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We combined quantitative proteomics with N terminome profiling and substrate trapping AP-MS to identify new ClpXP substrates. Mitochondrial protein N termini isolated from mouse hearts revealed frequent aminopeptidase processing after MTS cleavage and many internal protease-generated neo-N termini. CLPP-deficient mice showed perturbed processing patterns with overall accumulation of protease-generated neo-N termini. New high-confidence candidate ClpXP substrates were identified by strong changes in N termini abundance or pull-downs with inactive CLPP and validated using immunoblotting and cycloheximide-chase experiments., Graphical Abstract Highlights • Mitochondrial heart N terminome shows aminopeptidase processing after MTS cleavage. • CLPP-deficiency alters protein processing patterns in mouse heart mitochondria. • Candidate substrates identified by N termini accumulation and interaction with inactive ClpXP. • UQCRC1, HSPA9 and OAT validated biochemically as high confidence ClpXP substrates., The mammalian mitochondrial proteome consists of more than 1100 annotated proteins and their proteostasis is regulated by only a few ATP-dependent protease complexes. Technical advances in protein mass spectrometry allowed for detailed description of the mitoproteome from different species and tissues and their changes under specific conditions. However, protease-substrate relations within mitochondria are still poorly understood. Here, we combined Terminal Amine Isotope Labeling of Substrates (TAILS) N termini profiling of heart mitochondria proteomes isolated from wild type and Clpp−/− mice with a classical substrate-trapping screen using FLAG-tagged proteolytically active and inactive CLPP variants to identify new ClpXP substrates in mammalian mitochondria. Using TAILS, we identified N termini of more than 200 mitochondrial proteins. Expected N termini confirmed sequence determinants for mitochondrial targeting signal (MTS) cleavage and subsequent N-terminal processing after import, but the majority were protease-generated neo-N termini mapping to positions within the proteins. Quantitative comparison revealed widespread changes in protein processing patterns, including both strong increases or decreases in the abundance of specific neo-N termini, as well as an overall increase in the abundance of protease-generated neo-N termini in CLPP-deficient mitochondria that indicated altered mitochondrial proteostasis. Based on the combination of altered processing patterns, protein accumulation and stabilization in CLPP-deficient mice and interaction with CLPP, we identified OAT, HSPA9 and POLDIP2 and as novel bona fide ClpXP substrates. Finally, we propose that ClpXP participates in the cooperative degradation of UQCRC1. Together, our data provide the first landscape of the heart mitochondria N terminome and give further insights into regulatory and assisted proteolysis mediated by ClpXP.

Most manipulations that extend lifespan also increase resistance to various stress factors and environmental cues in a range of animals from yeast to mammals. However, the underlying molecular mechanisms regulating stress resistance during aging are still largely unknown. Here we identify Krüppel-like factor 1 (KLF-1) as a mediator of a cytoprotective response that dictates longevity induced by reduced mitochondrial function. A redox-regulated KLF-1 activation and transfer to the nucleus coincides with the peak of somatic mitochondrial biogenesis that occurs around a transition from larval stage L3 to D1. We further show that KLF-1 activates genes involved in the xenobiotic detoxification programme and identified cytochrome P450 oxidases, the KLF-1 main effectors, as longevity-assurance factors of mitochondrial mutants. Collectively, these findings underline the importance of the xenobiotic detoxification in the mitohormetic, longevity assurance pathway and identify KLF-1 as a central factor in orchestrating this response., Cytochrome P450 oxidases (CYPs) are enzymes that participate in the xenobiotic detoxification and their expression is enhanced in long-lived model organisms. Here the authors show that KLF-1 promotes cyp expression and ensures lifespan extension in C. elegans mitomutants by activating mitohormesis.

Upon mitochondrial dysfunction, CHOP acts as a rheostat that attenuates prolonged stress and delays the onset of cardiomyopathy., In response to disturbed mitochondrial gene expression and protein synthesis, an adaptive transcriptional response sharing a signature of the integrated stress response (ISR) is activated. We report an intricate interplay between three transcription factors regulating the mitochondrial stress response: CHOP, C/EBPβ, and ATF4. We show that CHOP acts as a rheostat that attenuates prolonged ISR, prevents unfavorable metabolic alterations, and postpones the onset of mitochondrial cardiomyopathy. Upon mitochondrial dysfunction, CHOP interaction with C/EBPβ is needed to adjust ATF4 levels, thus preventing overactivation of the ATF4-regulated transcriptional program. Failure of this interaction switches ISR from an acute to a chronic state, leading to early respiratory chain deficiency, energy crisis, and premature death. Therefore, contrary to its previously proposed role as a transcriptional activator of mitochondrial unfolded protein response, our results highlight a role of CHOP in the fine-tuning of mitochondrial ISR in mammals.

Cellular proteostasis describes all processes, which are involved in synthesis, folding and degradation of proteins. The balance between protein translation and degradation is strictly regulated in the cell to ensure its viability. For this purpose, the activity of the protein synthesis and degradation machinery can be adapted according to cellular needs. Disturbance of proteostasis leads for example to accumulation of misfolded or damaged proteins in the cell and is linked to aging and conformational diseases (proteinopathies) such as neurodegenerative, cardiovascular and pulmonary diseases. The ubiquitin-proteasome system plays a central role for the balanced protein turnover in the cell as it is responsible for the degradation of up to 80 % of all cellular proteins. The proteasome is a large protein complex, with catalytically active cleavage sites located within the 20S core proteasome. Ubiquitin-dependent degradation of folded proteins is mainly performed by the 26S proteasome, which is formed by the assembly of 20S core particle and 19S regulatory particles. Assembly and activity of the 26S proteasome are fine-tuned according to cellular needs such as growth and differentiation. Regulation of protein synthesis via the mammalian target of rapamycin (mTOR) has for example direct effects on 26S proteasome function. As 26S proteasome function is strictly dependent on energy in form of ATP, mitochondria – the powerhouses of the cell – are also involved in the regulation of protein degradation by the proteasome. Additionally, reactive oxygen species (ROS), which are mainly produced by dysfunctional mitochondria, negatively influence 26S proteasome assembly and activity. However, mitochondria are not only the main source of cellular ATP and ROS but also provide important metabolites and precursors generated by the tricarboxylic acid cycle (TCA), which are involved in central cellular processes such as proliferation. While a variety of regulatory mechanisms for protein translation and 26S proteasome mediated protein degradation have already been described, a metabolic regulation of cellular proteostasis mediated by mitochondria has not been demonstrated so far. Therefore, the main focus of the present study was to dissect a possible connection between mitochondrial metabolism and cellular proteostasis. For that three different models for mitochondrial respiratory chain dysfunction were used: mouse embryonic fibroblasts (MEFs) derived from the so-called mtDNA mutator mouse model, primary human skin fibroblasts with a mutation in the ND5 subunit of respiratory chain complex I and primary human skin and lung fibroblasts treated with the complex I inhibitor and anti-diabetic drug metformin. The different models are all characterized by respiratory chain complex I deficiency in the absence of increased ROS production. Cells maintained cellular viability and did not show signs of severe stress despite respiratory chain dysfunction. Mitochondrial complex I deficiency in mutator MEFs caused metabolic reprogramming of the TCA cycle resulting in diminished aspartate biosynthesis. Reduced aspartate levels caused downregulated proteostasis as both protein translation and 26S proteasome assembly and activity was decreased in respiration deficient cells but could be rescued by supplementation of aspartate. Furthermore, aspartate supplementation induced mTORC1 mediated protein synthesis and mTORC1-dependent transcriptional activation of defined proteasome assembly factors, which were involved in activation of 26S proteasome assembly and activity in cells with complex I deficiency. Similar data were obtained in ND5 mutant skin fibroblasts and upon metformin treatment. In contrast to diminished proteasome function, chronic respiratory chain impairment in mutator MEFs led to strongly induced immunoproteasome expression and activity. Upregulation of the immunoproteasome was accompanied by increased MHC class I antigen presentation during chronic mitochondrial dysfunction representing a so far unknown stress response which may probably serve to alert the immune system. This finding requires further analysis. These results thus uncover a novel concept of how mitochondrial metabolism adaptively adjusts protein synthesis and degradation by the proteasome to the metabolic condition of the cell. These data extend the knowledge about proteasomal regulation in the cell and have therapeutic implications for diseases and drug-targeted mitochondrial reprogramming.

Protein homeostasis involving protein synthesis and degradation by the proteasome system is intricately balanced in the cell and fine-tuned according to cellular needs and upon stress. Metabolic regulation of protein synthesis and proteasome activity by respiratory chain dysfunction has not been investigated so far. In our study, we used mouse embryonic fibroblasts (MEFs) isolated from the mitochondrial (mt)DNA mutator mouse model that is characterized by genetic respiratory chain dysfunction. Cellular proteostasis including protein synthesis and proteasome activity was significantly decreased in mutator MEFs. In particular, mTOR signaling was downregulated and the formation of 26S proteasome complexes was impaired. Electron microscopy and proteomic analysis of isolated mitochondria revealed massive respiratory chain complex I deficiency in mutator mitochondria resulting in the accumulation of NADH and altered Krebs cycle metabolism. Targeted metabolomics of mutator MEFs identified impaired biosynthesis of aspartate as a consequence of disturbed Krebs cycle function. Notably, supplementation with aspartate restored both mTOR mediated protein translation and proteasome activity in mutator cells. Furthermore, we delineated impairment of respiratory chain complex I activity as the driving factor of this metabolic regulation in primary human lung fibroblasts treated with the complex I inhibitor Metformin. Metformin downregulated both protein synthesis and proteasome activity, a phenotype that was rescued by aspartate supplementation. Our data thus unravel a previously unrecognized adaptation of proteostasis to metabolic alterations of the cell, which might be of therapeutic relevance for the treatment of lung diseases.

The mitochondrial matrix protease CLPP plays a central role in the activation of the mitochondrial unfolded protein response (UPRmt) in Caenorhabditis elegans . Far less is known about mammalian UPRmt signaling, although similar roles were assumed for central players, including CLPP. To better understand the mammalian UPRmt signaling, we deleted CLPP in hearts of DARS2‐deficient animals that show robust induction of UPRmt due to strong dysregulation of mitochondrial translation. Remarkably, our results clearly show that mammalian CLPP is neither required for, nor it regulates the UPRmt 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 UPRmt signaling in mammals, while introducing CLPP as a possible novel target for therapeutic intervention in mitochondrial diseases. ![][1] Loss of mammalian ClpP does not affect the mitochondrial unfolded protein response (UPRmt) under proteotoxic stress. Instead, loss of ClpP alleviates mitochondrial cardiomyopathy by partially rescuing the mitochondrial translation defect and reducing the level of potentially toxic peptides. [1]: /embed/graphic-1.gif

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.

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.

An increase in mtDNA mutation load leads to a loss of critical cells in different tissues thereby contributing to the physiological process of organismal ageing. Additionally, the accumulation of senescent cells that display changes in metabolic function might act in an active way to further disrupt the normal tissue function. We believe that this could be the important link missing in our understanding of the molecular mechanisms of premature ageing in the mtDNA mutator mice. We tested proliferation capacity of mtDNA mutator cells in vitro. When cultured in physiological levels of oxygen (3%) their proliferation capacity is somewhat lower than wild-type cells. Surprisingly, in conditions of increased oxidative stress (20% O(2)) mtDNA mutator mouse embryonic fibroblasts exhibit continuous proliferation due to spontaneous immortalization, whereas the same conditions promote senescence in wild-type cells. We believe that an increase in aerobic glycolysis observed in mtDNA mutator mice is a major mechanism behind this process. We propose that glycolysis promotes proliferation and allows a fast turnover of metabolites, but also leads to energy crisis due to lower ATP production rate. This could lead to compromised replication and/or repair and therefore, in rare cases, might lead to mutations in tumor suppressor genes and spontaneous immortalization.

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., Author Summary Mitochondria produce the majority of the energy needed for numerous cell functions through oxidative phosphorylation. However, this comes with the cost in the form of potentially harmful reactive oxygen species (ROS) that could damage all kinds of biological macromolecules. Changes in mitochondrial membrane potential through mild uncoupling could alter ROS production in the cell (“uncoupling to survive”). Mitochondrial uncoupling proteins (UCPs) are believed to play a central role in this process. We detected increased amounts of UCP2 in mtDNA mutator mice, a model for premature aging. Depletion of UCP2 in mtDNA mutator mice led to further shortening of the lifespan with earlier signs of mitochondrial cardiomyopathy accompanied with high systemic lactic acidosis, often used as a marker of mitochondrial diseases. Remarkably, our results demonstrate that the presence of UCP2 wields beneficial effect on respiratory deficient mitochondria without affecting ROS production or uncoupling. Instead, UCP2 protein seems to mediate a valuable upregulation of fatty acid metabolism detected in mtDNA mutator hearts. Our results provide a novel mechanism of adaptation of mitochondria to respiratory deficiency mediated by UCP2 that clearly argues against the “uncoupling to survive” theory.

Advanced glycation end products (AGEs) accumulate during ageing with reactive oxygen species from the mitochondrial respiratory chain discussed as a driving force. To determine the role of mitochondrial activity for AGE formation, a rho0 derivative of the 143B.TK- osteosarcoma cell line lacking the respiratory chain, was analysed. These cells exhibit decreased superoxide formation but unchanged mitochondrial SOD expression as well as unchanged antioxidative free sulfhydryl (SH) levels. Whereas total protein content shows no differences in AGE levels, cell fractionation and Western blotting demonstrates some changes in the AGE pattern. Thus, the absence of functional respiration has only a negligible impact on AGE accumulation.

Mitochondria play a critical role in the life of the cell as they control their metabolic rate, energy production and cell death. Mitochondria have long been appreciated as causative to aging. The age-associated respiratory chain deficiency is typically unevenly distributed and affects only a subset of cells in various human tissues, such as heart, skeletal muscle, colonic crypts and neurons. Studies of mtDNA mutator mice has provided the first direct evidence that accelerating the mtDNA mutation rate can result in premature aging, consistent with the view that loss of mitochondrial function is a major causal factor in aging. New, controversial data have arisen from the studies on molecular mechanisms that drive premature aging in mtDNA mutator mice. Our results suggest that the accumulation of high levels of mtDNA point mutations, causing amino acid substitutions, combined with their clonal expansion is probably the main driving force behind premature aging in mtDNA mutator mice.

Eukaryotic cells devoid of mitochondrial DNA (rho0 cells) were originally generated under artificial growth conditions utilizing ethidium bromide. The chemical is known to intercalate preferentially with the mitochondrial double-stranded DNA thereby interfering with enzymes of the replication machinery. Rho0 cell lines are highly valuable tools to study human mitochondrial disorders because they can be utilized in cytoplasmic transfer experiments. However, mutagenic effects of ethidium bromide onto the nuclear DNA cannot be excluded. To foreclose this mutagenic character during the development of rho0 cell lines, we developed an extremely mild, reliable and timesaving method to generate rho0 cell lines within 3-5 days based on an enzymatic approach. Utilizing the genes for the restriction endonuclease EcoRI and the fluorescent protein EGFP that were fused to a mitochondrial targeting sequence, we developed a CMV-driven expression vector that allowed the temporal expression of the resulting fusion enzyme in eukaryotic cells. Applied on the human cell line 143B.TK- the active protein localized to mitochondria and induced the complete destruction of endogenous mtDNA. Mouse and rat rho0 cell lines were also successfully created with this approach. Furthermore, the newly established 143B.TK- rho0 cell line was characterized in great detail thereby releasing interesting insights into the morphology and ultra structure of human rho0 mitochondria.