A cell model to study different degrees of Hsp60 deficiency in HEK293 cells

TY - JOUR

T1 - A cell model to study different degrees of Hsp60 deficiency in HEK293 cells

AU - Bie, Anne Sigaard

AU - Palmfeldt, Johan

AU - Hansen, Jakob

AU - Christensen, Rikke

AU - Gregersen, Niels

AU - Corydon, Thomas Juhl

AU - Bross, Peter

PY - 2011/6/30

Y1 - 2011/6/30

N2 - 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.

AB - 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.

U2 - 10.1007/s12192-011-0275-5

DO - 10.1007/s12192-011-0275-5

M3 - Journal article

C2 - 21717087

SN - 1355-8145

VL - 16

SP - 633

EP - 640

JO - Cell Stress & Chaperones

JF - Cell Stress & Chaperones

IS - 6

ER -