Enhanced J-protein interaction and compromised protein stability of mtHsp70 variants lead to mitochondrial dysfunction in Parkinson's disease

Abstract

Parkinson's disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multifactorial in nature, a recent genetic screen involving PD patients identified two mitochondrial Hsp70 variants (P509S and R126W) that are suggested in PD pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD variants are centrally involved in PD progression is totally elusive. In this article, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD variants. Biochemically, the R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, the P509S variant exhibits significantly enhanced interaction with J-protein cochaperones involved in folding and import machinery, thus altering the overall regulation of chaperone-mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD mutations at the cellular level, we developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with ‘mitochondrial dysfunction’, including compromised growth, impairment in protein translocation, reduced functional mitochondrial mass, mitochondrial DNA loss, respiratory incompetency and increased susceptibility to oxidative stress. In addition to that, R103W protein is prone to aggregate in vivo due to reduced stability, whereas P486S showed enhanced interaction with J-proteins, thus remarkably recapitulating the cellular defects that are observed in human PD variants. Taken together, our findings provide evidence in favor of direct involvement of mtHsp70 as a susceptibility factor in PD.

Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability

“…Measurement of Mitochondrial and Cellular ROS-The extent of superoxide generation in the WT and mutant mitochondria was measured by FACS analysis, utilizing MitoSOX Red dye (Molecular Probes) according to a previously described protocol in both the yeast model system and HeLa cell lines (24,25). The respiratory inhibitor rotenone (1 mM) was used as a positive control, which produces higher superoxide levels by inhibiting complex I.…”

“…Miscellaneous-Analysis of mitochondrial mass and membrane potential was performed following previously reported techniques (24,25). Overall cellular and mitochondrial iron levels were estimated using iron-specific colorimetric assay and atomic absorption spectroscopy according to earlier described procedures (24,29,30).…”

Heat shock proteins in neurodegenerative disorders and aging

“…Furthermore, several variants of the gene that codes for mtHsp70 (HSPA9) have been found in a small cohort of late-onset Parkinson's patients (De Mena et al 2009). Some of the phenotypes associated with mtHsp70 mutations are characterized by mitochondrial dysfunction, such as Njemini et al 2007 respiratory incompetency and increased susceptibility to oxidative stress (Burbulla et al 2010;Goswami et al 2012). On the other hand, mutations in HSPA9 are not common in earlyonset Parkinson's disease (Freimann et al 2013).…”

Molecular Insights Revealing Interaction of Tim23 and Channel Subunits of Presequence Translocase

“…Analysis of mitochondrial membrane potential and mitochondrial mass was accomplished by previously described protocols (49). Briefly, to test for the loss in membrane potential, roughly 50 g of mitochondria was first subjected to heat shock for 15 min, followed by incubation with JC-1 dye for 10 min at room temperature (25°C) in the dark.…”

“…Fluorescence anisotropy analysis was performed with fluorescein-labeled peptide corresponding to the presequence of cytochrome c oxidase 4 (Cox4) (MLSL RQSIRFFKPTRRLC), as described previously, with minor modifications (49). Briefly, increasing concentrations of Tim23 were incubated with 25 nM fluorescein-labeled Cox4 (F-Cox4) in a buffer containing 25 mM Tris-Cl, pH 7.5, 100 mM KCl, and 10% glycerol for 1 h at room temperature.…”