Obiettivo In eukaryotic cells, the most dramatic events of cellular stress response and virus-induced transformation occur within the nuclear compartment. The previously obtained data suggested that(i) heat stress and drug-induced inhibition of proteasomes trigger in the cell nucleus a special mechanism of redistribution (sorting) of a set of proteins between intranuclear micro-compartments such as the nucleoli and small granules or PML bodies, and;(ii) some chaperones (Hsp70, Hsp27) and their co-factors or inhibitors (Hsp40, Bag-1) are involved in and affect this mechanism.As the intranuclear sorting was shown to involve, besides the chaperones, a stress-damaged protein (heat-denatured nls-luciferase), an aberrant protein (mutant p53) and a viral protein (EBNA-5), it was supposed that this mechanism is based on recognizing by chaperones of nuclear-localized proteins with abnormal folding and it serves for compartmentalization of these proteins within special sites for storage, refolding or degradation. Presumably, the chaperones are able to modulate the process of protein sorting and its consequences for either protein involved. If so, the chaperone-dependent intranuclear protein sorting is of importance as a component of cellular response to proteotoxic stress or viral infection.The present project is aimed at resolving two major issues:(i) how are the chaperones Hsp70, Hsp27 and their cofactors involved in the process of protein sorting between the nucleolus and the small granules or PML bodies? and;(ii) how does their involvement in the process of intranuclear protein sorting affects cellular responses to stresses and viral infection?For this purpose, special objectives and research activities are outlined in terms of the project:- To characterize in details the redistribution of a set of certain nuclear-localized or nuclear-translocated proteins such as chaperones (Hsp70, Hsp27, Hsp40, others), nls-luciferase, p53, beta-polymerase, transcription regulators, virus-encoded proteins within distinct intranuclear micro-compartments (the nucleoli, small foci or granules, proteasomes and PML bodies) of stressed or virus-infected cells. This will be done using biochemical, immunocytochemical and ultra-structural (microscopy) studies in situ- Combining methods of molecular biology and protein biochemistry with fluorescence analyses in situ, to elucidate the functional state (biological activity) and fate (storage or degradation?) of these proteins undergoing the intranuclear micro-compartmentalization following cellular stresses or viral infection:- Employing plasmid- and virus-based vectors for in vivo over expression or inhibition of some chaperones (Hsp70, Hsp27, Hsp40, their mutant or antisense constructs, Bag-1), to reveal functional roles of either chaperone in mechanisms of the stress- or virus-induced intranuclear micro-compartmentalization of these proteins (e.g. how either chaperone affects the biological activity and fate of the compartmentalized proteins)- Artificially modulating the intranuclear protein sorting by the overexpressed chaperones, their inhibitors or some drugs (e.g. proteasomal inhibitors, As2O3, retinoic acid, interferon), to establish causal links between its machinery and over-all cellular response to stresses (resistance and recovery or cell death?) and viral infection (transformation or apoptosis, or presentation of viral antigens?).It is planned that the major part of all experimental work will be performed in Russia, under a tight cooperation between the team of Dr Kabakov (Obninsk, Kaluga Region) and the team of Professor Polyakov (Moscow). Herein, all the well-tested model systems including plasmid constructs and cell lines will be adopted by both the Russian teams from the team of Professor Szekely (Stockholm, Sweden) and the team of Professor Kampinga (Groningen, The Netherlands); Professor Kampinga will also be responsible for general coordinating of the research. Upon fulfilment of the project, it is expected to find out how the cell nuclei respond to stresses by changing localization, migration and turnover of certain nuclear proteins. The specific intranuclear sites for protein storage and degradation will be established and characterized at the molecular and ultra structure levels. Also, the understanding of the roles of chaperones and its co-factors in regulation of accumulation and processing of some endogenous and viral proteins within the nuclear compartment will be achieved. The novelty of data to be generated from this comparative study on the cellular response to stress and viral infection lies in the possible distinction between storage and refolding or, instead, enhanced degradation of affected nuclear proteins resulting from the chaperone-dependent protein sorting between intranuclear micro-compartments. As a whole, successful fulfilment of this project will hopefully yield new fundamental knowledge in the field of molecular mechanisms of cell regulation. Programma(i) IC-INTAS - International Association for the promotion of cooperation with scientists from the independent states of the former Soviet Union (INTAS), 1993- Argomento(i) 4 - Life Sciences OPEN - OPEN Call Invito a presentare proposte Data not available Meccanismo di finanziamento Data not available Coordinatore University of Groningen Contributo UE Nessun dato Indirizzo A. Deusinglaan 1 9713AV Groningen Paesi Bassi Mostra sulla mappa Costo totale Nessun dato Partecipanti (3) Classifica in ordine alfabetico Classifica per Contributo UE Espandi tutto Riduci tutto Karolinska Institute Svezia Contributo UE Nessun dato Indirizzo Nobelsvag, 16 17177 Stockholm Mostra sulla mappa Costo totale Nessun dato M.V. Lomonosov Moscow State University Russia Contributo UE Nessun dato Indirizzo Building A 119899 Moscow Mostra sulla mappa Costo totale Nessun dato Medical Radiology Research Center of the RAMS Russia Contributo UE Nessun dato Indirizzo Ulitsa Koroleva 4 249020 Obninsk, Kaluga Region Mostra sulla mappa Costo totale Nessun dato
