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Ochtrop, Philipp
Publications (7 of 7) Show all publications
Ochtrop, P., Ernst, S., Itzen, A. & Hedberg, C. (2019). Exploring the Substrate Scope of the Bacterial Phosphocholine Transferase AnkX for Versatile Protein Functionalization. ChemBioChem (Print), 20(18), 2336-2340
Open this publication in new window or tab >>Exploring the Substrate Scope of the Bacterial Phosphocholine Transferase AnkX for Versatile Protein Functionalization
2019 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 20, no 18, p. 2336-2340Article in journal (Refereed) Published
Abstract [en]

Site-specific protein functionalization has become an indispensable tool in modern life sciences. Here, tag-based enzymatic protein functionalization techniques are among the most versatilely applicable approaches. However, many chemo-enzymatic functionalization strategies suffer from low substrate scopes of the enzymes utilized for functional labeling probes. We report on the wide substrate scope of the bacterial enzyme AnkX towards derivatized CDP-choline analogues and demonstrate that AnkX-catalyzed phosphocholination can be used for site-specific one- and two-step protein labeling with a broad array of different functionalities, displaying fast second-order transfer rates of 5x10(2) to 1.8x10(4) m(-1) s(-1). Furthermore, we also present a strategy for the site-specific dual labeling of proteins of interest, based on the exploitation of AnkX and the delabeling function of the enzyme Lem3. Our results contribute to the wide field of protein functionalization, offering an attractive chemo-enzymatic tag-based modification strategy for in vitro labeling.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2019
Keywords
CDP-choline, dual labeling, phosphocholination, protein modifications, transferases
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-164139 (URN)10.1002/cbic.201900200 (DOI)000486565800006 ()31054261 (PubMedID)
Available from: 2019-10-17 Created: 2019-10-17 Last updated: 2019-10-17Bibliographically approved
Paulsen, M. H., Karlsen, E. A., Ausbacher, D., Anderssen, T., Bayer, A., Ochtrop, P., . . . Strøm, M. B. (2018). An amphipathic cyclic tetrapeptide scaffold containing halogenated β2,2-amino acids with activity against multiresistant bacteria. Journal of Peptide Science, 24(10), Article ID UNSP e3117.
Open this publication in new window or tab >>An amphipathic cyclic tetrapeptide scaffold containing halogenated β2,2-amino acids with activity against multiresistant bacteria
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2018 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 24, no 10, article id UNSP e3117Article in journal (Refereed) Published
Abstract [en]

The present study describes the synthesis and biological studies of a small series of head-to-tail cyclic tetrapeptides of the general structure c(Lys‐β2,2‐Xaa‐Lys) containing one lipophilic β2,2-amino acid and Lys, Gly, Ala, or Phe as the Xaa residue in the sequence. The peptides were investigated for antimicrobial activity against gram-positive and gram-negative reference strains and 30 multiresistant clinical isolates including strains with extended spectrum β-lactamase-carbapenemase (ESBL-CARBA) production. Toxicity was determined against human red blood cells. The most potent peptides showed high activity against the gram-positive clinical isolates with minimum inhibitory concentrations of 4-8μg/mL and low haemolytic activity. The combination of high antimicrobial activity and low toxicity shows that these cyclic tetrapeptides containing lipophilic β2,2-amino acids form a valuable scaffold for designing novel antimicrobial agents.

Place, publisher, year, edition, pages
John Wiley & Sons, 2018
Keywords
antimicrobial peptides, beta-amino acids, CARBA, cyclic tetrapeptides, ESBL, MRSA, multiresistant bacteria, peptidomimetics, SMAMPs, synthetic mimics of antimicrobial peptides
National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:umu:diva-153717 (URN)10.1002/psc.3117 (DOI)000445732400005 ()30112781 (PubMedID)
Available from: 2018-11-27 Created: 2018-11-27 Last updated: 2018-11-27Bibliographically approved
Ochtrop, P. (2017). Selective protein functionalisation via enzymatic phosphocholination. (Doctoral dissertation). Umeå: Umeå universitet
Open this publication in new window or tab >>Selective protein functionalisation via enzymatic phosphocholination
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Proteins are the most abundant biomolecules within a cell and are involved in all biochemical cellular processes ultimately determining cellular function. Therefore, to develop a complete understanding of cellular processes, obtaining knowledge about protein function and interaction at a molecular level is critical. Consequently, the investigation of proteins in their native environment or in partially purified mixtures is a major endeavour in modern life sciences. Due to their high chemical similarity, the inherent problem of studying proteins in complex mixtures is to specifically differentiate one protein of interest from the bulk of other proteins. Site-specific protein functionalisation strategies have become an indispensable tool in biochemical- and cell biology studies. This thesis presents the development of a new enzymatic site-specific protein functionalisation strategy that is based on the reversible covalent phosphocholination of short amino acid sequences in intact proteins. A synthetic strategy has been established that allows access to functionalised CDP-choline derivatives carrying fluorescent reporter groups, affinity tags or bioorthogonal handles. These CDP-choline derivatives serve as co-substrates for the bacterial phosphocholinating enzyme AnkX from Legionella pneumophila, which transfers a phosphocholine moiety to the switch II region of its native target protein Rab1b during infection. We identified the octapeptide sequence TITSSYYR as the minimum recognition sequence required to direct the AnkX catalysed phosphocholination and demonstrated the functionalisation of proteins of interest carrying this recognition tag at the N- or C-terminus as well as in internal loop regions. Moreover, this covalent modification can be hydrolytically reversed by the action of the Legionella enzyme Lem3, which makes the labeling strategy the first example of a covalent and reversible approach that is fully orthogonal to current existing methodologies. Thus, the here presented protein functionalisation approach holds the potential to increase the scope of possible labeling strategies in complex biological systems. In addition to the labeling of tagged target proteins, a CDP-choline derivative equipped with a biotin affinity-tag was synthesised and used in pull-down experiments to investigate the substrate scope of AnkX and to elucidate the role of protein phosphocholination during Legionella pneumophila infection.

Abstract [sv]

Proteiner utgör huvudbeståndsdelen av alla biomolekyler i en cell. Dessa är involverade i alla cellulära processer som bestämmer cellens egenskaper. För att förstå de cellulära processerna är det nödvändigt att förstå proteinernas funktion på molekylär nivå. Att studera proteiner i deras naturliga omgivning, det vill säga inuti en cell eller i ett cellextrakt, är en stor utmaning i dagens livsvetenskaper. Eftersom proteiner är kemiskt lika varandra så är det svårt att skilja ett från tusentals andra. Att specifikt märka proteiner för att skilja ut dem från bakgrunden har blivit ett viktigt arbetssätt i modern biokemi och cellbiologi. Avhandlingen beskriver utvecklandet av en ny metod för reversibel och kovalent enzymatisk märkning baserat på fosfokolinering/defosfokolinering av en kort aminosyrasekvens i intakta proteiner. En syntesmetod för att framställa onaturliga CDP-kolinderivat har etablerats vilket tillåter oss att framställa CDP-kolin som bär en funktionalitet, vilket kan vara ett färgämne eller en affinitetstagg. Dessa onaturliga CDP-kolinderivat accepteras som co-substrat av enzymet AnkX från Legionella pneumophila vilket transfererar den funktionaliserade delen av CDP-kolinderivatet till en kort aminosyrasekvens baserad på AnkX’s naturliga substrat vid infektion, det lilla GTPaset Rab1. Under avhandlingsarbetets gång identifierades den kortaste aminosyrasekvensen som känns igen av AnkX, endast de åtta aminosyrorna TITSSYYR är nödvändiga för igenkänning av AnkX. Dessa åtta aminosyror kan genetiskt infogas i början, slutet eller mitt i ett protein för igenkänning och funktionalisering via AnkX och våra syntetiska CDP-kolinderivat. Vid Legionellainfektion i eukaryota celler klyvs fosfokolineringen efter en viss tid, eftersom Legionella pneumophila producerar ett fosfodiesteras, Lem3, som tar bort de fosfokolineringar som AnkX har installerat när de inte längre behövs. Vi har använt Lem3 för att ta bort märkning i sekvensen TITSS(PC)YYR, vilket gör vår strategi helt reversibel. Vi har kunnat demonstrera att AnkX-Lem3 systemet accepterar ett brett spektrum av CDP-kolinderivat, vilket gör metoden till den första av sitt slag, eftersom den är fullt reversibel. Vi har vidare undersökt vilka proteiner AnkX reagerar med inuti celler, vi använde oss av ett CDP-kolinderivat funktionaliserat med biotin, vilket har tillåtit oss att fiska ut alla de proteiner som fosfokolineras av AnkX. Förutom de små GTPaserna i Rab-familjen så identifierade vi även IMPDH2, ett enzym som reglerar det hastighetsbestämmande steget i syntesen av guanosin-nukleotider. Detta är mycket intressant, eftersom det leder till frågan ifall Legionella pneumophila manipulerar sin värdcell genom att förändra mängden GTP i förhållande till ATP.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2017. p. 101
Keywords
chemical biology, organic synthesis, site-selective protein labeling, PTM, phosphocholination, nucleotides, bioorthogonal chemistry, proteomics, IMPDH2
National Category
Organic Chemistry Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-140349 (URN)978-91-7601-785-2 (ISBN)
Public defence
2017-11-03, KB.E3.01 (Lilla Hörsalen), Kemiska Institutionen, Umeå Universitet, KBC-huset, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2017-10-13 Created: 2017-10-05 Last updated: 2018-06-09Bibliographically approved
Heller, K., Ochtrop, P., Albers, M. F., Zauner, F. B., Itzen, A. & Hedberg, C. (2015). Covalent Protein Labeling by Enzymatic Phosphocholination. Angewandte Chemie International Edition, 54(35), 10327-10330
Open this publication in new window or tab >>Covalent Protein Labeling by Enzymatic Phosphocholination
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2015 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 54, no 35, p. 10327-10330Article in journal (Refereed) Published
Abstract [en]

We present a new protein labeling method based on the covalent enzymatic phosphocholination of a specific octapeptide amino acid sequence in intact proteins. The bacterial enzyme AnkX from Legionella pneumophila has been established to transfer functional phosphocholine moieties from synthetically produced CDP-choline derivatives to N-termini, C-termini, and internal loop regions in proteins of interest. Furthermore, the covalent modification can be hydrolytically removed by the action of the Legionella enzyme Lem3. Only a short peptide sequence (eight amino acids) is required for efficient protein labeling and a small linker group (PEG-phosphocholine) is introduced to attach the conjugated cargo.

Keywords
enzymes, nucleotides, phosphocholination, protein modifications
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-109454 (URN)10.1002/anie.201502618 (DOI)000360216800050 ()26147231 (PubMedID)
Funder
German Research Foundation (DFG)Knut and Alice Wallenberg FoundationMax Planck Society
Available from: 2015-10-08 Created: 2015-09-28 Last updated: 2018-06-07Bibliographically approved
Heller, K., Ochtrop, P., Albers, M., Hedberg, C. & Itzen, A. (2015). Enzymatic phosphocholination as a tool for protein labeling. Paper presented at 40th Congress of the Federation-of-European-Biochemical-Societies (FEBS) - The Biochemical Basis of Life, Berlin, July 4-9, 2015. The FEBS Journal, 282, 12-12
Open this publication in new window or tab >>Enzymatic phosphocholination as a tool for protein labeling
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2015 (English)In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 282, p. 12-12Article in journal, Meeting abstract (Other academic) Published
Abstract [en]

Posttranslational modification (PTM) of proteins is a versatile cellular process to regulate the activities of proteins. The high regioselectivity and catalysis rate of posttranslationally modifying enzymes utilizing high-energy precursors can potentially be exploited to equip proteins or peptide sequences with a label of choice site selectively and efficiently. We and others have recently described and analyzed a new reversible PTM called phosphocholination in which a phosphocholine group is transferred from a cytidine diphosphate choline (CDP-choline) to a serine residue of the small GTPase Rab1 [1–3]. The enzymes AnkX and Lem3 catalyze the modification and the corresponding demodification reactions, respectively. Interestingly, we could demonstrate that the modifying enzyme AnkX only requires a short amino acid sequence for substrate recognition. Therefore, we envision AnkX as a tool for the site directed labeling of target proteins. Here we report on the progress of developing a novel reversible protein labeling strategy based on the enzymes AnkX and Lem3 and on derivatives of CDP-choline. We demonstrate the optimization of AnkX and Lem3 enzyme activities and the identification of optimal and minimal peptide target sequences. Results indicate that indeed arbitrary proteins of interest can be functionalized with phosphocholine derivatives. In summary, this work yields first insights into the development of a CDP-choline based fully reversible protein labeling strategy.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-111160 (URN)10.1111/febs.13320 (DOI)000362570600031 ()
Conference
40th Congress of the Federation-of-European-Biochemical-Societies (FEBS) - The Biochemical Basis of Life, Berlin, July 4-9, 2015
Note

Supplement: 1

Special Issue: SI

Meeting Abstract: P14-004-SH

Available from: 2015-11-10 Created: 2015-11-06 Last updated: 2018-06-07Bibliographically approved
Ochtrop, P., Ernst, S., Itzen, A. & Hedberg, C.Exploring the substrate scope of the phosphocholine transferase AnkX for versatile protein functionalisation.
Open this publication in new window or tab >>Exploring the substrate scope of the phosphocholine transferase AnkX for versatile protein functionalisation
(English)Manuscript (preprint) (Other academic)
National Category
Organic Chemistry Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-140346 (URN)
Available from: 2017-10-05 Created: 2017-10-05 Last updated: 2018-06-09Bibliographically approved
Ochtrop, P., Swart, L., Simon, S., Janning, P., Dickhut, C., Zahedi, R. P., . . . Hedberg, C.Identification of cellular protein targets for the Legionella pneumophila phosphocholinating effector AnkX.
Open this publication in new window or tab >>Identification of cellular protein targets for the Legionella pneumophila phosphocholinating effector AnkX
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(English)Manuscript (preprint) (Other academic)
National Category
Biochemistry and Molecular Biology Microbiology
Identifiers
urn:nbn:se:umu:diva-140348 (URN)
Available from: 2017-10-05 Created: 2017-10-05 Last updated: 2018-06-09Bibliographically approved
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