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Adenylylation, MS, and proteomics-Introducing a "new" modification to bottom-up proteomics
Abt. Chemische Biologie, Max-Planck-Institut für molekulare Physiologie.
2013 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 13, no 6, 955-963 p.Article in journal (Refereed) Published
Abstract [en]

Although the addition of a 5'-adenosine phosphodiester group to proteins, called adenylylation, has been known for decades, the possibility that adenylylation could be a molecular switch in cellular signaling pathways has emerged recently. The distinct mass shift upon adenylation of threonine or tyrosine residues renders it a good target for MS detection and identification; however, the fragmentation of adenylylated peptides derived from proteolytic digestion of adenylylated proteins has not yet been systematically investigated. Here, we demonstrate that adenylylated peptides show loss of parts of the adenosine monophosphate (AMP) upon different fragmentation techniques. As expected, causing the least fragmentation of the AMP group, electron transfer dissociation yields less complicated spectra. In contrast, CID and higher energy collision (HCD) fragmentation caused AMP to fragment, generating characteristic ions that could be utilized in the specific identification of adenylylated peptides. The characteristic ions and losses upon CID and higher energy collision fragmentation from the AMP group turned out to be highly dependent on which amino acid was adenylylated, with different reporter ions for adenylylated threonine and tyrosine. We also investigated how adenylylation is best incorporated into search engines, exemplified by Mascot and showed that it is possible to identify adenylylation by search engines.

Place, publisher, year, edition, pages
2013. Vol. 13, no 6, 955-963 p.
Keyword [en]
Adenylylation, Adenylation, AMPylation, MS, Technology
National Category
Bioinformatics (Computational Biology)
Identifiers
URN: urn:nbn:se:umu:diva-114329DOI: 10.1002/pmic.201200344ISI: 000316150500007PubMedID: 23335384OAI: oai:DiVA.org:umu-114329DiVA: diva2:895053
Available from: 2016-01-18 Created: 2016-01-18 Last updated: 2016-01-27Bibliographically 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

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Albers, Michael
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