Laboratories

Teresa Żołądek, PhD, DSc, Prof.

Laboratory of Yeast Genetics and Molecular Biology

ORCID: 0000-0002-9881-0516

Research Scope

We study the mechanisms of protein transport between subcellular compartments to understand how defects of transport contribute to human disease. We utilize yeast models of neurodegenerative diseases to discover ways to overcome defects that are caused by pathogenic mutations, learn more about the function of disease-related proteins and discover new drugs and drug targets to treat selected diseases.

Research

Main Scientific Achievements

  • Using a yeast model, we discovered several new phenotypes of the vps13A-I2771R mutation, which corresponds to this found in patient suffering from ChAc.
  • We discovered that the vps13 mutation causes the upregulation of calcineurin, and we found that its inhibition by FK506 can reverse the growth phenotype, suggesting a first drug target for the treatment of VPS13A-D-dependent diseases.
  • We discovered that the APT1 domain that is present in yeast and human Vps13 and Atg2 proteins binds PI3P and/or PI5P, and this binding is regulated by calcium ions and important for protein function.
  • We discovered the new pathway of import of peroxisomal matrix proteins, dependent on the PTS3 import signal.

Research Description

Neurodegenerative diseases (NDs) are a group of age-related diseases that are characterized by progressive neuronal loss, which manifests as a decline of motor and cognitive functions. Most common diseases, such as Alzheimer’s disease and Parkinson’s disease, are the most investigated NDs. However, some NDs are very rare and attract much less attention. Such NDs include chorea-acanthocytosis (ChAc), which is caused by mutations of the VPS13A gene, and other diseases that are caused by mutations of VPS13B-D genes. To investigate these NDs and find effective treatments, we are using the yeast Saccharomyces cerevisiae as a model organism, which enables large-scale genetic and chemical screenings and functional genetics. We modeled in yeast the vps13-I2749R mutation, equivalent to VPS13A-I2771R in ChAc patients, and showed that it caused an amino acid substitution in the APT1 domain and found several phenotypes that indicated a loss of function (1). We also showed that the human APT1 domain of chorein (VPS13A protein) binds phosphatidylinositol phosphates (PI3P and PI5P) in a calcium-dependent manner, and the VPS13A-I2771R mutation diminished this binding (2). Thus, we found that chorein function is regulated by calcium.

One new phenotype, hypersensitivity to SDS (sodium dodecyl sulfate), a common household detergent, was successfully used to screen for multicopy suppressors, second site mutations and repurposed drugs, to determine how defects that are caused by VPS13 mutations can be overcome. The analysis of multicopy suppressors, such as MYO3-N (which encodes myosin), PMC1 (which encodes the calcium transporter), and cmd1-228 (which encodes calmodulin), and further experiments demonstrated an increase in the activation of calcineurin, a calcium-dependent phosphatase that is crucial for calcium signaling, in vps13Δ cells. Moreover, finding that the calcineurin inhibitor FK506 is a chemical suppressor of the vps13Δ growth phenotype indicated that calcineurin is a potential target for the treatment of ChAc and other VPS13-dependent diseases (3). Analyses of multicopy suppressors and second site suppressor will be continued to provide new information about the physiology of cells that are devoid of VPS13 proteins.

The phenotype of the hypersensitivity to SDS of vps13Δ was also used to screen the Prestwick library of repurposed drugs. Three groups of drugs that restored growth were found. One was the group of flavonoids, such as luteolin, which are able to bind iron, and this turned our attention to iron transport and metabolism in vps13Δ. This correlated with the finding that the FET4 gene, which encodes the iron transporter, acts as a vps13Δ suppressor (4). Studies of the identified drugs will be continued, and other models, including cells from patients, can be used. To date, there are no specific treatments for VPS13-related diseases; only symptomatic treatment is used.

Other types of rare NDs affect the peripheral nervous system, so-called Charcot-Marie-Tooth (CMT) diseases. This group of disorders is characterized by similar clinical manifestations and high genetic heterogeneity. Excessive diversity presents many problems. First, proper genetic diagnosis is very difficult. Second, the molecular mechanisms that underlie the observed symptoms are extremely diverse. Third, there is no cure. There is an urgent need to develop new CMT models. We started to develop a yeast-based model system that is dedicated to studying CMT diseases (5). We initiated a model of GDAP1 gene-related pathology and found that GDAP1 pathogenic variants, when expressed in yeast cells, influence mitochondrial morphology and function. Moreover, we found that wildtype GDAP1 gene expression can overcome the calcium sensitivity of strains that do not produce complex sphingolipids (6). In the future, we plan to elucidate the mechanism of this finding and apply this knowledge to better understand the pathology of GDAP1-related CMT diseases and identify potential drug target.

Budding yeast Hsp31 protein belongs to DJ-1/ThiJ/PfpI superfamily together with human Parkinson’s disease-associated DJ-1 protein. We previously demonstrated that is a stress response protein protecting the cell against environmental stresses. It protects the cell against methylglyoxal toxicity thanks to its glyoxalase III enzymatic activity [7]. Recently, we discovered that it is secreted to the periplasmic space via unconventional route. We found that Ser209 and Phe17 aa residues of Hsp31, as well as the C-terminal 8 amino acids, all located on the surface of the native protein molecule, are crucial for its secretion and for the functionality of the protein. Unconventional secretion pathways have recently attracted a lot of attention, as they are implicated in neurodegenerative, cardiovascular and cancer diseases yet they are poorly understood. Budding yeast cells and Hsp31 comprise an attractive model for studying these pathways.

The import of peroxisomal matrix proteins depends on C-terminal PTS1 and N-terminal PTS2 targeting signals recognized by the Pex5 and Pex7 receptors, respectively. However, the budding yeast acyl-CoA oxidase (AOx) have none of these signals yet it is efficiently imported into peroxisomes, which suggests the existence of yet another import pathway, which we now call the PTS3 pathway. In our studies we identified both components of this new pathway: the signal and receptor that recognizes it. We determined the PTS3 import signal in AOx as the Signal Patch, i.e. a domain on the surface of the natively folded molecule composed of amino acids dispersed on the polypeptide chain. The import of AOx depends on the recognition of PTS3 signal by N-terminal FXXXW motif in Pex5 receptor [8]. We also demonstrated that in addition to AOx three other peroxisomal proteins, Cat2, Fox2 and Cta1 are imported exclusively or partially via this new pathway [9].

  • Bibliography
  1. Rzepnikowska et al. Hum Mol Genet. 2017. doi: 10.1093/hmg/ddx054
  2. Kolakowski et al. Biochim Biophys Acta Biomembr. doi: 10.1016/j.bbamem.2020.183349
  3. Soczewka et al. Dis Model Mech. 2019. Doi: 10.1242/dmm.036830
  4. Soczewka et al. Genes.2020. doi: 10.3390/genes11070828
  5. Rzepnikowska et al. Int J Mol Sci. 2020. doi: 10.3390/ijms21124277
  6. Rzepnikowska et al. Genes. 2020. doi: 10.3390/genes11030310.
  7. Natkańska et al. Biochim Biophys Acta Mol Cell Res. doi: 10.1016/j.bbamcr.2016.10.016
  8. Kempiński et al. Front Cell Dev Biol. 2020. doi: 10.3389/fcell.2020.00198.
  9. Rymer et al. J Cell Sci. 2018. doi: 10.1242/jcs.216986

Methodology

  • We use classic and molecular genetics and molecular biology methods in yeast, including classic genetic tetrad analysis, multicopy and second site suppressors screens, drug screens, gene cloning, in vitro site-directed mutagenesis, two-hybrid screens, two-hybrid protein interaction analysis, analyses of subcellular protein localization using fluorescence and confocal microscopy, Western blot analyses of proteins and their posttranslational modification by ubiquitination or phosphorylation, protein production in E. coli cells, and analyses of protein interactions with lipids on filters using protein lipid overlay assays or liposomes in vitro.
  • We also employ the large scale techniques involving the use of Yeast Deletion Collection, as well as whole-genome and whole transcriptome analyses using the on-site Agilent microarray platform. Our expertise in these techniques serves both our studies and those of other groups on a collaborative basis.

Selected Publications

  • Flavonoids as potential drugs for VPS13-dependent rare neurodegenerative diseases. Soczewka P, Flis K, Tribouillard-Tanvier D, di Rago J-P, Santos CN, Menezes R, Kaminska J, Zoladek T. Genes. 2020. doi:10.3390/genes11070828.
  • The binding of the APT1 domains to phosphoinositides is regulated by metal ions in vitro. Kolakowski D, Kaminska J, Zoladek T. BBA – Biomembranes. 2020. doi:10.1016/j.bbamem.2020.183349
  • Yeast-model-based study identified myosin- and calcium-dependent calmodulin signalling as a potential target for drug intervention in chorea-acanthocytosis. Soczewka P, Kolakowski D, Smaczynska-de Rooij I, Rzepnikowska W, Ayscough KR, Kaminska J, Zoladek T. Dis Model Mech. 2019. doi: 10.1242/dmm.036830.
  • Amino acid substitution equivalent to human chorea-acanthocytosis I2771R in yeast Vps13 protein affects its binding to phosphatidylinositol 3-phosphate. Rzepnikowska W, Flis K, Kaminska J, Grynberg M, Urbanek A, Ayscough KR, Zoladek T. Human Mol. 2017. doi: 10.1093/hmg/ddx054.
  • Phosphatidylinositol-3-phosphate regulates response of cells to proteotoxic stress. Kaminska J, Rzepnikowska W, Polak A, Flis K, Soczewka P, Bala K, Sienko M, Grynberg M, Kaliszewski P, Urbanek A, Ayscough K, Zoladek T. Int. J. Bioch. Cell Biol. 2016. doi: 10.1016/j.biocel.2016.08.007.

Collaborations

Publications (IBB PAS affiliated)

RZEPNIKOWSKA W., KAMINSKA J., KOCHAŃSKI A., The molecular mechanisms that underlie IGHMBP2-related diseases. Neuropathology and Applied Neurobiology (2024) 50(4): e13005 (13 p.) DOI: 10.1111/nan.13005 IF 4.0 (2023)
ENKHBAATAR T., SKONECZNY M., STĘPIEŃ K., MOŁOŃ M., SKONECZNA A., Live while the DNA lasts. The role of autophagy in DNA loss and survival of diploid yeast cells during chronological aging. Aging-US (2023) 15(19): 9967-9992 DOI: 10.18632/aging.205102 IF 3.9 (2023)
KOSIOREK K., KORYSZEWSKA-BAGIŃSKA A., SKONECZNY M., ALEKSANDRZAK-PIEKARCZYK T., Control of Bacterial Phenotype and Chromosomal Gene Expression by Single Plasmids of Lactococcus lactis IL594. International Journal of Molecular Sciences (2023) 24(12): 9877 (13 p.) DOI: 10.3390/ijms24129877 IF 4.9 (2023)
ANTONIUK-MAJCHRZAK J., ENKHBAATAR T., DŁUGAJCZYK A., KAMINSKA J., SKONECZNY M., KLIONSKY D.J., SKONECZNA A., Stability of Rad51 recombinase and persistence of Rad51 DNA repair foci depends on post-translational modifiers, ubiquitin and SUMO. Biochimica et Biophysica Acta-Molecular Cell Research (2023) 1870(7): 119526 (18 p.) DOI: 10.1016/j.bbamcr.2023.119526 IF 4.6 (2023)
KOSIOREK K., KORYSZEWSKA-BAGIŃSKA A., SKONECZNY M., STASIAK-RÓŻAŃSKA L., ALEKSANDRZAK-PIEKARCZYK T., The Presence of Plasmids in Lactococcus lactis IL594 Determines Changes in the Host Phenotype and Expression of Chromosomal Genes. International Journal of Molecular Sciences (2023) 24(1): 793 (15 p.) DOI: 10.3390/ijms24010793 IF 4.9 (2023)
DĄBROWSKA M., KĘPCZYŃSKA A., GOŹDZIK K., CAŁKA-KRESA M., SKONECZNY M., ZIELINSKI Z., DOLIGALSKA M., SIKORA E., Novel Role of Mammalian Cell Senescence-Sustenance of Muscle Larvae of Trichinella spp. Oxidative Medicine and Cellular Longevity (2022) 2022: 1799839 (13 p.) DOI: 10.1155/2022/1799839 IF - (2022)
RZEPNIKOWSKA W., KAMINSKA J., KOCHAŃSKI A., Validation of the Pathogenic Effect of IGHMBP2 Gene Mutations Based on Yeast S. cerevisiae Model. International Journal of Molecular Sciences (2022) 23(17): 9913 (17 p.) DOI: 10.3390/ijms23179913 IF 5.6 (2022)
KABZINSKA D., CHABROS K., KAMINSKA J., KOCHAŃSKI A., The GDAP1 p.Gly222Lys Variant-Weak Pathogenic Effect, Cumulative Effect of Weak Sequence Variants, or Synergy of Both Factors? Genes (2022) 13(9): 1546 (13 p.) DOI: 10.3390/genes13091546 IF 3.5 (2022)
KAMINSKA J., SOCZEWKA P., RZEPNIKOWSKA W., ZOLADEK T., Yeast as a Model to Find New Drugs and Drug Targets for VPS13-Dependent Neurodegenerative Diseases. International Journal of Molecular Sciences (2022) 23(9): 5106 (24 p.) DOI: 10.3390/ijms23095106 IF 5.6 (2022)
KOŁAKOWSKI D., RZEPNIKOWSKA W., KANIAK-GOLIK A., ZOLADEK T., KAMINSKA J., The GTPase Arf1 is a determinant of yeast Vps13 localization to the Golgi apparatus. International Journal of Molecular Sciences (2021) 22(22): 12274 (21 p.) DOI: 10.3390/ijms222212274 IF 6.208 (2021)
KAMINSKA J., STOPIŃSKI M., MUCHA K., PAC M., GOŁĘBIOWSKI M., NIEWCZAS M., PĄCZEK L., FORONCEWICZ B., Circulating osteoprotegerin in chronic kidney disease and all-cause mortality. International Journal of General Medicine (2021) 14: 2413–2420 DOI: 10.2147/IJGM.S302251 IF 2.145 (2021)
KLIM J., ZIELENKIEWICZ U., SKONECZNY M., SKONECZNA A., KURLANDZKA A., KACZANOWSKI S., Genetic interaction network has a very limited impact on the evolutionary trajectories in continuous culture-grown populations of yeast. BMC Ecology and Evolution (2021) 21(1): 99 DOI:10.1186/s12862-021-01830-9 IF - (2021)
KLIONSKY D.J., ZOLADEK T., SIRKO A., KAMINSKA J., WAWRZYŃSKA A., ET AL. Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy (2021) 17(1): 1-382 DOI:10.1080/15548627.2020.1797280 IF 13.391 (2021)
YU Z., GAO J., IGBALAJOBI O., SKONECZNY M., SIENKO M., MACIEJEWSKA A.M., BRZYWCZY J., FISCHER R., The sulfur metabolism regulator MetR is a global regulator controlling phytochrome-dependent light responses in Aspergillus nidulans. Science Bulletin (2021) 66(6): 592-602 DOI: 10.1016/j.scib.2020.11.001 IF 20.577 (2021)
SOCZEWKA P., TRIBOUILLARD-TANVIER D., DI RAGO J.-P., ZOLADEK T., KAMINSKA J., Targeting copper homeostasis improves functioning of vps13Δ yeast mutant cells, a model of VPS13-related diseases. International Journal of Molecular Sciences (2021) 22(5): 2248 DOI: 10.3390/ijms22052248 IF 6.208 (2021)
WARDASZKA P., SOCZEWKA P., SIENKO M., ZOLADEK T., KAMINSKA J., Partial inhibition of calcineurin activity by Rcn2 as a potential remedy for Vps13 deficiency. International Journal of Molecular Sciences (2021) 22(3): 1193 DOI:10.3390/ijms22031193 IF 6.208 (2021)
BINIĘDA K., RZEPNIKOWSKA W., KOŁAKOWSKI D., KAMINSKA J., SZCZEPANKIEWICZ A.A., NIEZNAŃSKA H., KOCHAŃSKI A., KABZINSKA D., Mutations in GDAP1 influence structure and function of the Trans-Golgi network. International Journal of Molecular Sciences (2021) 22(2): 914 DOI: 10.3390/ijms22020914 IF 6.208 (2021)
SOCZEWKA P., FLIS K., TRIBOUILLARD-TANVIER D., DI RAGO J.-P., SANTOS C.N., MENEZES R., KAMINSKA J., ZOLADEK T., Flavonoids as potential drugs for VPS13-dependent rare neurodegenerative diseases. Genes (2020) 11(7): 828 (17 p.) DOI: 10.3390/genes11070828 IF 4.096 (2020)
SU X., DAUTANT A., GODARD F., BOUHIER M., ZOLADEK 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)
KOŁAKOWSKI D., KAMINSKA J., ZOLADEK T., The binding of the APT1 domains to phosphoinositides is regulated by metal ions in vitro. BBA - Biomembranes (2020) 1862(9): 183349 (12 p.) DOI: 10.1016/j.bbamem.2020.183349 IF 3.747 (2020)

Team