Lefave C.V.,Sloan Kettering Cancer Center |
Lefave C.V.,The New School |
Squatrito M.,Brain Tumor Center |
Vorlova S.,Sloan Kettering Cancer Center |
And 8 more authors.
EMBO Journal | Year: 2011
In tumours, aberrant splicing generates variants that contribute to multiple aspects of tumour establishment, progression and maintenance. We show that in glioblastoma multiforme (GBM) specimens, death-domain adaptor protein Insuloma-Glucagonoma protein 20 (IG20) is consistently aberrantly spliced to generate an antagonist, anti-apoptotic isoform (MAP-kinase activating death domain protein, MADD), which effectively redirects TNF-Î ±/TRAIL-induced death signalling to promote survival and proliferation instead of triggering apoptosis. Splicing factor hnRNPH, which is upregulated in gliomas, controls this splicing event and similarly mediates switching to a ligand-independent, constitutively active Recepteur dĝ€2Origine Nantais (RON) tyrosine kinase receptor variant that promotes migration and invasion. The increased cell death and the reduced invasiveness caused by hnRNPH ablation can be rescued by the targeted downregulation of IG20/MADD exon 16-or RON exon 11-containing variants, respectively, using isoform-specific knockdown or splicing redirection approaches. Thus, hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as the centre of a splicing oncogenic switch, which might reflect reactivation of stem cell patterns and mediates multiple key aspects of aggressive tumour behaviour, including evasion from apoptosis and invasiveness. © 2011 European Molecular Biology Organization | All Rights Reserved. Source
Roux K.J.,University of South Dakota |
Roux K.J.,Florida College |
Kim D.I.,University of South Dakota |
Kim D.I.,Florida College |
And 2 more authors.
Journal of Cell Biology | Year: 2012
We have developed a new technique for proximity-dependent labeling of proteins in eukaryotic cells. Named BioID for proximitydependent biotin identification, this approach is based on fusion of a promiscuous Escherichia coli biotin protein ligase to a targeting protein. BioID features proximitydependent biotinylation of proteins that are near-neighbors of the fusion protein. Biotinylated proteins may be isolated by affinity capture and identified by mass spectrometry. We apply BioID to lamin-A (LaA), a well-characterized intermediate filament protein that is a constituent of the nuclear lamina, an important structural element of the nuclear envelope (NE). We identify multiple proteins that associate with and/or are proximate to LaA in vivo. The most abundant of these include known interactors of LaA that are localized to the NE, as well as a new NE-associated protein named SLAP75. Our results suggest BioID is a useful and generally applicable method to screen for both interacting and neighboring proteins in their native cellular environment. © 2012 Roux et al. Source
Wong J.S.,p53 Laboratory |
Wong J.S.,A-Life Medical |
Warbrick E.,A-Life Medical |
Vojtesk B.,Masaryk Memorial Cancer Institute |
And 2 more authors.
Oncotarget | Year: 2013
c-Met is a tyrosine receptor kinase which is activated by its ligand, the hepatocyte growth factor. Activation of c-Met leads to a wide spectrum of biological activities such as motility, angiogenesis, morphogenesis, cell survival and cell regeneration. c-Met is abnormally activated in many tumour types. Aberrant c-Met activation was found to induce tumour development, tumour cell migration and invasion, and the worst and final step in cancer progression, metastasis. In addition, c-Met activation in cells was also shown to confer resistance to apoptosis induced by UV damage or chemotherapeutic drugs. This study describes the development of monoclonal antibodies against c-Met as therapeutic molecules in cancer treatment/diagnostics. A panel of c-Met monoclonal antibodies was developed and characterised by epitope mapping, Western blotting, immunoprecipitation, agonist/antagonist effect in cell scatter assays and for their ability to recognise native c-Met by flow cytometry. We refer to these antibodies as Specifically Engaging Extracellular c-Met (seeMet). seeMet 2 and 13 bound strongly to native c-Met in flow cytometry and reduced SNU-5 cell growth. Interestingly, seeMet 2 binding was strongly reduced at 4°C when compared to 37°C. Detail mapping of the seeMet 2 epitope indicated a cryptic binding site hidden within the c-Met a-chain. Source
Say E.,SGSK Group |
Tay H.-G.,Singapore Institute of Medical Biology |
Zhao Z.-s.,SGSK Group |
Baskaran Y.,SGSK Group |
And 4 more authors.
Molecular Cell | Year: 2010
Loss of fragile X mental retardation protein FMR1 is the most common genetic cause of mental deficiency in man. We find that both FMR1 and the related FXR1 serve as direct binding partners for the Cdc42 effector PAK1. This involves an 11 residue segment in the PAK1 autoinhibitory domain that is exposed upon kinase activation and binds the FXR1 KH2 domain. Active PAK1 can phosphorylate FXR1 at Ser420; antibodies to this site show increased phosphorylation when fragile X proteins are recruited to stress granules. During zebrafish muscle development, FXR1 Ser420 phosphorylation is needed for protein function. The familial FMR1(I304N) mutation is biologically inactive, and FXR1(I304N) fails to bind PAK1. A different PAK1 binding-deficient mutant, FXR1(Q348K/E352A), fails to rescue loss of Zf-FXR1 unless combined with a gain-of-function S420D phosphomimetic. This is the first documented protein partner for the KH(2) domain of FMR1 or FXR1, and it has several implications for signaling by fragile X proteins. © 2010 Elsevier Inc. All rights reserved. Source
Arslan Yildiz A.,Institute of Materials Research and Engineering of Singapore |
Kang C.,Experimental Therapeutics Center |
Sinner E.-K.,University of Natural Resources and Life Sciences, Vienna
Analyst | Year: 2013
The hERG (human ether-à-go-go-related gene) potassium channel has been extensively studied by both academia and industry because of its relation to inherited or drug-induced long QT syndrome (LQTS). Unpredicted hERG and drug interaction affecting channel activity is of main concern for drug discovery. Although there are several methods to test hERG and drug interaction, it is still necessary to develop some efficient and economic ways to probe hERG and drug interactions. To contribute this aim, we have developed a biomimetic lipid membrane platform into which the hERG channel can be folded. Expression and integration of the hERG channel was achieved using a cell-free (CF) expression system. The folding of hERG in the biomimetic membrane system was investigated using Surface Plasmon Enhanced Fluorescence Spectroscopy (SPFS) and Imaging Surface Plasmon Resonance (iSPR). In addition, the hERG channel folded into our biomimetic membrane platform was used for probing the channel and drug interactions through fluorescence polarization (FP) assay. Our results suggest that the biomimetic system employed is capable of detecting the interaction between hERG and different channel blockers at varied concentrations. We believe that our current approach could be applied to other membrane proteins for drug screening or other protein-related interactions. © 2013 The Royal Society of Chemistry. Source