A cell model to study different degrees of Hsp60 d

Authors: Anne Sigaard Bie Johan Palmfeldt Jakob Hansen Rikke Christensen Niels Gregersen Thomas Juhl Corydon Peter Bross

Publish Date: 2011/06/30

Volume: 16, Issue: 6, Pages: 633-640

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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 barrelshaped 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 tetracyclinecontrolled 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 mitochondrialtargeted green fluorescent protein a wellknown 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 48h induction and mitochondrial membrane potential decreased in a timedependent 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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