Molecular and cellular mechanisms to protect against the accumulation of protein aggregates in mitochondria
Final Report Abstract
Mitochondria as essential eukaryotic organelles play a major role in human pathologies, ranging from aging processes to neurodegenerative diseases. The identification and characterization of the molecular mechanisms involved in maintaining mitochondrial function and protein homeostasis under normal and stress conditions are the main topic of the research group. The focus of the DFG-funded project was the characterization of aggregation-protective processes that are specific for mitochondria. The project comprised three major approaches: A) a biochemical characterization of the endogenous substrate interaction of the mitochondrial chaperone Hsp78, using yeast as a model system, B) the identification of novel aggregation-protective processes in yeast mitochondria by artificially increasing the load of misfolding and aggregation-prone polypeptides, and C) the analysis of the mitochondrial aggregation reactions in higher eukaryotes. The endogenous substrate range and interaction properties of Hsp78, the major mitochondrial chaperone shown to be involved in disaggregation reactions, were analyzed by two quantitative proteomic approaches. By analyzing the protein amounts accumulating in aggregates under different heat-stress conditions, we were able to identify the general sensitivity of a large number of mitochondrial polypeptides to stress-induced aggregation. We also identified polypeptides that were aggregated but recovered to a soluble state by a Hsp78-dependent disaggregation reaction. This analysis was combined with a direct proteomic screen for interacting polypeptides that utilized a newly generated substrate-trap mutant of Hsp78. Using these approaches we were able to define the role of Hsp78 in mitochondrial stress protection and to extend the information on its endogenous substrate proteins under in vivo conditions that was previously limited to a few model substrates. The expression of artificial aggregation-prone reporter proteins in the mitochondrial matrix of yeast cells yielded a surprising observation, a complete sequestration of mitochondrial aggregates in a single cellular compartment, termed IMiQ. This sequestration process correlated with the absence of phenotypic defects in wild type cells, demonstrating the importance of IMiQ formation for the protection of mitochondrial functions against aggregation-based proteotoxicity. We could also establish that the protective effect of IMiQ formation was dependent on a functional mitochondrial fusion and fission machinery. Previously, similar sequestration processes have only been observed with cytosolic aggregates, indicating that mitochondria are able to employ similar active protection mechanisms. Future studies will address the cell-biological details of the IMiQ formation process and its cooperation with other organellar PQC mechanisms. A proteomic characterization of heat-stress induced aggregation reactions in mammalian mitochondria resulted in the observation of a surprisingly high stability of most mitochondrial polypeptides. Interestingly, the mitochondrial translation factors Tufm and Tsfm exhibited a very quick and complete stress-dependent aggregation behavior in contrast to most other mitochondrial polypeptides. Correlating with this phenomenon we observed a reduction of mitochondrial translation efficiency and also preprotein import, the two processes responsible for mitochondrial protein biogenesis. We conclude that a meta-stabile folding state of the translation factor might represent a sensor mechanism for shutting down mitochondrial protein biogenesis under stress conditions, preventing a further accumulation of misfolding-prone nascent polypeptides. We also performed preliminary studies concerning the behavior of reporter proteins generating amorphous and amyloid aggregates in mammalian cells. These experiments showed a differential effect on mitochondrial morphologies, depending on aggregate properties. We have preliminary evidence that also mammalian mitochondria are able to perform aggregate sequestration reactions, similar to the yeast IMiQ compartment. The impact of aggregation reactions on mitochondrial homeostasis and the underlying protective mechanisms need to be addressed in future follow-up studies. Taken together, we have now obtained a comprehensive picture of aggregation processes in mitochondria, an organellar system that is very important for activity and survival of eukaryotic cells. We were able to extend the analysis of mitochondrial protein aggregation, which has been basically performed in model systems using lower eukaryotes like yeast, to the characterization of the situation in higher eukaryotes like human cells. These studies will have a future direct impact on the elucidation of pathologic mechanisms, in particular for neurodegenerative diseases.
Publications
- (2016). Amyloid β-peptides interfere with mitochondrial preprotein import competence by a co-aggregation process. Mol. Biol. Cell 27, 3257-3272
Cenini, C., Rüb, C., Bruderek, M. & Voos, W.
(See online at https://doi.org/10.1101/050617) - (2016). Quality control at the mitochondrion. Essays Biochem. 60, 213-225
Voos, W., Jaworek, W., Wilkening, J. & Bruderek M.
(See online at https://doi.org/10.1042/EBC20160009) - (2016). Role of mitochondrial protein quality control in oxidative stress-induced neurodegenerative diseases. Curr. Alzheimer Res. 13, 164-173
Cenini, G. & Voos, W.
- (2018). High aggregation sensitivity of mammalian mitochondrial elongation factor Tu (Tufm) as a sensor for organellar stress. J. Bio. Chem.
Wilkening, A., Rüb, C., Sylvester, M. & Voos, W.
(See online at https://doi.org/10.1101/253153) - (2018). IMiQ: a novel protein quality control compartment protecting mitochondrial functional integrity. Mol. Biol. Cell 29, 256-269
Bruderek, M., Jaworek, W., Wilkening, A., Förtsch, A., Rüb, C., Cenini, G., Sylvester, M. & Voos, W.
(See online at https://doi.org/10.1101/075127)
