Final Report Summary - MITOUPR (Mitochondrial unfolded protein response and the role in ageing)
Mitochondria are essential organelles for almost all eukaryotic cells; thus, sustaining the integrity of their proteome is crucial for cell viability. Any misfolded, impaired, or no longer needed proteins are recognized and degraded by the organelle’s proteolytic machinery to avoid further damage that ultimately might lead to cell death. Protein quality control is commonly maintained by ATP dependent proteases present in different cellular compartments, including mitochondria. Until recently, CLPP was one of the least studied mitochondrial proteases, but gained substantial interest after it was suggested that is essential for the mitochondrial unfolded protein response (UPRmt) in nematode Caenorhabditis elegans. ClpXP is highly conserved proteasome-like machinery present in all bacterial species that is also found in the mitochondria and chloroplasts of eukaryotic cells. Despite being the most studied protease in bacteria, very little is know about it function in higher eukaryotes. Through MitoUPR project we have now uncovered a number of important roles for CLPP in mammalian physiology.
We created CLPP deficient mice that faithfully recapitulate Perrault syndrome caused by CLPP deficiency in humans, characterized by sensorineural hearing loss and a premature ovarian failure. Remarkably, the loss of CLPP results in a lean phenotype with improved glucose homeostasis. CLPP-deficient mice are protected from diet-induced obesity and insulin-resistance, possibly due to higher fatty acid oxidation and browning of white adipose tissue. However, CLPP ablation also leads to a decline in brown adipocytes function leaving mice unable to cope with a cold-induced stress due to non-functional adaptive thermogenesis.
On the molecular level, we uncovered an unforeseen role of CLPP in the regulation of ribosomal biogenesis and mitochondrial translation. As a result of impaired mitochondrial translation, respiratory chain dysfunction seems to be the major effect of CLPP deficiency in mammalian mitochondria. Through proteomic approach and the use of a catalytically inactive CLPP we produced the first comprehensive list of possible ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation and a number of metabolic pathways. The analysis of potential CLPP substrates highlighted ERAL1, a putative 12 rRNA chaperone, as a likely candidate mediating the effect on mitochondrial protein synthesis. High association of ERAL1 with 28S small ribosomal subunit in the absence of CLPP prevents its full maturation and assembly with 39S large subunit into functional mitoribosome. Therefore, ClpP seems to be essential for the regulation of mitochondrial ribosomal assembly through timely removal of ERAL1 that allows proper maturation of mitoribosome.
In parallel with these studies, we refuted the role of mammalian CLPP in regulation and propagation of UPRmt signal. We further show that ClpP is also not necessary for the upregulation of integrated stress response or for the suppression of autophagy. In the same time, loss of ClpP strongly alleviates the observed mitochondrial cardiomyopathy and remarkably prolongs severely shorten lifespan in this model. The observed improvement is a result of increased number of full-length mtDNA-encoded subunits synthesized inside mitochondria and a significantly less abortive protein synthesis. This is quite striking, as the loss of CLPP in the wild type background actually slows down the rate of mitochondrial protein synthesis and in humans causes Perrault syndrome.
In conclusion, these results demonstrate a critical role for CLPP in either mitochondrial, cellular or organismal physiology, thus providing tools to understand pathologies with deregulated Clpp expression. In the same time our results opened a possibility for exploration of therapeutic intervention targeting CLPP activity in the large group of mitochondrial diseases that directly affect mitochondrial protein synthesis and novel insights into therapeutic approaches against metabolic dysfunctions linked to mitochondrial diseases.
We created CLPP deficient mice that faithfully recapitulate Perrault syndrome caused by CLPP deficiency in humans, characterized by sensorineural hearing loss and a premature ovarian failure. Remarkably, the loss of CLPP results in a lean phenotype with improved glucose homeostasis. CLPP-deficient mice are protected from diet-induced obesity and insulin-resistance, possibly due to higher fatty acid oxidation and browning of white adipose tissue. However, CLPP ablation also leads to a decline in brown adipocytes function leaving mice unable to cope with a cold-induced stress due to non-functional adaptive thermogenesis.
On the molecular level, we uncovered an unforeseen role of CLPP in the regulation of ribosomal biogenesis and mitochondrial translation. As a result of impaired mitochondrial translation, respiratory chain dysfunction seems to be the major effect of CLPP deficiency in mammalian mitochondria. Through proteomic approach and the use of a catalytically inactive CLPP we produced the first comprehensive list of possible ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation and a number of metabolic pathways. The analysis of potential CLPP substrates highlighted ERAL1, a putative 12 rRNA chaperone, as a likely candidate mediating the effect on mitochondrial protein synthesis. High association of ERAL1 with 28S small ribosomal subunit in the absence of CLPP prevents its full maturation and assembly with 39S large subunit into functional mitoribosome. Therefore, ClpP seems to be essential for the regulation of mitochondrial ribosomal assembly through timely removal of ERAL1 that allows proper maturation of mitoribosome.
In parallel with these studies, we refuted the role of mammalian CLPP in regulation and propagation of UPRmt signal. We further show that ClpP is also not necessary for the upregulation of integrated stress response or for the suppression of autophagy. In the same time, loss of ClpP strongly alleviates the observed mitochondrial cardiomyopathy and remarkably prolongs severely shorten lifespan in this model. The observed improvement is a result of increased number of full-length mtDNA-encoded subunits synthesized inside mitochondria and a significantly less abortive protein synthesis. This is quite striking, as the loss of CLPP in the wild type background actually slows down the rate of mitochondrial protein synthesis and in humans causes Perrault syndrome.
In conclusion, these results demonstrate a critical role for CLPP in either mitochondrial, cellular or organismal physiology, thus providing tools to understand pathologies with deregulated Clpp expression. In the same time our results opened a possibility for exploration of therapeutic intervention targeting CLPP activity in the large group of mitochondrial diseases that directly affect mitochondrial protein synthesis and novel insights into therapeutic approaches against metabolic dysfunctions linked to mitochondrial diseases.