Pracownie badawcze

Prof. dr hab. Róża Kucharczyk

Pracownia Bioenergetyki i Mechanizmów Chorób Mitochondrialnych

ORCID: 0000-0002-8712-7535

Zakres badań

Our research focuses on mitochondrial adenosine triphosphate (ATP) synthase deficiencies and regulation in S. cerevisiae. We aim to understand how mitochondrial redox homeostasis is controlled by Fmp40 AMPylase and Abh1 depalmitoylase. We utilize yeast models of VPS13A-D-dependent neurodegenerative diseases and C. elegans models of PACS2-based disease to discover ways to overcome defects that are caused by pathogenic mutations. We seek to learn more about the function of VPS13 and PACS proteins and discover new drugs and drug targets to treat VPS13– and PACS2-related diseases.

Badania

Najważniejsze osiągnięcia badawcze

  • We discovered the biological role of Fmp40 AMPylase in yeast cells and its involvement in redox homeostasis by regulation of the redox cycles of Prx1 and Trx3 and the Trx3 maturation.
  • We described the mechanism of the pathology of 24 mutations of the mitochondrial ATP6 gene, eight of them at the molecular level.
  • We created a yeast strain that expresses the β1-10 non-fluorescent fragment of GFP from the mitochondrial genome, thus permitting the screening of matrix-localized pools of proteins that have dual/multiple localization in the cell.

Opis badań

Adenosine triphosphate (ATP) synthase is an enzyme located in the inner mitochondrial membrane, responsible for synthesizing ATP through oxidative phosphorylation. The enzyme is composed of 17 structural subunits. In yeast, three of these subunits (a, 8, and c), and in humans, two (a and 8), are encoded by the mitochondrial genome (mtDNA). The biogenesis of ATP synthase is a complex process that requires coordinated gene expression from both the nuclear and mitochondrial genomes, as well as proper assembly of the enzyme. Its activity is closely linked to the function of the respiratory chain and is regulated by factors such as the natural inhibitor peptide IF1, which suppresses its hydrolytic activity. Numerous post-translational modifications have been identified in ATP synthase subunits, including phosphorylation, acetylation, trimethylation, nitration, S-nitrosylation, and tryptophan oxidation. While some of these modifications are known to affect ATP synthase activity, in most cases, the signaling pathways that mediate these changes, the tissues or biological contexts in which they occur, and their functional consequences on the enzyme and its activity remain poorly understood. Mutations in genes encoding ATP synthase subunits or assembly factors can lead to severe neurodegenerative diseases, which currently lack effective treatments.

Our research is organized into several interconnected areas:

  • Molecular Mechanisms of ATP Synthase-Related Diseases

    Cells adjust energy production to meet their demands, and energy deficits can lead to mitochondrial diseases, diabetes, heart failure, cancer, and neurodegenerative disorders. We investigate the molecular mechanisms of ATP synthase-associated diseases caused by mutations in the mitochondrial ATP6 and ATP8 genes, which encode subunits a and 8, using yeast as a model organism. We have generated yeast strains carrying 24 ATP6 mutations and 3 ATP8 mutations linked to human diseases and have elucidated their pathogenic mechanisms.

  • AMPylation and Mitochondrial Redox Regulation

    We discovered that human selenoprotein O (SelO), along with its yeast (Fmp40) and E. coli (YdiU) homologs—classified as pseudokinases by our collaborators—exhibit AMPylase activity, catalyzing the attachment of AMP to threonine, tyrosine, or serine residues of protein substrates. One biological function of these proteins is to regulate protein S-glutathionylation through AMPylation of the Grx family and other proteins under oxidative stress. SelO is the only AMPylase described in yeast and only the second identified in humans. We have shown that three mitochondrial redox enzymes—Prx1, Trx3, and Grx2—are AMPylated by Fmp40, with AMPylation of Trx3 at threonine 66 playing a role in regulating its import to mitochondria and/or processing during oxidative stress. We are also characterizing the mitochondrial “AMPylome” and have found ATP synthase subunits among the AMPylated proteins. We are exploring the significance of these modifications for ATP synthase regulation and mitochondrial bioenergetics.

  • Redox Regulation by the Hydrolase Abh1

    The yeast hydrolase Abh1, homologous to human ABHD17, is predicted to have depalmitoylase activity. We discovered that Abh1 regulates cellular responses to oxidative stress, in part by modulating respiratory chain activity and oxygen consumption. Our data suggest that Abh1 interacts with Fmp40, and we are investigating how redox homeostasis is co-regulated by Fmp40 AMPylase and Abh1 depalmitoylase activity.

  • Split-GFP System to Study Protein Localization

    Many yeast proteins show dual cytosolic and mitochondrial localization. We adapted a split-green fluorescent protein (GFP) system developed by Cabantous et al. 2005. Split-GFP is composed of two parts: a larger β1–10 fragment and a smaller β11 fragment. We engineered a yeast strain with the β1–10 fragment encoded in the mitochondrial genome and expressed in the mitochondrial matrix. The β11 fragment is fused to nuclear-encoded proteins and translated in the cytosol. Fluorescence appears only when the β11-tagged protein enters mitochondria, allowing us to study mitochondrial localization dynamics.

  • Lipid Transport and Vps13 Regulation

    Mitochondria acquire lipids from the endoplasmic reticulum (ER) via ER-mitochondria encounter structures (ERMES). In the absence of ERMES, lipid transfer relies on Vps13, which localizes to multiple organelle contact sites and shifts location in response to growth conditions. We are investigating Vps13 regulation and have observed that abh1∆ mutant shows elevated Vps13 levels. Vps13 deficiency affects the abundance of the ubiquitin ligase Rsp5, which regulates transcriptional coactivators of lipid biosynthesis genes.

  • Stress Responses and SUMOylation in C. elegans

    Various cellular stresses increase protein SUMOylation. We are examining how stress responses are integrated at the whole-organism level using the nematode Caenorhabditis elegans, a cost-effective higher eukaryotic model for high-throughput compound screening. We are developing C. elegans models for drug repurposing screens targeting rare diseases, including PACS2 syndrome and L1CAM syndrome.

Metodologia

  • Molecular biology: polymerase chain reaction, DNA cloning, electrophoresis, etc.
  • Yeast genetics: site-directed mutagenesis of nuclear and mitochondrial DNA, transformation into mitochondria (Biolistics), drug screening.
  • Biochemical methods for the characterization of mitochondrial activity: oxygraphy, Clark electrode, spectrofluorometry, enzymatic activity.
  • Protein methods: immunoprecipitation, affinity chromatography, in-gel activities, blue-native gel electrophoresis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, expression and purification from bacteria, in vitro AMPylation activity, redox states of proteins.
  • C. elegans: genetics and proteomics, CRISPR/Cas9 transgenesis, compound screens in C. elegans.

Wybrane publikacje

  • Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP. Zuttion, S., Senger, B., Panja, C., Friant, S., Kucharczyk, R., Becker, H. D. Methods Enzymol. 2024:706:75-95. doi: 10.1016/bs.mie.2024.07.028
  • Fmp40 ampylase regulates cell survival upon oxidative stress by controlling Prx1 and Trx3 oxidation. Masanta, S., Wiesyk, A., Panja, C., Pilch, S., Ciesla, J., Sipko, M., De, A., Enkhbaatar, T., Maslanka, R., Skoneczna, A., Kucharczyk, R. Redox Biol. 2024 Jul:73:103201. doi: 10.1016/j.redox.2024.103201.
  • Arf1 coordinates fatty acid metabolism and mitochondrial homeostasis. Enkler, L., Szentgyörgyi, V., Pennauer, M., Prescianotto-Baschong, C., Riezman, I., Wiesyk, A., Avraham, R. E., Spiess, M., Zalckvar, E., Kucharczyk, R., Riezman, H., Spang, A. Nat Cell Biol. 2023 Aug;25(8):1157-1172. doi: 10.1038/s41556-023-01180-2.
  • ATP synthase interactome analysis identifies a new subunit l as a modulator of permeability transition pore in yeast. Panja, C., Wiesyk, A., Niedźwiecka, K., Baranowska, E., Kucharczyk, R. Sci Rep. 2023 Mar 7;13(1):3839. doi: 10.1038/s41598-023-30966-5.
  • Molecular basis of diseases induced by the mitochondrial DNA mutation m.9032T>C. Baranowska, E., Niedzwiecka, K., Panja, C., Charles, C., Dautant, A., di Rago, J. P., Tribouillard-Tanvier, D., Kucharczyk, R. Hum Mol Genet. 2023 Apr 6;32(8):1313-1323. doi: 10.1093/hmg/ddac292.

Współpraca

Nagrody i wyróżnienia

  • Róża Kucharczyk. Award of the Minister of Education and Science for entire scientific achievements. 2023, Poland.

Publikacje (z afiliacją IBB PAN)

ZUTTION S., SENGER B., PANJA CH., FRIANT S., KUCHARCZYK R., BECKER H.D., Monitoring mitochondrial localization of dual localized proteins using a Bi-Genomic Mitochondrial-Split-GFP. Chapter 5 in: Methods in Enzymology. Mitochondrial Translocases. Part A, Ed. by Nils Wiedemann, ISBN 978-0-443-29330-6, Elsevier (2024) 706: 75-95, DOI: 10.1016/bs.mie.2024.07.028
MASANTA S., WIĘSYK A., PANJA CH., PILCH S., CIEŚLA J., SIPKO M., DE A., ENKHBAATAR T., MAŚLANKA R., SKONECZNA A., KUCHARCZYK R., Fmp40 ampylase regulates cell survival upon oxidative stress by controlling Prx1 and Trx3 oxidation. Redox Biology (2024) 73: 103201 (19 p.) DOI: 10.1016/j.redox.2024.103201 IF 10.7 (2023)
ENKLER L., SZENTGYÖRGYI V., PENNAUER M., PRESCIANOTTO-BASCHONG C., RIEZMAN I., WIĘSYK A., AVRAHAM R.E., SPIESS M., ZALCKVAR E., KUCHARCZYK R., RIEZMAN H., SPANG A., Arf1 coordinates fatty acid metabolism and mitochondrial homeostasis. Nature Cell Biology (2023) 25(8): 1157–1172 DOI: 10.1038/s41556-023-01180-2 IF 17.3 (2023)
PANJA CH., NIEDŹWIECKA K., BARANOWSKA E., POZNAŃSKI J., KUCHARCZYK R., Analysis of MT-ATP8 gene variants reported in patients by modeling in silico and in yeast model organism. Scientific Reports (2023) 13(1): 9972 (13 p.) DOI: 10.1038/s41598-023-36637-9 IF 3.8 (2023)
BARANOWSKA E., NIEDŹWIECKA K., PANJA CH., CHARLES C., DAUTANT A., POZNAŃSKI J., DI RAGO J.-P., TRIBOUILLARD-TANVIER D., KUCHARCZYK R., Probing the pathogenicity of patient-derived variants of MT-ATP6 in yeast. Disease Models and Mechanisms (2023) 16(4): dmm049783 (12 p.) DOI: 10.1242/dmm.049783 IF 4.0 (2023)
BARANOWSKA E., NIEDŹWIECKA K., PANJA CH., CHARLES C., DAUTANT A., DI RAGO J.-P., TRIBOUILLARD-TANVIER D., KUCHARCZYK R., Molecular basis of diseases induced by the mitochondrial DNA mutation m.9032T > C. Human Molecular Genetics (2023) 32(8): 1313-1323 DOI: 10.1093/hmg/ddac292 IF 3.1 (2023)
PANJA CH., WIĘSYK A., NIEDŹWIECKA K., BARANOWSKA E., KUCHARCZYK R., ATP synthase interactome analysis identifies a new subunit l as a modulator of permeability transition pore in yeast. Scientific Reports (2023) 13(1): 3839 (18 p.) DOI: 10.1038/s41598-023-30966-5 IF 3.8 (2023)
HEMMERLE M., SENGER B., KUCHARCZYK R., BECKER H.D., Visualizing Mitochondrial Importability of a Protein Using the Yeast Bi-Genomic Mitochondrial-Split-GFP Strain and an Ordinary Fluorescence Microscope. Chapter 16 in: Mitochondria. Methods and Protocols, Methods in Molecular Biology, vol 2497. Humana New York, NY. Ed. N. Tomar. ISBN 978-1-0716-2308-4, 2022, p. 255-267 DOI: 10.1007/978-1-0716-2309-1_16
TRIBOUILLARD-TANVIER D., DAUTANT A., GODARD F., PANJA CH., DI RAGO J.-P., KUCHARCZYK R., Creation of Yeast Models for Evaluating the Pathogenicity of Mutations in the Human Mitochondrial Gene MT-ATP6 and Discovering Therapeutic Molecules. Chapter 14 in: Mitochondria. Methods and Protocols, Methods in Molecular Biology, vol 2497. Humana New York, NY. Ed. N. Tomar. ISBN 978-1-0716-2308-4, 2022, p. 221-242 DOI: 10.1007/978-1-0716-2309-1_14
KABALA A.M., BIŃKO K., GODARD F., CHARLES C., DAUTANT A., BARANOWSKA E., SKOCZEŃ N., GOMBEAU K., BOUHIER M., BECKER H.D., ACKERMAN S.H., STEINMETZ L.M., TRIBOUILLARD-TANVIER D., KUCHARCZYK R., DI RAGO J.-P., Assembly-dependent translation of subunits 6 (Atp6) and 9 (Atp9) of ATP synthase in yeast mitochondria. Genetics (2022) 220(3): iyac007 (15 p.) DOI: 10.1093/genetics/iyac007 IF 3.3 (2022)
PAPROCKI D., WINIEWSKA-SZAJEWSKA M., SPEINA E., KUCHARCZYK R., POZNAŃSKI J., 5,6‑diiodo‑1H‑benzotriazole: new TBBt analogue that minutely afects mitochondrial activity. Scientific Reports (2021) 11(1): 23701 (10 p.) DOI: 10.1038/s41598-021-03136-8 IF 4.996 (2021)
SU X., DAUTANT A., RAK M., GODARD F., EZKURDIA N., BOUHIER M., BIETENHADER M., MUELLER D.M., KUCHARCZYK R., DI RAGO J.-P., TRIBOUILLARD-TANVIER D., The pathogenic m.8993T>G mutation in mitochondrial ATP6 gene prevents proton release from the subunit c-ring rotor of ATP synthase. Human Molecular Genetics (2021) 30(5): 381-392 DOI: 10.1093/hmg/ddab043 IF 5.121 (2021)
DING Q., KUCHARCZYK R., ZHAO W., DAUTANT A., XU S., NIEDŹWIECKA K., SU X., GIRAUD M.-F., GOMBEAU K., ZHANG M., XIE H., ZENG C., BOUHIER M., DI RAGO J.-P., LIU Z., TRIBOUILLARD-TANVIER D., CHEN H., Case report: identification of a novel variant (m.8909T>C) of human mitochondrial ATP6 gene and its functional consequences on yeast ATP synthase. Life-Basel ( 2020) 10(9): 215 (20 p.) DOI: 10.3390/life10090215 IF 3.817 (2020)
CARRARO M., JONES K., SARTORI G., SCHIAVONE M., ANTONUCCI S., KUCHARCZYK R., DI RAGO J.-P., FRANCHIN C., ARRIGONI G., FORTE M., BERNARDI P., The unique cysteine of F-ATP synthase OSCP subunit participates in modulation of the permeability transition pore. Cell Reports (2020) 32(9): 108095 DOI: 10.1016/j.celrep.2020.108095 IF 9.423 (2020)
BADER G., ENKLER L., ARAISO Y., HEMMERLE M., BIŃKO K., BARANOWSKA E., DE CRAENE J-O., RUER-LAVENTIE J., PIETERS J., TRIBOUILLARD-TANVIER D., SENGER B., DI RAGO J.-P., FRIANT S., KUCHARCZYK R., BECKER H.D., Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP. eLife (2020) 9: e56649 (24 p.) DOI: 10.7554/eLife.56649 IF 8.146 (2020)
SU X., DAUTANT A., GODARD F., BOUHIER M., ŻOŁĄDEK T., KUCHARCZYK R., DI RAGO J.-P., TRIBOUILLARD-TANVIER D., Molecular basis of the pathogenic mechanism induced by the m.9191T>C mutation in mitochondrial ATP6 gene. International Journal of Molecular Sciences (2020) 21(14): 5083 (16 p.) DOI: 10.3390/ijms21145083 IF 5.923 (2020)
NIEDŹWIECKA K., BARANOWSKA E., PANJA CH., KUCHARCZYK R., ATP synthase subunit a supports permeability transition in yeast lacking dimerization subunits and modulates yPTP conductance. Cellular Physiology and Biochemistry (2020) 54(2): 211-229 DOI: 10.33594/000000215 IF - (2020)
KUCHARCZYK R., DAUTANT A., GOMBEAU K., GODARD F., TRIBOUILLARD-TANVIER D., DI RAGO J.-P., The pathogenic MT-ATP6 m.8851T>C mutation prevents proton movements within the n-side hydrophilic cleft of the membrane domain of ATP synthase. Biochimica et Biophysica Acta - Bioenergetics (2019) 1860(7): 562-572 DOI: 10.1016/j.bbabio.2019.06.002 IF 3.465 (2019)
KUCHARCZYK R., DAUTANT A., GODARD F., TRIBOUILLARD-TANVIER D., DI RAGO J.-P., Functional investigation of an universally conserved leucine residue in subunit a of ATP synthase targeted by the pathogenic m.9176 T > G mutation. Biochimica et Biophysica Acta - Bioenergetics (2019) 1860(1): 52-59 DOI: 10.1016/j.bbabio.2018.11.005 IF 3.465 (2019)
CARRARO M., CHECCHETTO V., SARTORI G., KUCHARCZYK R., DI RAGO J.-P., MINERVINI G., FRANCHIN C., ARRIGONI G., GIORGIO V., PETRONILLI V., TOSATTO S.C.E., LIPPE G., SZABÓ I., BERNARDI P., High-conductance channel formation in yeast mitochondria is mediated by F-ATP synthase e and g subunits. Cellular Physiology and Biochemistry (2018) 50(5): 1840–1855 DOI: 10.1159/000494864 IF - (2018)

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