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Covalent Protein Labeling by Enzymatic Phosphocholination
Center for Integrated Protein Science Munich, Technische Universität München, Department Chemistry, Lichtenbergstrasse 4, 85748 Garching, Germany.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Center for Integrated Protein Science Munich, Technische Universität München, Department Chemistry, Lichtenbergstrasse 4, 85748 Garching, Germany.
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2015 (English)In: Angewandte Chemie International Edition, ISSN 1433-7851, E-ISSN 1521-3773, Vol. 54, no 35, 10327-10330 p.Article 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.

Place, publisher, year, edition, pages
2015. Vol. 54, no 35, 10327-10330 p.
Keyword [en]
enzymes, nucleotides, phosphocholination, protein modifications
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-109454DOI: 10.1002/anie.201502618ISI: 000360216800050PubMedID: 26147231OAI: oai:DiVA.org:umu-109454DiVA: diva2:859709
Funder
German Research Foundation (DFG)Knut and Alice Wallenberg FoundationMax Planck Society
Available from: 2015-10-08 Created: 2015-09-28 Last updated: 2017-10-09Bibliographically approved
In thesis
1. Synthesis and investigation of bacterial effector molecules
Open this publication in new window or tab >>Synthesis and investigation of bacterial effector molecules
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

During infections, bacterial microorganisms initiate profound interactions with mammalian host cells. Usually defense mechanisms of the host destroy intruding bacteria in rapid manner. However, many bacterial pathogens have evolved in a way to avoid these mechanisms. By use of effector molecules, which can be small organic molecules or proteins with enzymatic activity, the host is manipulated on a molecular level. Effectors mediating post-translational modifications (PTMs) are employed by many pathogens to influence the biological activity of host proteins. In the presented thesis, two related PTMs are investigated in detail: Adenylylation, the covalent transfer of an adenosine monophosphate group from adenosine triphosphate onto proteins, and phosphocholination, the covalent transfer of a phosphocholine moiety onto proteins. Over the past years, enzymes mediating these modifications have been discovered in several pathogens, especially as a mechanism to influence the signaling of eukaryotic cells by adenylylating or phosphocholinating small GTPases. However, the development of reliable methods for the isolation and identification of adenylylated and phosphocholinated proteins remains a vehement challenge in this field of research. This thesis presents general procedures for the synthesis of peptides carrying adenylylated or phosphocholinated tyrosine, threonine and serine residues. From the resulting peptides, mono-selective polyclonal antibodies against adenylylated tyrosine and threonine have been raised. The antibodies were used as tools for proteomic research to isolate unknown substrates of adenylyl transferases from eukaryotic cells. Mass spectrometric fragmentation techniques have been investigated to ease the identification of adenylylated proteins. Furthermore, this work presents a new strategy to identify adenylylated proteins. Additionally, small effector molecules are involved in the regulation of infection mechanisms. In this work, the small molecule LAI-1 (Legionella autoinducer 1) from the pathogen Legionella pneumophila, the causative agent of the Legionnaire’s disease, was synthesised together with its amino-derivatives. LAI-1 showed are a clear pharmacological effect on the regulation of the life cycle of L. pneumophila, initiating transmissive traits like motility and virulence. Furthermore, LAI-1 was shown to have an effect on eukaryotic cells as well. Directed motility of the eukaryotic cells was significantly reduced and the cytoskeletal architecture was reorganised, probably by interfering with the small GTPase Cdc42.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2016. 110 p.
Keyword
bacterial effectors, organic synthesis, Legionella, PTM, peptide synthesis, nucleotide chemistry
National Category
Organic Chemistry
Research subject
Biorganic Chemistry
Identifiers
urn:nbn:se:umu:diva-114698 (URN)978-91-7601-411-0 (ISBN)
Public defence
2016-02-19, KB3A9, KBC-huset, Umeå University, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2016-01-29 Created: 2016-01-26 Last updated: 2016-01-27Bibliographically approved
2. Selective protein functionalisation via enzymatic phosphocholination
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. 101 p.
Keyword
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: 2017-10-20Bibliographically approved

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