Identification and characterization of the nuclear localization/retention signal in the EWS proto-oncoprotein.

Ewing sarcoma (EWS) protein, a member of a large family of RNA-binding proteins, contains an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD). Due to its multifunctional properties EWS protein is involved in processes such as gene expression, RNA processing and transport, and cell signaling. Chimeric EWS proteins generated by chromosomal translocations cause malignant tumors. EWS protein is located predominantly in the nucleus, but was found also in the cytosol and associated with the cell membrane. The determinants responsible for the nuclear localization of the protein were as yet unknown. We identified the nuclear localization signal of EWS protein at its C terminus (C-NLS), which is required for the nuclear import and retention of the protein. The C-NLS sequence is conserved in related proto-oncoproteins suggesting an NLS function also in these proteins. Two arginine residues, due to their positive charge, a proline residue and a tyrosine residue are essential for C-NLS function. The nuclear localization of EWS protein is independent of the regions in RBD containing numerous arginine methylation sites, RNA-recognition and zinc finger motifs. Regions in EAD guide the subnuclear partition of EWS protein and contain another but different NLS that allows nucleocytoplasmic shuttling of the N-terminal domain

Protein arginine methylation: Cellular functions and methods of analysis.

During the last few years, new members of the growing family of protein arginine methyltransferases (PRMTs) have been identified and the role of arginine methylation in manifold cellular processes like signaling, RNA processing, transcription, and subcellular transport has been extensively investigated. In this review, we describe recent methods and findings that have yielded new insights into the cellular functions of arginine-methylated proteins, and we evaluate the currently used procedures for the detection and analysis of arginine methylation

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

Revelation of Immune Memory at the First Stage of Antigen-Specific Cell Response after Second Introduction of the Live Plague Vaccine

Background. The study of immune memory is necessary to evaluate the effectiveness of immunization against infection, including plague and to make a choice of vaccination scheme.Goals. The goal is to study the possible role of immune memory in the early stage of the antigen-specific response – the formation of cells with receptors for capsular (F1) and lipopolysaccharide (LPS) antigens of plague live EV vaccine.Methodology. Volunteers vaccinated with live plague vaccine EV for the first time (6 persons – group 1) and again (6 persons – group 2) were examined. In the mononuclear fraction of the blood of volunteers the cells binding antigens F1 and LPS Y. pestis (CBA) were determined.Results. In the volunteers group 2, the content of CBA at 2 days after vaccination was higher than in group 1. Between the 5th day and the end of the CBA detection, their content in group 2 decreased, and in group 1, it increased, but remained significantly less than in group 2 two days after immunization.Сonclusions. It is shown that the previous vaccination accelerates the first stage of the antigen-specific human response to second vaccination against plague. This reflects the role of immune memory in the formation of this stage of the immune response at vaccination against plague

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