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Our preliminary and unpublished data show that by removing the major mitochondrial matrix protease CLPXP, and therefore stabilizing CI, we could ameliorate the symptoms of respiratory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).

Fig. 1. Loss of CLLP ameliortes: (A) lifespan and heart hypertrophy in cardiac model for mitochondrial deficiency; (B) Neurodegeneration and neuroinflamation in neuro-specific model for mitochondrial deficiency: (C) Respiratory chain defect in  cell culture model for premature aging; (D) Development defect in C. elegans model for Complex I deficiency; (E) and acts in a dose-dependent manner opening prospect for therapeutic interventions.

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.

  • 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.    
  • 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.
  • 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
  • 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
  • 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
  • Croon M, Szczepanowska K, Popovic M, Lienkamp C, Senft K, Brandscheid CP, Bock T, Gnatzy-Feik L, Ashurov A, Acton RJ, Kaul H, Pujol C, Rosenkranz S, Kruger M, and Trifunovic A (2022). FGF21 modulates mitochondrial stress response in cardiomyocytes only under mild mitochondrial dysfunction. Sci Adv. 2022 Apr 8;8(14):eabn7105. doi: 10.1126/sciadv.abn7105. Epub 2022 Apr 6.
  • Gonzalez-Franquesa A, Gama-Perez P, Kulis M, Szczepanowska K, Dahdah N, Moreno-Gomez S, Latorre-Pellicer A, Fernandez-Ruiz R, Aguilar-Mogas A, Hoffman A, Monelli E, Samino S, Miro-Blanch J, Oemer G, Duran X, Sanchez-Rebordelo E, Schneeberger M, Obach M, Montane J, Castellano G, Chapaprieta V, Sun W, Navarro L, Prieto I, Castano C, Novials A, Gomis R, Monsalve M, Claret M, Graupera M, Soria G, Wolfrum C, Vendrell J, Fernandez-Veledo S, Enriquez JA, Carracedo A, Perales JC, Nogueiras R, Herrero L, Trifunovic A, Keller MA, Yanes O, Sales-Pardo M, Guimera R, Bluher M, Martin-Subero JI, and Garcia-Roves PM (2022). Remission of obesity and insulin resistance is not sufficient to restore mitochondrial homeostasis in visceral adipose tissue. Redox Biol. 2022 Aug;54:102353. doi: 10.1016/j.redox.2022.102353. Epub 2022 Jun 24.
  • Hoehne MN, Jacobs L, Lapacz KJ, Calabrese G, Murschall LM, Marker T, Kaul H, Trifunovic A, Morgan B, Fricker M, Belousov VV, and Riemer J (2022). Spatial and temporal control of mitochondrial H2 O2 release in intact human cells. EMBO J. 2022 Apr 4;41(7):e109169. doi: 10.15252/embj.2021109169. Epub 2022 Feb 11.
  • Rumyantseva A, Popovic M, and Trifunovic A (2022). CLPP deficiency ameliorates neurodegeneration caused by impaired mitochondrial protein synthesis. Brain. 2022 Mar29; 145(1):92-104. doi: 10.1093/brain/awab303.
  • Schatton D, Di Pietro G, Szczepanowska K, Veronese M, Marx MC, Braunohler K, Barth E, Muller S, Giavalisco P, Langer T, Trifunovic A, and Rugarli EI (2022). CLUH controls astrin-1 expression to couple mitochondrial metabolism to cell cycle progression. Elife. 2022 May 13;11:e74552. doi: 10.7554/eLife.74552.
  • 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 Commun. 2021 Aug 12;12(1):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 Adv. 2021 Jun 25;7(26):eabg3012. doi: 10.1126/sciadv.abg3012. Print 2021 Jun. 
  • 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 Cell2021 Oct 21;81(20):4191-4208.e8. 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 Adv. 2021 May 26;7(22):eabf0971. doi: 10.1126/sciadv.abf0971. PMID: 34039602; PMCID: PMC8153728.
  • 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 Res. 2021 May 21;49(9):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 Metab. 2021 Dec 7;33(12):2398-2414.e9. doi: 10.1016/j.cmet.2021.10.004. Epub 2021 Oct 28. 
  • 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 Rep. 2021 May 27;11(1):11185. doi: 10.1038/s41598-021-90801-7. PMID: 34045646; PMCID: PMC8160014.
  • Szczepanowska K, and Trifunovic A (2021). Mitochondrial matrix proteases: quality control and beyond. FEBS J. 2021 May 10. doi: 10.1111/febs.15964. Online ahead of print.
  • Szczepanowska K, and Trifunovic A (2021). Tune instead of destroy: How proteolysis keeps OXPHOS in shape. Biochim Biophys Acta Bioenerg. 2021 Apr 1;1862(4):148365. doi: 10.1016/j.bbabio.2020.148365. Epub 2021 Jan 6.
  • Silva-Pinheiro P, Pardo-Hernández C, Reyes A, Tilokani L, Mishra A, Cerutti R, Li S, Rozsivalova DH, Valenzuela S, Dogan SA, Peter B, Fernández-Silva P, Trifunovic A, Prudent J, Minczuk M, Bindoff L, Macao B, Zeviani M, Falkenberg M, Viscomi C. Correction to 'DNA polymerase gamma mutations that impair holoenzyme stability cause catalytic subunit depletion'. Nucleic Acids Res. 2021 Oct 11;49(18):10803. doi: 10.1093/nar/gkab837. Erratum for: Nucleic Acids Res. 2021 May 21;49(9):5230-5248. PMID: 34520541; PMCID: PMC8501975.
  • 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. 2020 Aug;19(8):1330-1345. doi: 10.1074/mcp.RA120.002082. Epub 2020 May 28.
  • 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. 2020 Nov 4;48(19):10973-10985. doi: 10.1093/nar/gkaa795. PMID: 33021672; PMCID: PMC7641758.
  • 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. 2020 Feb 26;40(9):1975-1986. doi: 10.1523/JNEUROSCI.1345-19.2019. Epub 2020 Jan 31.
  • 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. doi: 10.1093/hmg/ddaa176.
  • 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. 2020 Apr 2;11(1):1643. doi: 10.1038/s41467-020-15467-7.
  • 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. 2020 Jun 2;31(6):1189-1205.e13. doi: 10.1016/j.cmet.2020.05.001. Epub 2020 May 19. 
Prof. Dr. Aleksandra Trifunovic CMMC Cologne
Prof. Dr. Aleksandra Trifunovic

Inst. for Mitochondrial Diseases and Ageing | CECAD Research Center

CMMC - PI - B 11

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+49 221 478 32400

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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