Regulation of the turnover of complex I (CI), the largest mitochondrial respiratory chain complex, remains enigmatic despite huge advancement in understanding its structure and the assembly. Here, we report that the NADH-oxidizing N-module of CI is turned over at a higher rate and largely independently of the rest of the complex by mitochondrial matrix protease ClpXP, which selectively removes and degrades damaged subunits. The observed mechanism seems to be a safeguard against the accumulation of dysfunctional CI arising from the inactivation of the N-module subunits due to attrition caused by its constant activity under physiological conditions. This CI salvage pathway maintains highly functional CI through a favorable mechanism that demands much lower energetic cost than de novo synthesis and reassembly of the entire CI. Our results also identify ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.

Mitochondria are fundamental for cellular metabolism as they are both a source and a target of nutrient intermediates originating from converging metabolic pathways, and their role in the regulation of systemic metabolism is increasingly recognized. Thus, maintenance of mitochondrial homeostasis is indispensable for a functional energy metabolism of the whole organism. Here, we report that loss of the mitochondrial matrix protease CLPP results in a lean phenotype with improved glucose homeostasis. Whole-body CLPP-deficient mice are protected from diet-induced obesity and insulin resistance, which was not present in mouse models with either liver- or muscle-specific depletion of CLPP 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. These results demonstrate a critical role for CLPP in different metabolic stress conditions such as high-fat diet feeding and cold exposure providing tools to understand pathologies with deregulated Clpp expression and novel insights into therapeutic approaches against metabolic dysfunctions linked to mitochondrial diseases.

Systemic immune cell dysfunction is a typical feature of liver diseases and increases the risk of bacterial infection, especially spontaneous bacterial peritonitis. We evaluated functional properties of neutrophil granulocytes in blood and ascites of patients both with and without decompensated cirrhosis. We collected blood and ascites samples from 63 patients with cirrhosis and eight without cirrhosis. Phagocytosis activity (PA) and oxidative burst activity (OBA) were evaluated after ex vivo stimulation with E. coli , while fluorescence signals were measured by flow cytometry. Ascites’ neutrophil function tests were repeated after incubation with autologous plasma. Ascites’ neutrophils showed an impaired PA and OBA (median blood PA 98.1% (86.8–99.8) vs. ascites’ PA 50.5% (0.4–97.3), p < 0.0001; median blood OBA 98.7% (27.5–100) vs. ascites’ OBA 27.5% (0.3–96.7), p < 0.0001). Patients with non-cirrhotic ascites showed higher PA but equally suppressed OBA. Ascites’ neutrophil function could be partially restored after incubation with autologous plasma (median increase PA: 22.5% (−49.7 – +93.2), p = 0.002; OBA: 22.8% (−10.4 – +48.8), p = 0.002). Ascites’ neutrophils of patients with cirrhosis are functionally impaired, but could be partially restored after incubation with plasma. Further investigations are needed to identify the factors in ascites that are associated with neutrophils’ function.

Despite being one of the most studied proteases in bacteria, very little is known about the role of ClpXP in mitochondria. We now present evidence that mammalian CLPP has an essential role in determining the rate of mitochondrial protein synthesis by regulating the level of mitoribosome assembly. Through a proteomic approach and the use of a catalytically inactive CLPP, we produced the first comprehensive list of possible mammalian ClpXP substrates involved in the regulation of mitochondrial translation, oxidative phosphorylation, and a number of metabolic pathways. We further show that the defect in mitoribosomal assembly is a consequence of the accumulation of ERAL1, a putative 12S rRNA chaperone, and novel ClpXP substrate. The presented data suggest that the timely removal of ERAL1 from the small ribosomal subunit is essential for the efficient maturation of the mitoribosome and a normal rate of mitochondrial translation.

The mitochondrial matrix protease CLPP plays a central role in the activation of the mitochondrial unfolded protein response (UPR(mt)) in Caenorhabditis elegans Far less is known about mammalian UPR(mt) signaling, although similar roles were assumed for central players, including CLPP To better understand the mammalian UPR(mt) signaling, we deleted CLPP in hearts of DARS2-deficient animals that show robust induction of UPR(mt) due to strong dysregulation of mitochondrial translation. Remarkably, our results clearly show that mammalian CLPP is neither required for, nor it regulates the UPR(mt) in mammals. Surprisingly, we demonstrate that a strong mitochondrial cardiomyopathy and diminished respiration due to DARS2 deficiency can be alleviated by the loss of CLPP, leading to an increased de novo synthesis of individual OXPHOS subunits. These results question our current understanding of the UPR(mt) signaling in mammals, while introducing CLPP as a possible novel target for therapeutic intervention in mitochondrial diseases.

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.