Center for Molecular Medicine Cologne

Trifunovic, Aleksandra | Szczepanowska, Karolina - C 15

Manipulation of CLPP protease to complement mitochondrial respiratory deficiency


Despite a steady increase in the number of novel genes implicated in the pathogenesis of mitochondrial diseases, an effective treatment for any mitochondrial disorder is still missing.

We recently discovered a novel respiratory complex I salvage pathway that maintains fully functional CI and thereby healthy mitochondria through a favourable mechanism requiring lower energetic expenditure. Our results also shed light in ClpXP activity as unexpected potential target for therapeutic interventions in the large group of mitochondrial disorders characterized by the CI instability that we will explore in this project.

Mitochondria are essential for maintaining numerous fundamental cell functions. Mutations in either mtDNA or nDNA genes coding for mitochondrial proteins are known to lead to major and catastrophic diseases in humans. They are one of the most common inborn errors of metabolism with a frequency of about 1 in 5000 and come with an impressive variability of symptoms, organ involvement, and clinical course, which considerably impact the quality of life and often shorten the lifespan expectancy.

Unfortunately, currently no treatment is available for myriad of diseases caused by mutations in mitochondrial genes and therapies are mainly aimed to alleviate symptoms and/or slow down the progression of the diseases.

Our preliminary and unpublished data show that by removing the major mitochondrial matrix protease CLPXP, and therefore stabilizing CI, we could ameliorate the symptoms of respiratory deficiency in different cellular models of mitochondrial dysfunction (Figure 1.A-D). The loss of CLPP in these models resulted not only in increased stability of CI (Figure 1D), but also normalized NAD+/NADH ratios. Remarkably, even partial loss of CLPXP activity in respiratory deficient cells led to mild increase in the CI levels, opening an exciting prospect for therapeutic interventions (Figure 1E).

Therefore, the overall goal of this project is to explore the possibility of targeting CLPP activity to ameliorate symptoms of mitochondrial diseases in in vivo models through genetic interventions and usage of specific protease inhibitors. To this end we will use a panel of patient derived cell lines with documented CI deficiency. We will further explore a possible beneficial effect of CLPP deficiency in mouse models for mitochondrial diseases through genetic interventions and usage of protease inhibitors in vivo.

Our Aims

  1. Explore the therapeutical potential of CLPP depletion in cell lines derived from patients with mitochondrial diseases.
  2. Investigate the effect of CLPP deficiency on in vivo murine models of mitochondrial deficiency.
  3. Analyse molecular mechanism of specific CLPP inhibitors and test their efficiency in cello and in vivo models

Previous Work

We demonstrated 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.

We discovered a novel CI salvage pathway that maintains highly functional CI through an energetically favourable mechanism that demands much lower cost than de novo synthesis and reassembly of the entire CI. 

In this pathway 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.

Our results also illuminate ClpXP activity as an unforeseen target for therapeutic interventions in the large group of mitochondrial diseases characterized by the CI instability.

  1. Dogan SA, Pujol C, Maiti P, Kukat A, Hermans S, Senft K, Wibom R, Rugarli EI, Trifunovic A: Tissue-specific loss of DARS2 activates stress responses independently of respiratory chain deficiency in the heart. Cell Metab. 2014 Mar 4;19(3):458-69.    
  2. Seiferling D, Szczepanowska K, Becker C, Senft K, Hermans S, König T, Kukat A, Trifunovic A: Loss of CLPP alleviates mitochondrial cardiomyopathy without affecting the mammalian UPRmt. EMBO Reports 2016 May 6. pii: e201642077.
  3. Becker C, Kukat A, Szczepanowska K, Hermans S, Senft K, Brandscheid CP, Maiti P,  and Trifunovic A. (2018) CLPP deficiency protects against metabolic syndrome but hinders adaptive thermogenesis. EMBO Rep. 2018 Mar 27. pii: e45126. doi:10.15252/embr.201745126. PMID: 29588285
  4. Szczepanowska K, Senft K, Heidler J, Herholz M, Kukat A, Höhne MN, Hofsetz E, Becker C, Kaspar S, Giese H, Zwicker K, Guerrero-Castillo S, Baumann L, Kauppila J, Rumyantseva A, Müller S, Frese C, Brandt U, Riemer J, Wittig, Trifunovic A: A salvage pathway maintains highly functional respiratory complex I. Nature Communications 2020 ; in press
  5. Szczepanowska K, Maiti P, Kukat A, Hofetz E, Nolte H, Senft K, Becker C, Ruzzenente B, Hornig-Do HT, Wibom R, Wiesner RJ, Krüger M and Trifunovic A CLPP coordinates mitoribosomal assembly through the regulation of ERAL1 levels. EMBO J 2016 Dec 1;35(23):2566-2583
  • Aravamudhan S, Turk C, Bock T, Keufgens L, Nolte H, Lang F, Krishnan RK, Konig T, Hammerschmidt P, Schindler N, Brodesser S, Rozsivalova DH, Rugarli E,Trifunovic A, Bruning J, Langer T, Braun T, and Kruger M (2021). Phosphoproteomics of the developing heart identifies PERM1 - An outer mitochondrial membrane protein. J Mol Cell Cardiol154, 41-59. doi:10.1016/j.yjmcc.2021.01.010.
                                  • Bock T, Turk C, Aravamudhan S, Keufgens L, Bloch W, Rozsivalova DH, Romanello V, Nogara L, Blaauw B, Trifunovic A, Braun T, and Kruger M (2021). PERM1 interacts with the MICOS-MIB complex to connect the mitochondria and sarcolemma via ankyrin B. Nat Commun12, 4900. doi:10.1038/s41467-021-25185-3.
                                                        • Calculli G, Lee HJ, Shen K, Pham U, Herholz M, Trifunovic A, Dillin A, and Vilchez D (2021). Systemic regulation of mitochondria by germline proteostasis prevents protein aggregation in the soma of C. elegans. Sci Adv7. doi:10.1126/sciadv.abg3012.
                                                                                                                                                                        • Jobava R, Mao Y, Guan BJ, Hu D, Krokowski D, Chen CW, Shu XE, Chukwurah E, Wu J, Gao Z, Zagore LL, Merrick WC, Trifunovic A, Hsieh AC, Valadkhan S, Zhang Y, Qi X, Jankowsky E, Topisirovic I, Licatalosi DD, Qian SB, and Hatzoglou M (2021). Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress. Mol Cell81, 4191-4208 e4198. doi:10.1016/j.molcel.2021.09.029.
                                                                                                                                                                                    • Kaspar S, Oertlin C, Szczepanowska K, Kukat A, Senft K, Lucas C, Brodesser S, Hatzoglou M, Larsson O, Topisirovic I, and Trifunovic A (2021). Adaptation to mitochondrial stress requires CHOP-directed tuning of ISR. Sci Adv7. doi:10.1126/sciadv.abf0971.
                                                                                                                                                                                                                                                                                                                                                        • Silva-Pinheiro P, Pardo-Hernandez C, Reyes A, Tilokani L, Mishra A, Cerutti R, Li S, Rozsivalova DH, Valenzuela S, Dogan SA, Peter B, Fernandez-Silva P, Trifunovic A, Prudent J, Minczuk M, Bindoff L, Macao B, Zeviani M, Falkenberg M, and Viscomi C (2021). DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion. Nucleic Acids Res49, 5230-5248. doi:10.1093/nar/gkab282.
                                                                                                                                                                                                                                                                                                                                                                                                                                        • Willenborg S, Sanin DE, Jais A, Ding X, Ulas T, Nuchel J, Popovic M, MacVicar T, Langer T, Schultze JL, Gerbaulet A, Roers A, Pearce EJ, Bruning JC, Trifunovic A, and Eming SA (2021). Mitochondrial metabolism coordinates stage-specific repair processes in macrophages during wound healing. Cell Metab33, 2398-2414 e2399. doi:10.1016/j.cmet.2021.10.004.
                                                                                                                                                                                                                                                                                                                                                                                                                                        • Messner M, Mandl MM, Hackl MW, Reinhardt T, Ardelt MA, Szczepanowska K, Fradrich JE, Waschke J, Jeremias I, Fux A, Stahl M, Vollmar AM, Sieber SA, and Pachmayr J (2021). Small molecule inhibitors of the mitochondrial ClpXP protease possess cytostatic potential and re-sensitize chemo-resistant cancers. Sci Rep11, 11185. doi:10.1038/s41598-021-90801-7.
                                                                                                                                                                                                                                                                                                                                                                                                                                        • Szczepanowska K, and Trifunovic A (2021). Mitochondrial matrix proteases: quality control and beyond. FEBS J. doi:10.1111/febs.15964.
                                                                                                                                                                                                                                                                                                                                                                                                                                        • Szczepanowska K, and Trifunovic A (2021). Tune instead of destroy: How proteolysis keeps OXPHOS in shape. Biochim Biophys Acta Bioenerg1862, 148365. doi:10.1016/j.bbabio.2020.148365.
                                                                                                                                                                                                                                                                                                                                                                                                                                        • Hofsetz E, Demir F, Szczepanowska K, Kukat A, Kizhakkedathu JN, Trifunovic A, and Huesgen PF (2020). The Mouse Heart Mitochondria N Terminome Provides Insights into ClpXP-Mediated Proteolysis. Mol Cell Proteomics 19, 1330-45.
                                                                                                                                                                                                                                                                                                                                                                                                                                        • Lopes AFC, Bozek K, Herholz M, Trifunovic A, Rieckher M, Schumacher B. (2020) A C. elegans model for neurodegeneration in Cockayne syndrome. Nucleic Acids Res. 48(19):10973-10985. doi: 10.1093/nar/gkaa795.PMID: 33021672
                                                                                                                                                                                                                                                                                                                                                                                                                                        • Nemeth CL, Tomlinson SN, Rosen M, O'Brien BM, Larraza O, Jain M, Murray CF, Marx JS, Delannoy M, Fine AS, Wu D, Trifunovic A, and Fatemi A (2020). Neuronal ablation of mt-AspRS in mice induces immune pathway activation prior to severe and progressive cortical and behavioral disruption. Exp Neurol 326, 113164.
                                                                                                                                                                                                                                                                                                                                                                                                                                        • Ricke KM, Pass T, Kimoloi S, Fahrmann K, Jungst C, Schauss A, Baris OR, Aradjanski M, Trifunovic A, Eriksson Faelker TM, Bergami M, and Wiesner RJ (2020). Mitochondrial Dysfunction Combined with High Calcium Load Leads to Impaired Antioxidant Defense Underlying the Selective Loss of Nigral Dopaminergic Neurons. J Neurosci 40, 1975-86.
                                                                                                                                                                                                                                                                                                                                                                                                                                        • Rumyantseva A, Motori E, Trifunovic A. (2020) DARS2 is indispensable for Purkinje cell survival and protects against cerebellar ataxia. Hum Mol Genet. 2020 Oct 10;29(17):2845-2854.
                                                                                                                                                                                                                                                                                                                                                                                                                                        • Szczepanowska K, Senft K, Heidler J, Herholz M, Kukat A, Hohne MN, Hofsetz E, Becker C, Kaspar S, Giese H, Zwicker K, Guerrero-Castillo S, Baumann L, Kauppila J, Rumyantseva A, Muller S, Frese CK, Brandt U, Riemer J, Wittig I, and Trifunovic A (2020). A salvage pathway maintains highly functional respiratory complex I. Nat Commun 11, 1643.
                                                                                                                                                                                                                                                                                                                                                                                                                                        • Timper K, Del Rio-Martin A, Cremer AL, Bremser S, Alber J, Giavalisco P, Varela L, Heilinger C, Nolte H, Trifunovic A, Horvath TL, Kloppenburg P, Backes H, and Bruning JC (2020). GLP-1 Receptor Signaling in Astrocytes Regulates Fatty Acid Oxidation, Mitochondrial Integrity, and Function. Cell Metab 31, 1189-205 e13.
                                                                                                                                                                                                                                                                                                                                                                                                                                        Prof. Dr. Aleksandra Trifunovic CMMC Cologne
                                                                                                                                                                                                                                                                                                                                                                                                                                        Prof. Dr. Aleksandra Trifunovic

                                                                                                                                                                                                                                                                                                                                                                                                                                        Inst. for Mitochondrial Diseases and Ageing | CECAD Research Center

                                                                                                                                                                                                                                                                                                                                                                                                                                        CMMC - PI - C 15

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                                                                                                                                                                                                                                                                                                                                                                                                                                        Inst. for Mitochondrial Diseases and Ageing | CECAD Research Center

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                                                                                                                                                                                                                                                                                                                                                                                                                                        Dr. Karolina Szczepanowska CMMC Cologne
                                                                                                                                                                                                                                                                                                                                                                                                                                        Dr. Karolina Szczepanowska

                                                                                                                                                                                                                                                                                                                                                                                                                                        Inst. of Molecular Mechanisms and Machines | Polish Academy of Science

                                                                                                                                                                                                                                                                                                                                                                                                                                        CMMC - Co-PI -  C 15

                                                                                                                                                                                                                                                                                                                                                                                                                                        Inst. of Molecular Mechanisms and Machines | Polish Academy of Science

                                                                                                                                                                                                                                                                                                                                                                                                                                        ul. B. Smetany 2


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