiation. Stem Cells 2017;35:1958–1972.
      131Scopus© Citations 68
  • Publication
    RASSF1A uncouples Wnt from Hippo signalling and promotes YAP mediated differentiation via p73
    Transition from pluripotency to differentiation is a pivotal yet poorly understood developmental step. Here, we show that the tumour suppressor RASSF1A is a key player driving the early specification of cell fate. RASSF1A acts as a natural barrier to stem cell self-renewal and iPS cell generation, by switching YAP from an integral component in the β-catenin-TCF pluripotency network to a key factor that promotes differentiation. We demonstrate that epigenetic regulation of the Rassf1A promoter maintains stemness by allowing a quaternary association of YAP-TEAD and β-catenin-TCF3 complexes on the Oct4 distal enhancer. However, during differentiation, promoter demethylation allows GATA1-mediated RASSF1A expression which prevents YAP from contributing to the TEAD/β-catenin-TCF3 complex. Simultaneously, we find that RASSF1A promotes a YAP-p73 transcriptional programme that enables differentiation. Together, our findings demonstrate that RASSF1A mediates transcription factor selection of YAP in stem cells, thereby acting as a functional "switch" between pluripotency and initiation of differentiation.
      107Scopus© Citations 55
  • Publication
    FIH Regulates Cellular Metabolism through Hydroxylation of the Deubiquitinase OTUB1
    The asparagine hydroxylase, factor inhibiting HIF (FIH), confers oxygen-dependence upon the hypoxia-inducible factor (HIF), a master regulator of the cellular adaptive response to hypoxia. Studies investigating whether asparagine hydroxylation is a general regulatory oxygen-dependent modification have identified multiple non-HIF targets for FIH. However, the functional consequences of this outside of the HIF pathway remain unclear. Here, we demonstrate that the deubiquitinase ovarian tumor domain containing ubiquitin aldehyde binding protein 1 (OTUB1) is a substrate for hydroxylation by FIH on N22. Mutation of N22 leads to a profound change in the interaction of OTUB1 with proteins important in cellular metabolism. Furthermore, in cultured cells, overexpression of N22A mutant OTUB1 impairs cellular metabolic processes when compared to wild type. Based on these data, we hypothesize that OTUB1 is a target for functional hydroxylation by FIH. Additionally, we propose that our results provide new insight into the regulation of cellular energy metabolism during hypoxic stress and the potential for targeting hydroxylases for therapeutic benefit.
      225Scopus© Citations 60
  • Publication
    Signalling mechanisms regulating phenotypic changes in breast cancer cells
    In MCF-7 breast cancer cells epidermal growth factor (EGF) induces cell proliferation, whereas heregulin (HRG)/neuregulin (NRG) induces irreversible phenotypic changes accompanied by lipid accumulation. Although these changes in breast cancer cells resemble processes that take place in the tissue, there is no understanding of signalling mechanisms regulating it. To identify molecular mechanisms mediating this cell-fate decision process, we applied different perturbations to pathways activated by these growth factors. The results demonstrate that phosphoinositide 3 (PI3) kinase (PI3K) and mammalian target of rapamycin (mTOR) complex (mTORC)1 activation is necessary for lipid accumulation that can also be induced by insulin, whereas stimulation of the extracellular-signal-regulated kinase (ERK) pathway is surprisingly dispensable. Interestingly, insulin exposure, as short as 4 h, was sufficient for triggering the lipid accumulation, whereas much longer treatment with HRG was required for achieving similar cellular response. Further, activation patterns of ATP citratelyase (ACLY), an enzyme playing a central role in linking glycolytic and lipogenic pathways, suggest that lipids accumulated within cells are produced de novo rather than absorbed from the environment. In the present study, we demonstrate that PI3K pathway regulates phenotypic changes in breast cancer cells, whereas signal intensity and duration is crucial for cell fate decisions and commitment. Our findings reveal that MCF-7 cell fate decisions are controlled by a network of positive and negative regulators of both signalling and metabolic pathways.
      327Scopus© Citations 9