Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We discovered the biological role of Fmp40 ampylase in yeast cells and its involvement in redox homeostasis.
• We described mechanism of the pathology of 11 mutations of the mitochondrial ATP6 gene, four 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 localization in the cell.

Opis badań

Adenosine triphosphate (ATP) synthase is an enzyme of the inner mitochondrial membrane that is responsible for ATP synthesis in the process of oxidative phosphorylation. ATP synthase consists of 17 structural subunits. Three subunits in yeast (a, 8, and c) and two in humans (a and 8) are encoded by mitochondrial DNA (mtDNA). The biogenesis of this enzyme is a sophisticated process that requires the coordination of gene expression in both nuclear and mitochondrial genomes with assembly of the enzyme. Activity of the enzyme is closely related to activity of the respiratory chain and controlled, for example, by the natural hydrolytic activity inhibitor peptide IF1. Many modifications have been identified in various subunits of ATP synthase, including phosphorylation, acetylation, trimethylation, nitration, S-nitrosylation, and tryptophan oxidation. Some modifications were reported to affect the enzymatic activity of ATP synthase, but in most cases remaining unknown are the signaling pathways that are responsible for these modifications, the tissues where or biological conditions under which these modifications occur, or the ways in which they impact biochemical activity of the target protein and holoenzyme. Mutations of genes that encode ATP synthase subunits or assembly factors lead to neurodegenerative diseases, which are currently untreatable.

Our research is divided into several interconnected axes:

1) The mitochondrial ATP synthase is composed of subunits of dual genetic origin, nuclear and mitochondrial. We are investigating the mechanisms that control the biogenesis of ATP synthase, including the expression of its mitochondrially encoded subunits and their assembly to the enzyme. We are looking for new proteins that are involved in these processes.

2) Cells adapt energy supplies to their demands. Deficits in energy production lead to metabolic diseases, such as mitochondrial disease, diabetes, heart failure, cancer, and neurodegenerative diseases. Our research seeks to identify molecular mechanisms of ATP synthase diseases that are caused by mutations of mitochondrial ATP6 and ATP8 genes, which encode ATP synthase subunits a and 8, in yeast as a model organism. We created yeast strains that bear 20 of 48 mutations of the ATP6 gene and three mutations of ATP8 that lead to diseases and deciphered their pathogenic mechanisms for five mutations at the molecular level. We extended our research to include mutations of conserved residues of the ATP8 gene to understand the function of subunit 8.

3) We discovered that human selenoprotein O (SelO) and its yeast (Fmp40) and E. coli (YdiU) homologues, classified by our collaborator as pseudokinases, have AMPylase activity. SelO proteins attach the AMP to the threonine, tyrosine, or serine residues in the protein substrates. One of the biological roles of these proteins is the regulation of protein S-glutathionylation levels by AMPylation of the grx family and other proteins during oxidative stress. This is the only AMPylase that is described in yeast to date and only the second in human cells. Our research, apart from understanding the significance of the ampylation of Grx2 for redox homeostasis, seeks to find Fmp40 substrates beyond redoxins and understand the role of this protein in the regulation of mitochondrial bioenergetics.

4) A growing number of proteins that have been studied in yeast have dual cytosolic and mitochondrial localization. We screened such mitochondrial proteins and explored a split-green fluorescent protein (GFP) method that was designed by Cabantous and co-workers such that the synthesis and localization of one of the two fragments of Split-GFP are restricted to the mitochondrial compartment. Split-GFP is based on the partition of 11β strand-composed GFP into two fragments: one long fragment that encompasses the first 10 β strands (β1-10) and one smaller fragment that consists of the remaining b strand (β11). We engineered a yeast strain that harbors the gene that encodes β1-10 fragments of Split-GFP in the mitochondrial genome and is thus translated inside the mitochondrial matrix. The second β11 Split-GFP fragment can be fused to any nuclear-encoded protein that will be translated by cytosolic translation machinery. The fluorescence signal in the mitochondrial network appears only from the mitochondrial pool of the protein that is fused to β11. We are working on engineering a yeast strain that expresses β1-10 from mitochondrial DNA, in which β1-10 would be located in the mitochondrial inter-membrane space (IMS). This strain will allow searching among non-mitochondrial proteins for those that transfer to the IMS under certain conditions.

• Bibliography

1. Carraro et al. Cell Rep. 2020. doi: 10.1016/j.celrep.2020.108095
2. Kucharczyk et al. BBA-BIO. doi: 10 10.18388/pb.2018_144
3. Carraro et al. Cell Physiol Biochem. 2018. doi: 10.1159/000494864
4. Kucharczyk et al. BBA-BIO 2019. doi: 10.1016/j.bbabio.2018.11.005
5. Niedzwiecka et al. BBA-MCR 2016. doi: 10.1016/j.bbamcr.2017.10.003
6. Wen et al. Sci Rep 2016. doi: 10.1038/srep36313
7. Żurawik et al. PLOS-One 2016. doi : 10.1371/journal.pone.0161353
8. Niedzwiecka et al. Mitochondrion 2016. doi: 10.1016/j.mito.2016.04.003
9. Lasserre et al. Dis Model Mech 2015. doi: 10.1242/dmm.020438
10. Aiyar et al. Nat Commun. 2014. doi: 10.1038/ncomms6585

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 (in collaboration with JP di Rago).
• Biochemical methods for the characterization of mitochondrial activity: oxygraphy, Clark electrode, spectrofluorometry, enzymatic activity.
• Protein methods: immunoprecipitation, affinity chromatography, in-gel activities, BN/CN-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, expression and purification from bacteria, in vitro activity.

Wybrane publikacje

• Assigning mitochondrial localization of dual localized proteins using a yeast Bi-Genomic Mitochondrial-Split-GFP. Bader G, Enkler L, Araiso Y, Hemmerle M, Binko K, Baranowska E, De Craene JO, Ruer-Laventie J, Pieters J, Tribouillard-Tanvier D, Senger B, di Rago JP, Friant S, Kucharczyk R*, Becker HD*. eLife. 2020, 9:e56649. doi: 10.7554/eLife.56649.
• Protein AMPylation by an Evolutionarily Conserved Pseudokinase. Sreelatha A, Yee SS, Lopez VA, Park BC, Kinch LN, Pilch S, Servage KA, Zhang J, Jiou J, Karasiewicz-Urbańska M, Łobocka M, Grishin NV, Orth K, Kucharczyk R, Pawłowski K, Tomchick DR, Tagliabracci VS. Cell. 2018, 175(3):809-821.e19. doi: 10.1016/j.cell.2018.08.046.
• ATP Synthase Subunit a Supports Permeability Transition in Yeast Lacking Dimerization Subunits and Modulates yPTP Conductance. Niedzwiecka K, Baranowska E, Panja C, Kucharczyk R. Cell Physiol Biochem. 2020, 54(2):211-229. doi: 10.33594/000000215.
• ATP Synthase Diseases of Mitochondrial Genetic Origin. Front Physiol. Dautant A, Meier T, Hahn A, Tribouillard-Tanvier D, di Rago JP, Kucharczyk. 2018, 9:329. doi: 10.3389/fphys.2018.00329.
• Two mutations in mitochondrial ATP6 gene of ATP synthase, related to human cancer, affect ROS, calcium homeostasis and mitochondrial permeability transition in yeast. Niedzwiecka K, Tisi R, Penna S, Lichocka M, Plochocka D, Kucharczyk R. Biochim Biophys Acta Mol Cell Res. 2018, 1865(1):117-131. doi: 10.1016/j.bbamcr.2017.10.003.

Współpraca

• Jean-Paul di Rago, Laboratory of Molecular Genetics of Mitochondrial Systems, IBGC du CNRS, France, http://www.ibgc.u-bordeaux2.fr/?page=equipe&eq=gmsm
• Hubert Dominique Becker, University of Strasbourg | UNISTRA · Génétique Moléculaire, Génomique, Microbiologie (GMGM), France, https://gmgm.unistra.fr/index.php?id=11217
• Maya Schuldiner, Dept. of Molecular Genetics, Weizmann Institute of Sciences, Israel, https://mayaschuldiner.wixsite.com/schuldinerlab
• Christos Chinopoulos, Department of Medical Biochemistry, Semmelweis University, Budapest, Hungary, https://scholar.semmelweis.hu/chinopoulos/
• Thomas Meier, Department of Life Sciences, Imperial College London, UK, https://www.imperial.ac.uk/people/t.meier