title: A cell model to study different degrees of Hsp60 deficiency in HEK293 cells.
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.
authors: Research Unit for Molecular Medicine (MMF), Aarhus University Hospital, Skejby, Aarhus N, Denmark. abie@svf.au.dk
authors: Anne Sigaard
authors: AS
authors: Bie
authors: Johan
authors: J
authors: Palmfeldt
authors: Jakob
authors: J
authors: Hansen
authors: Rikke
authors: R
authors: Christensen
authors: Niels
authors: N
authors: Gregersen
authors: Thomas Juhl
authors: TJ
authors: Corydon
authors: Peter
authors: P
authors: Bross
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D049109:Cell Proliferation; D018834:Chaperonin 60 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000378:metabolism; D057809:HEK293 Cells / Q000378:metabolism*; D006801:Humans; D053078:Membrane Potential, Mitochondrial; D024101:Mitochondrial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017354:Point Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D018834:Chaperonin 60; D024101:Mitochondrial Proteins; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate