An inventory of interactors of the human HSP60/HSP10 chaperonin in the mitochondrial matrix space

The HSP60/HSP10 chaperonin assists folding of proteins in the mitochondrial matrix space by enclosing them in its central cavity. The chaperonin forms part of the mitochondrial protein quality control system. It is essential for cellular survival and mutations in its subunits are associated with rare neurological disorders. Here we present the first survey of interactors of the human mitochondrial HSP60/HSP10 chaperonin. Using a protocol involving metabolic labeling of HEK293 cells, cross-linking, and immunoprecipitation of HSP60, we identified 323 interacting proteins. As expected, the vast majority of these proteins are localized to the mitochondrial matrix space. We find that approximately half of the proteins annotated as mitochondrial matrix proteins interact with the HSP60/HSP10 chaperonin. They cover a broad spectrum of functions and metabolic pathways including the mitochondrial protein synthesis apparatus, the respiratory chain, and mitochondrial protein quality control. Many of the genes encoding HSP60 interactors are annotated as disease genes. There is a correlation between relative cellular abundance and relative abundance in the HSP60 immunoprecipitates. Nineteen abundant matrix proteins occupy more than 60% of the HSP60/HSP10 chaperonin capacity. The reported inventory of interactors can form the basis for interrogating which proteins are especially dependent on the chaperonin

Effects of a mutation in the HSPE1 gene encoding the mitochondrial co-chaperonin HSP10 and its potential association with a neurological and developmental disorder

We here report molecular investigations of a missense mutation in the HSPE1 gene encoding the HSP10 subunit of the HSP60/ HSP10 chaperonin complex that assists protein folding in the mitochondrial matrix. The mutation was identified in an infant who came to clinical attention due to infantile spasms at three months of age. Clinical exome sequencing revealed heterozygosity for a HSPE1 NM_002157.2:c.217C>T de novo mutation causing replacement of leucine with phenylalanine at position 73 of the HSP10 protein. This variation has never been observed in public exome sequencing databases or the literature.To evaluate whether the mutation may be disease-associated we investigated its effects by in vitro and ex vivo studies. Our in vitro studies indicated that the purified mutant protein was functional, yet its thermal stability, spontaneous refolding propensity, and resistance to proteolytic treatment were profoundly impaired. Mass spectrometric analysis of patient fibroblasts revealed barely detectable levels of HSP10-p.Leu73Phe protein resulting in an almost 2-fold decrease of the ratio of HSP10 to HSP60 subunits. Amounts of the mitochondrial superoxide dismutase SOD2, a protein whose folding is known to strongly depend on the HSP60/HSP10 complex, were decreased to approximately 20% in patient fibroblasts in spite of unchanged SOD2 transcript levels. As a likely consequence, mitochondrial superoxide levels were increased about 2-fold. Although we cannot exclude other causative or contributing factors, our experimental data support the notion that the HSP10-p.Leu73Phe mutation could be the cause or a strong contributing factor for the disorder in the described patient

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

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