Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We described the sequence-specific reaction of peptide bond hydrolysis by Ni(II) ions and provided evidence of its key role in nickel allergy.
• We designed, synthesized, and commercialized a biopolymer (chitathione) that protects allergic skin from exposure to nickel.
• We demonstrated that fluorescent zinc sensors report ternary complexes with cellular ligands rather than “free zinc,” thus shifting the paradigm in cellular zinc trafficking.
• We described a new mechanism of stepwise Cu2+ binding to motifs that are present in key proteins that are responsible for extracellular copper metabolism, which explains how it is exchanged.

Opis badań

Transition metal elements are a fascinating element of nature, uniquely forming structures and catalyzing reactions that are inaccessible to light elements. The last three elements of the first transition row of the Periodic Table—nickel, copper, and zinc—are a particular interest of our laboratory. Nickel is a carcinogen and allergen in humans, whereas the other two are key biogenic metals of the human organism. This difference does not stem directly from their chemical properties, thus prompting interest in studying them in the context of human health.

In the first part of the reported period, we continued studies of the nickel-dependent hydrolysis of peptide bonds, supported by the FNP TEAM grant, with follow-up research that was performed until present. This reaction that was discovered previously by the laboratory head was thoroughly investigated, and its mechanism was fully described. The toxicological consequences were explored theoretically and experimentally, and target proteins were identified. The accumulated data gave rise to the concept that this reaction is the main source of molecules that elicit nickel allergy. In an associated study, a substance that protects against nickel allergy symptoms was designed and synthesized, and its efficacy was demonstrated. It was patented and licensed to a business partner that commercialized it as a series of dermocosmetics. We also demonstrated that nickel oxide nanoparticles can support the reaction, raising environmental concerns.

We demonstrated that fluorescent zinc sensors report ternary Zn(II) complexes with cellular ligands (rather than “free zinc”). Quantitative analysis of the data allowed us to calculate the molecular speciation of exchangeable zinc in the cell cytosol. This resulted in shifting the paradigm of cellular zinc trafficking by demonstrating that all cellular zinc is bound either to proteins or to small molecules that can act as previously unidentified zinc chaperones.

Studies of Cu(II) interactions with amyloid b (Aβ) peptides of Alzheimer’s disease that were previously initiated received a boost in 2015 when we demonstrated that Aβ species that were truncated specifically at position 4 (Aβ_4-x) bound Cu(II) ions in a tight, chemically inert form. A series of follow-up studies led to the concept that features of Aβ_4-x peptides predestine them to a physiological role of synaptic scavengers of toxic copper ions after their release during neurotransmission. Next, we searched for a mechanism of efficient relay of the captured copper back to cells, circumventing very slow Cu(II) release from Aβ_4-x. We found that certain small molecules can accelerate this process. Kinetic studies allowed us to describe a new general mechanism of stepwise Cu²⁺ ion binding to ATCUN/NTS motifs that are present in key proteins that are responsible for extracellular copper metabolism, such as Aβ_4-x, HSA (the main Cu[II] carrier in blood), and hCtr1 (the cell membrane copper transporter). This mechanism explains how Cu(II) ions can be exchanged between them and reduced to Cu(I) for cell entry at a millisecond timescale, which was scarcely investigated before our work. Studies of how this mechanism works in actual proteins and cell culture have been initiated.

Considerations of the effect of compartment volume on the course of the chemical reaction led us to notice that the concept of pH cannot be supported in subcellular structures, where water becomes essentially aprotic. Consequences of this finding include disqualifying the textbook knowledge of pH gradient as the driving force for adenosine triphosphate synthase. We supported this analysis by conducting an experimental study of isolated yeast mitochondria that also supported the concept of phosphates as catalysts of cellular acid/base chemistry.

These lines of research will be continued in the near future, with special attention to mechanisms of copper exchange in the context of Alzheimer’s disease. Our research will also be extended to molecular mechanisms of the toxicity of silver via interactions with copper and zinc and interactions between copper carriers and biological membranes.

Metodologia

The methodology of our chemical investigations includes the characterization of properties of molecules using numerous techniques, including ultraviolet-visible (UV-vis), circular dichroism (CD), and fluorescence spectroscopy to study complex formation and the coordination structure, plate readers for screening, potentiometry, isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) calorimetry and microscale thermophoresis to determine affinity constants, three high-performance liquid chromatography systems for the separation of chemical reaction products, a stopped-flow system with a UV-vis diode array and single wavelength CD and fluorescence detectors for studies of fast reactions, automated peptide synthesis and chemical synthesis facilities, and a workstation for studies of biomembranes. Nuclear magnetic resonance (NMR) studies of protein structures are performed in-house and in collaboration with others. An electron paramagnetic resonance (EPR) spectrometer will be acquired soon. Oxygen-sensitive compounds are studied using a glove box. Standard techniques of recombinant protein production in E. coli and P. pastoris are used, including isotopically enriched proteins for structural NMR studies. Cell cultures are also employed to study the effects of the studied complexes on the properties of living systems. Mass spectrometry (Dadlez Lab) is used to control the identity of products of chemical synthesis and the studied reactions. An integrated approach to study metal complexes with biomolecules includes the determination of stability constants of metal complexes using potentiometry for small molecules or calorimetry for proteins, corroboration of the obtained quantitative data by identifying characteristic spectral properties of specific complexes, the confirmation of their presence,  predicted on the basis of these quantitative data, by several spectroscopic techniques, and the determination of their association/dissociation rates. The resulting broad dataset is then used to formulate biological hypotheses regarding the presence and activity of individual complex species under biological conditions and propose further biological experiments. We recently began performing these experiments at the level of cell cultures and in C. elegans, a nematode that serves as a model multicellular organism. Theoretical methods of protein evolution are developed, with special attention to low-complexity sequences.

Wybrane publikacje

• Key intermediate species reveal the Cu(II) exchange pathway in biorelevant ATCUN/NTS complexes. Kotuniak R, Strampraad MJF, Bossak-Ahmad K, Ufnalska I, Wawrzyniak U, Hagedoorn P-L, Bal W. Angew. Chem. Int. Ed. 2020. doi: 10.1002/anie.202004264.
• Resistance of Cu(Aβ4-16) to copper capture by metallothionein 3 supports a function of Aβ4-42 peptide as a synaptic Cu^II Wezynfeld NE, Stefaniak E, Stachucy K, Drozd A, Płonka D, Drew SC, Krężel A, Bal W. Angew. Chem. Int. Ed. 2016. doi: 10.1002/anie.201511968.
• A Functional Role for Aβ in Metal Homeostasis? N-Truncation and High-Affinity Copper Binding. Mital M, Wezynfeld NE, Frączyk T, Wiloch MZ, Wawrzyniak UE, Bonna A, Tumpach C, Haigh CL, Barnham KJ, Bal W, Drew SC. Angew. Chem. Int. Ed. 2015. doi: 10.1002/anie.201502644.
• The Final Frontier of pH and the Undiscovered Country Beyond. Bal W, Kurowska E, Maret W. PLoS One 2012. doi:10.1371/journal.pone.0045832.
• Sequence-specific Ni(II)-dependent Peptide Bond Hydrolysis for Protein Engineering. Combinatorial Library Determination of Optimal Sequences. Krężel A, Kopera E, Protas AM, Poznański J, Wysłouch-Cieszyńska A, Bal W. J. Am. Chem. Soc. 2010. doi: 1021/ja907567r.

Współpraca

• Peter Faller, University of Strasbourg, France.
• Christelle Hureau, LCC CNRS, Toulouse, France.
• Peter-Leon Hagedoorn, Technical University Delft, The Netherlands.
• Andrea Hartwig, Karlsruhe Institute of Technology, Germany.
• Liming Ying, Imperial College London, UK.
• Wolfgang Maret, King’s College London, UK.
• Kathryn Haas, St. Mary’s College, Notre Dame IN, USA.
• Aurelien Deniaud, University of Grenoble, France.
• Bela Gyurcsik, University of Szeged, Hungary.
• Mi Hee Lim, KAIST, Daejeon, South Korea.
• Simon C. Drew, University of Melbourne, Australia.
• Francesco Tisato, CNR-ICMATE, Padova, Italy.
• Samantha J. Pitt, University of St. Andrews, UK.
• Graham Ogg, University of Oxford, UK.
• Artur Krężel, University of Wrocław, Poland.
• Wojciech Wróblewski, Urszula E. Wawrzyniak, Patrycja Wińska, Warsaw University of Technology, Poland.
• Maciej Kozak, Adam Mickiewicz University, Poznań, Poland.
• Agata Siwek, Department of Pharmacobiology, Faculty of Pharmacy, Jagiellonian University Medical College, Kraków, Poland
• Andrzej J. Bojarski (head), Grzegorz Satała, Maj Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland
• Marta K. Dudek, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, Łódź, Poland
• Joanna Wietrzyk (head), Marta Świtalska, Mateusz Psurski, Anna Nasulewicz-Goldeman, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland
• Marcin Wilczek, Krzysztof Woźniak, Damian Trzybiński, Roman Gajda, Joanna Trylska, Monika Wojciechowska, Anna Szakiel, University of Warsaw, Poland
• Zbigniew J. Leśnikowski, Laboratory of Molecular Virology & Biological Chemistry, Institute of Medical Biology, Polish Academy of Sciences, Łódź, Poland
• Aleksandra Gruca, The Faculty of Automatic Control, Electronics and Computer Science, The Silesian University of Technology in Gliwice, Poland.
• Miguel Andrade, The Faculty of Biology of the Johannes Gutenberg University in Mainz, Germany.

Nagrody i wyróżnienia

• Wojciech Bal, Prime Minister of Poland Award for Research Achievement 2020. The Prime Minister Office.