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001-es BibID:BIBFORM076180
035-os BibID:(cikkazonosító)e0204825 (WOS)000447417900013 (Scopus)85055079512
Első szerző:Fizil Ádám (biológus)
Cím:Calcium binding of the antifungal protein PAF: Structure, dynamics and function aspects by NMR and MD simulations / Ádám Fizil, Christoph Sonderegger, András Czajlik, Attila Fekete, István Komáromi, Dorottya Hajdu, Florentine Marx, Gyula Batta
Dátum:2018
ISSN:1932-6203
Megjegyzések:Calcium ions (Ca2+) play an important role in the toxicity of the cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum: high extracellular Ca2+ levels reduce the toxicity of PAF in the sensitive model fungus Neurospora crassa in a concentration dependent way. However, little is known about the mechanistic details of the Ca2+ ion impact and the Ca2+ binding capabilities of PAF outside the fungal cell, which might be the reason for the activity loss. Using nuclear magnetic resonance (NMR), isothermal titration calorimetry and molecular dynamics (MD) simulations we demonstrated that PAF weakly, but specifically binds Ca2+ ions. MD simulations of PAF predicted one major Ca2+ binding site at the C-terminus involving Asp53 and Asp55, while Asp19 was considered as putative Ca2+ binding site. The exchange of Asp19 to serine had little impact on the Ca2+ binding, however caused the loss of antifungal activity, as was shown in our recent study. Now we replaced the C-terminal aspartates and expressed the serine variant PAF{D53S/D55S}. The specific Ca2+ binding affinity of PAF{D53S/D55S} decreased significantly if compared to PAF, whereas the antifungal activity was retained. To understand more details of Ca2+ interactions, we investigated the NMR and MD structure/dynamics of the free and Ca2+-bound PAF and PAF{D53S/D55S}. Though we found some differences between these protein variants and the Ca2+ complexes, these effects cannot explain the observed Ca2+ influence. In conclusion, PAF binds Ca2+ ions selectively at the C-terminus; however, this Ca2+ binding does not seem to play a direct role in the previously documented modulation of the antifungal activity of PAF.
Tárgyszavak:Természettudományok Kémiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Megjelenés:Plos One. - 13 : 10 (2018), p. 1-19. -
További szerzők:Sonderegger, Christoph Czajlik András (1975-) (gyógyszerész) Fekete Attila (1983-) (vegyész) Komáromi István (1957-) (vegyész, molekuláris biológus, biokémikus) Hajdu Dorottya (1987-) (biológus) Marx, Florentine Batta Gyula (1953-) (molekula-szerkezet kutató)
Pályázati támogatás:ANN 110821
OTKA
TÁMOP-4.2.1/B-09/1/KONV-2010-0007
TÁMOP
GINOP-2.3.2-15-2016-00008
GINOP
GINOP-2.3.3-15-2016-00004
GINOP
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2.

001-es BibID:BIBFORM072198
035-os BibID:(Cikkazonosító)1751 (WOS)000423429800003 (Scopus)85041371349
Első szerző:Huber Anna
Cím:New Antimicrobial Potential and Structural Properties of PAFB: A Cationic, Cysteine-Rich Protein from Penicillium chrysogenum Q176 / Anna Huber, Dorottya Hajdu, Doris Bratschun-Khan, Zoltán Gáspári, Mihayl Varbanov, Stéphanie Philippot, Ádám Fizil, András Czajlik, Zoltán Kele, Christoph Sonderegger, László Galgóczy, Andrea Bodor, Florentine Marx, Gyula Batta
Dátum:2018
ISSN:2045-2322
Megjegyzések:Small, cysteine-rich and cationic proteins with antimicrobial activity are produced by diverse organisms of all kingdoms and represent promising molecules for drug development. The ancestor of all industrial penicillin producing strains, the ascomycete Penicillium chryosgenum Q176, secretes the extensively studied antifungal protein PAF. However, the genome of this strain harbours at least two more genes that code for other small, cysteine-rich and cationic proteins with potential antifungal activity. In this study, we characterized the pafB gene product that shows high similarity to PgAFP from P. chrysogenum R42C. Although abundant and timely regulated pafB gene transcripts were detected, we could not identify PAFB in the culture broth of P. chrysogenum Q176. Therefore, we applied a P. chrysogenum-based expression system to produce sufficient amounts of recombinant PAFB to address unanswered questions concerning the structure and antimicrobial function. Nuclear magnetic resonance (NMR)-based analyses revealed a compact [beta]-folded structure, comprising five [beta]-strands connected by four solvent exposed and flexible loops and an "abcabc" disulphide bond pattern. We identified PAFB as an inhibitor of growth of human pathogenic moulds and yeasts. Furthermore, we document for the first time an anti-viral activity for two members of the small, cysteine-rich and cationic protein group from ascomycetes.
Tárgyszavak:Természettudományok Kémiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Megjelenés:Scientific Reports. - 8 : 1 (2018), p. 1751. -
További szerzők:Hajdu Dorottya (1987-) (biológus) Bratschun-Khan, Doris Gáspári Zoltán Varbanov, Mihayl Philippot, Stéphanie Fizil Ádám (1988-) (biológus) Czajlik András (1975-) (gyógyszerész) Kele Zoltán Sonderegger, Christoph Galgóczy László (1950-) Bodor Andrea Marx, Florentine Batta Gyula (1953-) (molekula-szerkezet kutató)
Pályázati támogatás:ANN 110821
OTKA
GINOP-2.3.2-15-2016-00008
GINOP
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Intézményi repozitóriumban (DEA) tárolt változat
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3.

001-es BibID:BIBFORM066900
Első szerző:Sonderegger, Christoph
Cím:A Penicillium chrysogenum-based expression system for the production of small, cysteine-rich antifungal proteins for structural and functional analyses / Christoph Sonderegger, László Galgóczy, Sandra Garrigues, Ádám Fizil, Attila Borics, Paloma Manzanares, Nikoletta Hegedüs, Anna Huber, Jose F. Marcos, Gyula Batta, Florentine Marx
Dátum:2016
ISSN:1475-2859
Megjegyzések:Small, cysteine-rich and cationic antifungal proteins (APs) from filamentous ascomycetes, such as NFAP from Neosartorya fischeri and PAF from Penicillium chrysogenum, are promising candidates for novel drug development. A prerequisite for their application is a detailed knowledge about their structure-function relation and mode of action, which would allow protein modelling to enhance their toxicity and specificity. Technologies for structure analyses, such as electronic circular dichroism (ECD) or NMR spectroscopy, require highly purified samples and in case of NMR milligrams of uniformly 15N-/13C-isotope labelled protein. To meet these requirements, we developed a P. chrysogenum-based expression system that ensures sufficient amount and optimal purity of APs for structural and functional analyses.RESULTS:The APs PAF, PAF mutants and NFAP were expressed in a P. chrysogenum ?paf mutant strain that served as perfect microbial expression factory. This strain lacks the paf-gene coding for the endogenous antifungal PAF and is resistant towards several APs from other ascomycetes. The expression of the recombinant proteins was under the regulation of the strong paf promoter, and the presence of a paf-specific pre-pro sequence warranted the secretion of processed proteins into the supernatant. The use of defined minimal medium allowed a single-step purification of the recombinant proteins. The expression system could be extended to express PAF in the related fungus Penicillium digitatum, which does not produce detectable amounts of APs, demonstrating the versatility of the approach. The molecular masses, folded structures and antifungal activity of the recombinant proteins were analysed by ESI-MS, ECD and NMR spectroscopy and growth inhibition assays.CONCLUSION:This study demonstrates the implementation of a paf promoter driven expression cassettes for the production of cysteine-rich, cationic, APs in different Penicillium species. The system is a perfect tool for the generation of correctly folded proteins with high quality for structure-function analyses.KEYWORDS:Antifungal proteins; Electronic circular dichroism (ECD) spectroscopy; NFAP; Neosartorya fischeri; Nuclear magnetic resonance (NMR); PAF; Penicillium chrysogenum; Penicillium digitatum; Recombinant protein production
Tárgyszavak:Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Microbial Cell Factories 15 : 192 (2016), p. 1-14. -
További szerzők:Galgóczy László (1950-) Garrigues, Sandra Fizil Ádám (1988-) (biológus) Borics Attila Manzanares, Paloma Hegedűs Nikoletta Huber Anna Marcos, Jose F. Batta Gyula (1953-) (molekula-szerkezet kutató) Marx, Florentine
Pályázati támogatás:ANN110821
OTKA
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Intézményi repozitóriumban (DEA) tárolt változat
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4.

001-es BibID:BIBFORM066897
035-os BibID:(cikkazonosító)e0169920 (WOS)000391844200058 (Scopus)85009084271
Első szerző:Sonderegger, Christoph
Cím:D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function / Christoph Sonderegger, Ádám Fizil, Laura Burtscher, Dorottya Hajdu, Alberto Muñoz, Zoltán Gaáspári, Nick D. Read, Gyula Batta, Florentine Marx
Dátum:2017
ISSN:1932-6203
Megjegyzések:The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five ?-strands of PAF form a compact ?-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19). We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF.
Tárgyszavak:Természettudományok Kémiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Megjelenés:Plos One. - 12 : 1 (2017), p. e0169920-1 - e0169920-21. -
További szerzők:Fizil Ádám (1988-) (biológus) Burtscher, Laura Hajdu Dorottya (1987-) (biológus) Munoz, Alberto Gaáspári Zoltán Read, Nick D. Batta Gyula (1953-) (molekula-szerkezet kutató) Marx, Florentine
Pályázati támogatás:ANN110821
OTKA
Internet cím:Szerző által megadott URL
DOI
Intézményi repozitóriumban (DEA) tárolt változat
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