Skd3 (human CLPB) is a potent mitochondrial protein disaggregase that is inactivated by 3-methylglutaconic aciduria-linked mutations

Introduction

Protein aggregation is a challenge for any living cell, resulting in the loss of protein function and the generation of potentially toxic misfolded protein species. Non-metazoan eukaryotes such as yeast employ specialized molecular chaperones, Hsp104 in the cytosol and nucleus and Hsp78 in mitochondria, that are capable of untangling protein aggregates, allowing the native protein state to be restored. Curiously, these disaggregases seem to have been lost during evolution and are absent in metazoa such as ourselves. Recently, human chaperone complexes capable of disaggregation in the cytosol were identified (1, 2), but a mitochondrial disaggregase so far remained elusive. In this preprint, the authors investigate whether Skd3, an AAA+ ATPase that has some resemblance to Hsp78 and Hsp104, is in fact the long-sought mitochondrial disaggregase in metazoa.

 

The authors find that Skd3…

• … has ATPase and disaggregase activity. Using in vitro assays, the authors show that purified Skd3 causes aggregated luciferase to partially refold, and that this function is dependent on its ATPase activity. Mutation of a residue in the conserved pore loop that is thought to be critical for substrate binding abolishes the disaggregase activity of Skd3.

 

• … is regulated by an auto-inhibitory peptide. Skd3 contains a hydrophobic N-terminal stretch that is cleaved by the inner membrane protease PARL. When the authors express Skd3 without this region, mimicking PARL cleavage, they find that the disaggregase activity of Skd3 is boosted by over 10-fold compared to the full-length protein.

 

• … is capable of disaggregating α-synuclein fibrils. The authors note that the requirements for disaggregating amorphous aggregates versus highly structured amyloid fibrils may differ. However, using a sedimentation assay combined with a dot blot they observe that Skd3 can disaggregate α-synuclein fibrils in the presence of ATP.

 

• … disease mutations impair the function of the protein. Mutations in Skd3 are associated with the rare mitochondrial disorder 3-methylglutaconic aciduria, type VII (MGCA7). When testing four Skd3 mutants in their in vitro assay, the authors find that the ATPase activities vary but the disaggregase activity correlates with disease severity. Altogether the authors conclude that the function of Skd3 as a mitochondrial disaggregase is crucial for human health.

 

 

Why I chose this preprint

As the authors nicely outline in their introduction, it has so far remained a mystery how metazoa can do without the disaggregase proteins Hsp104 and Hsp78 that are present in yeast, but were lost during evolution. Machineries capable of disaggregating amorphous and fibrillar aggregates in the metazoan cytosol have been identified in recent years (1, 2) (see also my recent preLight on this topic), but a mitochondrial disaggregase analogous to yeast Hsp78 had remained elusive. The authors hypothesize that Skd3 could be the long-sought mitochondrial disaggregase, and demonstrate with an elegant series of experiments that this is indeed the case. These findings are disease-relevant, as the authors show that mutations associated with MGCA7 impair the disaggregation function of Skd3. Furthermore, the authors show that Skd3 can disaggregate preformed α-synuclein fibrils, which play a role in Parkinson’s disease and have been suggested to affect mitochondria. Given the importance of mitochondrial protein quality control, the implications of Skd3 activity may well extend more broadly to ageing and neurodegenerative diseases.

 

Questions

Did the authors test any other candidate mitochondrial disaggregase proteins, or was Skd3 the only obvious choice?

Are the authors aware of a correlation between Skd3 activity and ageing or neurodegenerative diseases?

 

References

1. Nillegoda NB, Kirstein J, Szlachcic A, Berynskyy M, Stank A, Stengel F, Arnsburg K, Gao X, Scior A, Aebersold R, Guilbride DL, Wade RC, Morimoto RI, Mayer MP and Bukau B (2015) Crucial HSP70 co-chaperone complex unlocks metazoan protein disaggregation. Nature 524: 247–251
2. Gao X, Carroni M, Nussbaum-Krammer C, Mogk A, Nillegoda NB, Szlachcic A, Guilbride DL, Saibil HR, Mayer MP and Bukau B (2015) Human Hsp70 Disaggregase Reverses Parkinson’s-Linked α-Synuclein Amyloid Fibrils. Mol. Cell 59: 781–793

Tags: chaperones, mitochondria, protein disaggregation

doi: https://doi.org/10.1242/prelights.16922

Read preprint (No Ratings Yet)