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Protein dephosphorylation group Leader:
Viktor Dombrádi, Ph.D., D. Sc., Professor
Members: Ilona Farkas, Ph.D., Associate professor Krisztina Szabó Tóth, PhD, Research associate Andrea Farkas Tanka, Technician
Research interest Protein phosphorylation is an essential post-synthetic protein modification in eukaryotic organisms. The balance between protein kinases and protein phosphatases determines the actual phosphorylation state of target proteins and has a profound effect on cellular regulation and signaling. While the kinases belong to a single superfamily the phosphatases are more diverse according to their structures and catalytic mechanism. Our group concentrates on the phosphatases that are specific for the Ser/Thr side chains. We have been studying the classical members of the phosphoprotein phosphatase enzyme family (like PP1 and PP2A) as well as the more recently uncovered novel phosphatases (like PPY and PPZ). By the combination of biochemistry, molecular biology, and genetics we are investigating the physiological functions of these enzymes in animals, plants, and fungi.
Past and current topics of investigation 1. Cloning, functional analysis and evolution of protein phosphatases in Drosophila In the 1990s at the University of Dundee, UK, the group leader mastered molecular biology methods and identified a number of novel members of the phosphoprotein phosphatase (PPP) family in Drosophila melanogaster by molecular cloning. Most importantly, with Dr. Myles Axton he demonstrated the essential role of protein phosphatase 1 (PP1 87) in the regulation of the onset of anaphase during the cell cycle. After returning to Hungary he established the Protein dephosphorylation research group and with his students and co-workers he continued the cloning of PPP catalytic and regulatory subunits from diverse sources. The investigations of the evolution of Ser/Thr specific PPP enzymes revealed additional phosphatase genes in several Drosophila species. We demonstrated the dynamic changes in the number, localization and structure of PPP genes in the Drosophilidae, and proved that the novel PPP retrogenes are predominantly expressed in the males. The sex specific expression of the new phosphatase genes supported the “out of testis hypothesis” suggesting that the testis was the main stage for the expression and selection of the fresh genes generated by gene duplication. 2. Cloning and functional analysis of plant protein phosphatases In collaboration with the Institute of Plant Physiology of the Hungarian Academy of Sciences during the first decade of the 21st century, the group members cloned the catalytic subunit of protein phosphatase 1 (PP1) as well as the regulatory subunits of protein phosphatase 2A (PP2A) from the plant Medicago sativa. With the aid of a cell-permeable inhibitor of serine/threonine-specific protein phosphatases, the involvement of PP2A in the regulation of the specific steps of plant cell division cycle was discovered. In addition, we identified the function of the inhibitor proteins of PP2A (NRPs) in Arabidopsis thaliana, and revealed the role of the PP2A B’’ subunit in the dephosphorylation of the plant retinoblastoma protein homologs in Oryza sativa. 3. Characterization of a fungus specific protein phosphatase In the past decades the interest of the group turned towards a fungus specific protein phosphatase. We cloned the genes and the corresponding cDNAs of the novel protein phosphatase Z (PPZ) from a number of fungal species (N. crassa, C. albicans, A. nidulans, and A. fumigatus). The physiological functions of these enzymes have been investigated by the complementation of known yeast mutants and by the direct disruption of the PPZ1 genes in Candida albicans and Aspergillus nidulans. These studies were carried out in cooperation with the research groups of the Universitat Autònoma de Barcelona, Spain and the Institute of Biotechnology, University of Debrecen, Hungary. Our results indicated that the CaPPZ1 gene was important in the cell wall biosynthesis, osmotic stability as well as for the hyphal outgrowth and the morphological changes of C. albicans. Furthermore, we uncovered novel functions for the CaPpz1 phosphatase in the modulation of the oxidative stress response and biofilm formation, and most importantly in the virulence of the opportunistic pathogen. We also identified the CaCab3 “moonlighting” protein as a physiological regulator of the phosphatase. Our recent RNA sequencing and quantitative RT-PCR based gene expression study demonstrated that the deletion of the CaPPZ1 gene exaggerated the effects of oxidative stress in C. albicans, and suggested the involvement of this phosphatase in the regulation of important physiological processes outlined in the following diagram:
Legend: The transcriptomic effects of CaPPZ1 gene deletion (KO vs WT), oxidative stress on the wild type (WTox vs WT), and on the deletion mutant (KOox vs KO) are depicted in two Venn diagrams. Up- and downregulated genes are indicated by their systematic names and are labeled according to the functional color code given below the figure. Source; Krisztina Szabó-Tóth: Investigation of a Fungus Specific Protein Phosphatase. PhD thesis. University of Debrecen, 2020. Since the CaPpz1 phosphatase is fungus specific and has important functions, it can be considered as a potential antifungal drug target. The synergistic interaction between the phosphatase deletion and oxidative stress conditions in terms of gene expression and cell proliferation suggest that the combination of a CaPpz1 specific phosphatase inhibitor with an oxidizing agent may be utilized for the development of potential drugs against the pathogenic fungus. From this respect it is important to note that our collaborators at the Brown University, Providence, RI, USA determined the three dimensional structure of the catalytic domain of CaPpz1 and pinpointed two specific structural elements that may be targeted by rational drug design. In addition, by site directed mutagenesis we have mapped an important N-terminal segment of the regulatory domain of the phosphatase that may also be utilized as a putative target. Taken together our results provide the bases of a promising strategy for a novel topical antifungal treatment, the feasibility of which still has to be substantiated by additional physiological tests.
Selected publications Axton JM, Dombrádi V, Cohen PTW, Glover DM: One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis. Cell 63 33-46 (1990) Miskei M, Ádám C, Kovács L, Karányi Z, Dombrádi V: Molecular evolution of phosphoprotein phosphatases in Drosophila. PLoS One. 6:e22218. (2011) Dombrádi V, Krieglstein J, Klumpp S: Regulating the regulators: Protein phosphorylation and protein phosphatases. EMBO Reports 3, 120-124 (2002) Farkas I, Dombrádi V, Miskei M., Szabados L, Koncz Cs: Arabidopsis PPP family of serine/threonine phosphatases. Trends Plant Sci. 12, 169-176 (2007) Ádám C, Erdei É, Casado C, Kovács L, González A, Majoros L, Petrényi K, Bagossi P, Farkas I, Molnár M, Pócsi I, Arino J, Dombrádi V: Protein phosphatase CaPpz1 is involved in cation homeostasis, cell wall integrity and virulence of Candida albicans. Microbiology-SGM 1581258-1267. (2012) Leiter É, González A, Erdei É, Casado C, Kovács L, Ádám Cs, Oláh J, Miskei M, Molnar M, Farkas I, Hamari Zs, Ariño J, Pócsi I, Dombrádi V: Protein phosphatase Z modulates oxidative stress response in fungi. Fungal Genetics and Biology 49, 708-716. (2012) Petrényi K, Molero C, Kónya Z, Erdődi F, Ariño J, Dombrádi V: Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles. PLoS One. 11(8):e0160965. (2016)
Chen E, Choy MS, Petrényi K, Kónya Z, Erdődi F, Dombrádi V, Peti W, Page R: Molecular insights into the fungus-specific serine/threonine protein phosphatase Z1 in Candida albicans. mBio 7(4):e00872-16. (2016)
Márkus B, Szabó K, Pfliegler WP, Petrényi K, Boros E,Pócsi I, Tőzsér J, Csősz É, Dombrádi V: Proteomic analysis of protein phosphatase Z1 from Candida albicans. PLoSOne. 24;12(8):e0183176. (2017)
Szabó K, Kónya Z, Erdődi F, Farkas I, Dombrádi V: Dissection of the regulatory role for the N-terminal domain in Candida albicans protein phosphatase Z1. PLoS One. 14(2):e0211426. (2019)
Szabó K, Jakab Á, Póliska Sz, Petrényi K, Kovács K, Issa LHB, Emri T, Pócsi I, Dombrádi V: Deletion of the fungus specific protein phosphatase Z1 exaggerates the oxidative stress response in Candida albicans. BMC Genomics. 20:873 (2019)
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