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Exploiting the HSP70 chaperone machine for novel therapeutic strategies in human diseases and for the engineering of productivecellular biomolecular factories

   
Project

QLK3-2000-00720

Cell factory area

3.3.1

EU Contribution

1 501 050 Euro

Duration

36 months

Type

Research project

Starting date

01-10-2000

Keywords
chaperone machine
Hsp70
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ABSTRACT
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Hsp70 is the central component of a multi-chaperone machine involved in many essential cellular processes and in the response of cells to proteotoxic stresses (e.g.) heat shock). Furthermore, Hsp70 is a potent anti-apoptotic protein that can enhance transformation and the tumorigenic potential of cells. This proposal aims to obtain detailed knowledge of the function of the Hsp720 machine and its regulation by co-chaperones. This knowledge will be used to gain insight into its exact role in disease pathogenesis (cancer and neurodegenerative diseases). The results are expected to reveal new possibilities for therapeutic interventions. The knowledge obtained will, furthermore, be exploited to improve the industrial use of mammalian cells as protein factories by attacking major problems in the large-scale production of therapeutic proteins in mammalian cells, i.e. increased apoptosis, sensitivity to temperature shifts, and incomplete protein folding.

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OBJECTIVES
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This project aims for the biochemical and functional characterisation of the Hsp70 chaperone machine. The role of (novel) co-chaperones on the Hsp70 chaperone machine will be defined and its, as yet unknown, mechanisms of action in the regulation of apoptosis and transformation will be investigated. Also the role of the Hsp70 machine in amyloidosis, and neurodegenerative diseases such as spinal bulbar muscular atrophy and spinocerebellar ataxia type 1 will be explored. Finally, the knowledge of the components and the functionality of the machine will be exploited to generate stable, stress and apoptosis resistant mammalian cell lines to be used as a factory for the efficient production of therapeutic proteins in an industrial setting.

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DESCRIPTION OF THE WORK
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The precise role of co-chaperones within the complex Hsp70 reaction cycle will be defined in vitro by an array of interaction and competition experiments between the various co-chaperones and Hsp70. The implications of these interactions for the functionality of the Hsp70 charperone machine will then be studied in mammalian cells using transfected reporter proteins such as GFP-tagged firefly luciferase, human disease related proteins, such as androgen receptor containing polyglutamine expansions and mutant rhodopsin. The fate of these proteins in chaperone expressing cells will be monitored by activity measurements or/and confocal immunofluorescence microscopy, biochemical analyses, and sub-cellular fractionation. The impact of the individual components of the chaperone machine on cellular sensitivity to apoptotic stimuli will be investigated by standard apoptosis assays following transfection of mutant and wild type cDNAs encoding co-chaperones together with cDNAs for wild type or mutant Hsp70 into apoptosis-sensitive cells. Thereafter, the tumorigenic potential of these cells as well as their sensitivity to transformation by SV40 large T antigen will be investigated to clarify the significance of these intercations to oncogene-induced transformation. A screening system for drugs affecting the anti-apoptotic and transforming functions of the Hsp70 machine will be designed susequently. Furthermore, we will exploit the scientific knowledge obtained on the in vivo function of the Hsp70 chaperone machine to develop a new generation of production cell lines with improved viability (low apoptotic proneness, resistance to temperature changes) and increased protein production when grown in large scale cultures. This will improve the cost-effectiveness of industrial-scale expression of proteins applied in research and treatment of human diseases.

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DELIVERABLES
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  • Improved understanding of the function of the Hsp70 chaperone machine.
  • Sufficient insight into the mechanism of action of the Hsp70 machine in apoptosis and transformation to develop novel strategies of therapeutic intervention in the field of cancer and neurodegenerative diseases.
  • Exploitation of the knowledge on the chaperone factory to engineer stable, stress resistant mammalian cell lines with the capacity to produce high levels correctly folded therapeutic proteins in an industrial setting.

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CONSORTIUM
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COORDINATOR
  Dr. Harm H. Kampinga
(Abbreviation: RUG)
Department of Radiobiology
University of Groningen
Loemsingel 1
9713 BZ Groningen, The Netherlands
Tel: +31 503632903
Fax: +31 503632913
h.h.kampinga@med.rug.nl

PARTNERS
  Dr. Wolfgang M.J. Obermann
(Abbreviation: MPG.IBCHEM)
Max-Planck-Institute for Biochemistry
Department of Cellular Biochemistry
Am Klopferspitz 18a
82152 Martinsried, Germany
Tel: +49 8985782224
Fax: +49 8985782211
obermann@libelle.biochem.mpg.de

Dr. Marja Jäättelä
(Abbreviation: DCS)
Apoptosis Laboratory
Institue of Cancer Biology
Danish Cancer Society
Stranboulevarden 49
2100 Copenhagen, Denmark
Tel: +45 35257500
Fax: +45 35257721
mhj@biobase.dk

Dr. Ioannis Lazaridis
(Abbreviation: UOI)
Laboratory of General biology
Medical School
University of Ioannina
45110 Ioannina, Greece
Tel: +30 65197752
Fax: +30 65167863
ilazarid@cc.uoi.gr

Dr. Michael E. Cheetham
(Abbreviation: UCL)
Department of Pathology
Institute of Ophtalmology
University College London
11-43 Bath Street
EC1V 9EL London, United Kingdom
Tel: +44 2076086944
Fax: +44 2076086862
michael.cheetham@ucl.ac.uk

Dr. Steffen Faisst
(Abbreviation: NN)
Department of Molecular Biology and Virology
Biologics Development
Novo Nordisk Health Care
Mørkhøj Bygade 28
2860 Søborg, Denmark
Tel: +45 44421134
Fax: +45 44421130
stfa@novo.dk
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