Dynamic subcellular localization of the EWS proto-oncoprotein and its association with and stabilization of microtubules

Ewing sarcoma (EWS) protein is a member of a large family of RNA-binding proteins. Chimeric EWS oncoproteins generated by chromosomal translocations between the EWS protein and several transcription factors cause various malignant tumors. Due to its multifunctional properties the EWS protein is involved in processes such as meiotic DNA pairing/recombination, cellular senescence, gene expression, RNA processing and transport, as well as cell signaling. The EWS protein is predominantly located in the nucleus. It was found in cytoplasm and associated with the cell membrane. In this study the analysis of the localization of endogenous and fluorescently labeled recombinant EWS protein in different phases of cell cycle in different cell lines revealed a very dynamic subcellular distribution of the EWS protein. In Cos7 and Hela cells an association of the EWS protein with the centrosomal compartments was shown. Furthermore, in HEK 293 (T) cells an interaction of the overexpessed recombinant EWS-YFP fusion protein with microtubules was demonstrated leading to their stabilization and cell cycle arrest. As an outlook, the present findings provide an important insight into temporally and spatially regulated functions of the EWS protein and, particularly, into its role in the regulation of the cell cycle and possibly cell differentiation

Securin and separase modulate membrane traffic by affecting endosomal acidification.

Securin and separase play a key role in sister chromatid separation during anaphase. However, a growing body of evidence suggests that in addition to regulating chromosome segregation, securin and separase display functions implicated in membrane traffic in Caenorhabditis elegans and Drosophila. Here we show that in mammalian cells both securin and separase associate with membranes and that depletion of either protein causes robust swelling of the trans-Golgi network (TGN) along with the appearance of large endocytic vesicles in the perinuclear region. These changes are accompanied by diminished constitutive protein secretion as well as impaired receptor recycling and degradation. Unexpectedly, cells depleted of securin or separase display defective acidification of early endosomes and increased membrane recruitment of vacuolar (V-) ATPase complexes, mimicking the effect of the specific V-ATPase inhibitor Bafilomycin A1. Taken together, our findings identify a new functional role of securin and separase in the modulation of membrane traffic and protein secretion that implicates regulation of V-ATPase assembly and function

Analysis of Ewing Sarcoma (EWS)-Binding Proteins: Interaction with hnRNP M, U, and RNA-Helicases p68/72 within Protein-RNA Complexes

The human Ewing Sarcoma (EWS) protein belongs to the TET family of RNA-binding proteins and consists of an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD), which is extensively methylated at arginine residues. This multifunctional protein acts in transcriptional co-activation, DNA-recombination, -pairing and -repair, in splicing, and mRNA transport. The role of arginine methylation in these processes as well as the time and place of methylation within cells is still unclear. In this study, we show that methylation of recombinant EWS protein in HEK cells occurs immediately after or even during translation. Pull-down experiments with recombinant EWS protein as bait, followed by mass spectrometric analysis identified more than 30 interacting proteins independent of whether the EWS protein was methylated or not. The EWS protein interacts via its RBD with RNase-sensitive protein complexes consisting of mainly heterogeneous nuclear ribonucleoproteins (hnRNPs) and RNA helicases. HnRNP M and U, the RNA-helicases p68 and p72, but also actin and tubulin were found to interact directly with the EWS protein. Co-precipitation experiments with recombinant proteins confirmed the interaction of the EWS protein with p68 via its RBD. Colocalization of the EWS protein and the RNA-helicases in the nucleus of HEK cells was visualized by expressing labeled EWS protein and p68 or p72. When co-expressed, the labeled proteins relocated from the nucleoplasm to nucleolar capping structures. As arginine methylation within the RBD of the EWS protein are neither needed for its subcellular localization nor for its protein-protein interaction, a role of EWS protein methylation in RNA-binding and affecting the activation/repression activity or even in the stabilization of the EWS protein seems very likely

Advances in understanding the molecular basis of frontotemporal dementia

Frontotemporal dementia (FTD) is a clinical syndrome with a heterogeneous molecular basis. Until recently, the underlying cause was known in only a minority of cases that were associated with abnormalities of the tau protein or gene. In 2006, however, mutations in the progranulin gene were discovered as another important cause of familial FTD. That same year, TAR DNA-binding protein 43 (TDP-43) was identified as the pathological protein in the most common subtypes of FTD and amyotrophic lateral sclerosis (ALS). Since then, substantial efforts have been made to understand the functions and regulation of progranulin and TDP-43, as well as their roles in neurodegeneration. More recently, other DNA/RNA binding proteins (FET family proteins) have been identified as the pathological proteins in most of the remaining cases of FTD. In 2011, abnormal expansion of a hexanucleotide repeat in the gene C9orf72 was found to be the most common genetic cause of both FTD and ALS. All common FTD-causing genes have seemingly now been discovered and the main pathological proteins identified. In this Review, we highlight recent advances in understanding the molecular aspects of FTD, which will provide the basis for improved patient care through the development of more-targeted diagnostic tests and therapies