Untersuchung von Klasse III und IV Lanthipeptidprozessierungsenzymen, Lanthipeptidmodifizierungsenzymen und von Lanthipeptiden im Epitope Grafting
Zusammenfassung der Projektergebnisse
In this project, the biosynthesis of class IV lanthipeptides as well as the use of lanthipeptides for drug development were investigated. From these studies a better understanding of the interaction of class IV lanthipeptide precursors and LanL processing enzymes were obtained. In particular, it was shown that the recognition site of the precursor peptide is situated in the N-terminal region of the leader peptide and that the central kinase domain is in charge of recognizing the substrate peptide. In addition, the first E. coli-based class IV lanthipeptide production system was established and the order of dehydration by LanL was demonstrated to follow a strict N- to C-terminal directionality. Furthermore, a systematic mutational analysis of the ProcA2.8 precursor peptide was performed to assess which changes are tolerated by the class II lanthipeptide processing enzyme ProcM. As these experiments revealed that most changes were tolerated well, ProcA2.8 was deemed to be a suitable scaffold for epitope grafting efforts, i.e. the incorporation of a small bioactive peptide sequence to generate a new compound with potentially interesting properties for drug development. As a proof-of-concept that this would indeed be possible with the ProcA2.8/ProcM pair and the resulting prochlorosin 2.8 lanthipeptide, the RGD sequence was incorporated at different positions of the core peptide to generate binders for the αvβ3 integrin; a target for treatment of certain tumor types. Of the three RGD peptides generated, one showed a low nanomolar binding affinity to αvβ3, proving prochlorosin 2.8 as suitable scaffold for epitope grafting studies. In addition to the study of lanthipeptides, additional lasso peptide-related projects were taken up. These projects established new methods to identify peptides with lasso topology and to discriminate between lasso and branched-cyclic peptides with identical amino acid sequences based on ion mobility mass spectrometry and fragmentation techniques as well as describing ways to predict the topology-stabilizing plug residues by mass spectrometry. Furthermore, new insights about lasso peptide biosynthesis were obtained, especially regarding the roles of the conserved residues in the precursor peptides during enzymatic processing, and the first example of production of an actinobacterial lasso peptide in E. coli was reported. In conclusion, the research during this DFG-funded scholarship helped to better understand RiPP biosynthesis and contributed to establishing such peptides as suitable scaffolds for drug development.
Projektbezogene Publikationen (Auswahl)
- Elucidation of the roles of conserved residues in the biosynthesis of the lasso peptide paeninodin, Chem. Commun. 2018, 54, 9007-9010
Julian D. Hegemann, Christopher J. Schwalen, Douglas A. Mitchell, Wilfred van der Donk
(Siehe online unter https://doi.org/10.1039/c8cc04411b) - General rules of fragmentation evidencing lasso structures in CID and ETD, Analyst 2018, 143, 1157-1170
Kevin Jeanne Dit Fouque, Hélène Lavanant, Séverine Zirah, Julian D. Hegemann, Chris D. Fage, Mohamed A. Marahiel, Sylvie Rebuffat, Carlos Afonso
(Siehe online unter https://doi.org/10.1039/c7an02052j) - Investigation of Substrate Recognition and Biosynthesis in Class IV Lanthipeptide Systems, J. Am. Chem. Soc. 2018, 140, 5743-5754
Julian D. Hegemann, Wilfred A. van der Donk
(Siehe online unter https://doi.org/10.1021/jacs.8b01323) - Investigation of the Biosynthesis of the Lasso Peptide Chaxapeptin Using an E. coli-Based Production System, J. Nat. Prod. 2018
Helena Martin-Gómez, Uwe Linne, Fernando Albericio, Judit Tulla-Puche, Julian D. Hegemann
(Siehe online unter https://doi.org/10.1021/acs.jnatprod.8b00392)
