A cell model to study different degrees of Hsp60 deficiency in HEK293 cells |
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Authors: | Anne Sigaard Bie Johan Palmfeldt Jakob Hansen Rikke Christensen Niels Gregersen Thomas Juhl Corydon Peter Bross |
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Institution: | (1) Research Unit for Molecular Medicine (MMF), Aarhus University Hospital, Skejby, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark;(2) Department of Forensic Medicine, Aarhus University Hospital, Skejby, Brendstrupgaardsvej 100, 8200 Aarhus N, Denmark;(3) Department of Clinical Genetics, Aarhus University Hospital, 8000 Aarhus C, Denmark;(4) Department of Human Genetics, Aarhus University, 8000 Aarhus C, Denmark |
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Abstract: | Mitochondrial dysfunction is associated with neurodegenerative diseases and mutations in the HSPD1 gene, encoding the mitochondrial Hsp60 chaperone, are the causative factors of two neurodegenerative diseases, hereditary
spastic paraplegia and MitChap60 disease. In cooperation with Hsp10, Hsp60 forms a barrel-shaped complex, which encloses unfolded
polypeptides and provides an environment facilitating folding. We have generated an Hsp60 variant with a mutation (Asp423Ala)
in the ATPase domain and established a stable human embryonic kidney (HEK293) cell line allowing tetracycline-controlled expression
of this mutant variant. We monitored expression of the Hsp60–Asp423Ala variant protein following induction and examined its
effects on cellular properties. We showed that the folding of mitochondrial-targeted green fluorescent protein, a well-known
substrate protein of Hsp60, was consistently impaired in cells expressing Hsp60–Asp423Ala. The level of the Hsp60–Asp423Ala
variant protein increased over time upon induction, cell proliferation stopped after 48-h induction and mitochondrial membrane
potential decreased in a time-dependent manner. In summary, we have established a stable cell line with controllable expression
of an Hsp60 variant, which allows detailed studies of different degrees of Hsp60 deficiency. |
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Keywords: | Hsp60 Mitochondrial dysfunction Mitochondrial protein folding Mitochondrial-targeted GFP Molecular chaperone |
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